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1
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Liotti F, Marotta M, Costanzo M, De Simone C, Zirpoli S, De Falco V, Melillo RM, Prevete N. Formyl peptide receptor 1 signaling strength orchestrates the switch from pro-inflammatory to pro-resolving responses: The way to exert its anti-angiogenic and tumor suppressor functions. Biomed Pharmacother 2025; 186:117961. [PMID: 40112515 DOI: 10.1016/j.biopha.2025.117961] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2024] [Revised: 02/24/2025] [Accepted: 03/04/2025] [Indexed: 03/22/2025] Open
Abstract
The well-paced trigger of inflammation resolution following an inflammatory response is crucial for tissue homeostasis and cancer. In gastrointestinal tumors the Formyl peptide receptor 1 (FPR1) stimulates an inflammation resolution response able to restrain cancer angiogenesis and growth. A preceding inflammatory signal is necessary for the induction of the pro-resolving response. However, if FPR1-induced inflammation resolution and tumor suppressor function require an early pro-inflammatory trigger and how this is achieved remains unknown. A ROS-dependent signaling is activated in response to FPR1 activation. In colorectal carcinoma (CRC) cells, we carefully analyzed this signal showing that FPR1 activation by the fMLF peptide induces biphasic ROS production: a first wave, early, mitochondrial (mROS), followed by a second, late, NADPH oxidase (NOX1)-dependent. mROS cause SHP2 phosphatase inactivation restraining its ability to dephosphorylate and inactivate SRC. SRC, in turn, allows the activation of RAS and Rac1 GTPases. RAS activates MAPK signaling, while Rac1 supports NOX1 activation, that causes the second wave of ROS, reinforcing this signaling cycle. Importantly, for the first time, we demonstrate that mROS production precedes and is necessary for pro-inflammatory mediators' release, while NOX1-dependent ROS are only required for pro-resolving mediators' synthesis. Pharmacological and genetic approaches and functional assays show that this signaling cascade is essential for the pro-resolving and anti-angiogenic properties of FPR1 in CRC. In conclusion, we show that FPR1 elicits pro-resolving effects in CRC activating two waves of ROS production characterized by different strength and kinetics, that parallel and are necessary for pro-inflammatory or pro-resolving mediators' production.
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Affiliation(s)
- Federica Liotti
- Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Italy
| | - Maria Marotta
- Institute of Endotypes in Oncology, Metabolism and Immunology (IEOMI), CNR, Naples, Italy
| | - Mattia Costanzo
- Department of Translational Medical Sciences, University of Naples Federico II, Italy
| | - Chiara De Simone
- Department of Translational Medical Sciences, University of Naples Federico II, Italy
| | - Sara Zirpoli
- Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Italy
| | - Valentina De Falco
- Institute of Endotypes in Oncology, Metabolism and Immunology (IEOMI), CNR, Naples, Italy
| | - Rosa Marina Melillo
- Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Italy.
| | - Nella Prevete
- Department of Translational Medical Sciences, University of Naples Federico II, Italy.
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2
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Casacuberta-Serra S, González-Larreategui Í, Capitán-Leo D, Soucek L. MYC and KRAS cooperation: from historical challenges to therapeutic opportunities in cancer. Signal Transduct Target Ther 2024; 9:205. [PMID: 39164274 PMCID: PMC11336233 DOI: 10.1038/s41392-024-01907-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2024] [Revised: 06/05/2024] [Accepted: 06/24/2024] [Indexed: 08/22/2024] Open
Abstract
RAS and MYC rank amongst the most commonly altered oncogenes in cancer, with RAS being the most frequently mutated and MYC the most amplified. The cooperative interplay between RAS and MYC constitutes a complex and multifaceted phenomenon, profoundly influencing tumor development. Together and individually, these two oncogenes regulate most, if not all, hallmarks of cancer, including cell death escape, replicative immortality, tumor-associated angiogenesis, cell invasion and metastasis, metabolic adaptation, and immune evasion. Due to their frequent alteration and role in tumorigenesis, MYC and RAS emerge as highly appealing targets in cancer therapy. However, due to their complex nature, both oncogenes have been long considered "undruggable" and, until recently, no drugs directly targeting them had reached the clinic. This review aims to shed light on their complex partnership, with special attention to their active collaboration in fostering an immunosuppressive milieu and driving immunotherapeutic resistance in cancer. Within this review, we also present an update on the different inhibitors targeting RAS and MYC currently undergoing clinical trials, along with their clinical outcomes and the different combination strategies being explored to overcome drug resistance. This recent clinical development suggests a paradigm shift in the long-standing belief of RAS and MYC "undruggability", hinting at a new era in their therapeutic targeting.
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Affiliation(s)
| | - Íñigo González-Larreategui
- Models of cancer therapies Laboratory, Vall d'Hebron Institute of Oncology, Cellex Centre, Hospital University Vall d'Hebron Campus, Barcelona, Spain
| | - Daniel Capitán-Leo
- Models of cancer therapies Laboratory, Vall d'Hebron Institute of Oncology, Cellex Centre, Hospital University Vall d'Hebron Campus, Barcelona, Spain
| | - Laura Soucek
- Peptomyc S.L., Barcelona, Spain.
- Models of cancer therapies Laboratory, Vall d'Hebron Institute of Oncology, Cellex Centre, Hospital University Vall d'Hebron Campus, Barcelona, Spain.
- Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain.
- Department of Biochemistry and Molecular Biology, Universitat Autonoma de Barcelona, Bellaterra, Spain.
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3
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Chen P, Chen X, Song X, He A, Zheng Y, Li X, Tian R. Dissecting phospho-motif-dependent Shc1 interactome using long synthetic protein fragments. Chem Sci 2024; 15:d4sc02350a. [PMID: 39184293 PMCID: PMC11342145 DOI: 10.1039/d4sc02350a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2024] [Accepted: 08/10/2024] [Indexed: 08/27/2024] Open
Abstract
Activated receptor tyrosine kinases (RTKs) rely on the assembly of signaling proteins into high-dimensional protein complexes for signal transduction. Shc1, a prototypical scaffold protein, plays a pivotal role in directing phosphotyrosine (pY)-dependent protein complex formation for numerous RTKs typically through its two pY-binding domains. The three conserved pY sites within its CH1 region (Shc1CH1) hold particular significance due to their substantial contribution to its functions. However, how Shc1 differentially utilizes these sites to precisely coordinate protein complex assembly remains unclear. Here, we employed multiple peptide ligation techniques to synthesize an array of long protein fragments (107 amino acids) covering a significant portion of the Shc1CH1 region with varying phosphorylation states at residues Y239, 240, 313, and S335. By combining these phospho-Shc1CH1 fragments with integrated proteomics sample preparation and quantitative proteomic analysis, we were able to comprehensively resolve the site-specific interactomes of Shc1 with single amino acid resolution. By applying this approach to different cancer cell lines, we demonstrated that these phospho-Shc1CH1 fragments can be effectively used as a diagnostic tool to assess cell type-specific RTK signaling networks. Collectively, these biochemical conclusions help to better understand the sophisticated organization of pY-dependent Shc1 adaptor protein complexes and their functional roles in cancer.
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Affiliation(s)
- Peizhong Chen
- Department of Chemistry, College of Science, Southern University of Science and Technology Shenzhen 518055 China
- Department of Chemistry, State Key Lab of Synthetic Chemistry, University of Hong Kong Pokfulam Road Hong Kong SAR P. R. China
| | - Xiong Chen
- Department of Chemistry, College of Science, Southern University of Science and Technology Shenzhen 518055 China
- Shenzhen Key Laboratory of Functional Proteomics, Guangming Advanced Research Institute, Southern University of Science and Technology Shenzhen 518055 China
| | - Xiaolei Song
- State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics Beijing 102206 China
| | - An He
- Department of Chemistry, College of Science, Southern University of Science and Technology Shenzhen 518055 China
| | - Yong Zheng
- State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics Beijing 102206 China
- Key Laboratory of Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases, Ministry of Education, School of Basic Medicine, School of Rehabilitation Medicine, Gannan Medical University Ganzhou 341000 China
| | - Xuechen Li
- Department of Chemistry, State Key Lab of Synthetic Chemistry, University of Hong Kong Pokfulam Road Hong Kong SAR P. R. China
| | - Ruijun Tian
- Department of Chemistry, College of Science, Southern University of Science and Technology Shenzhen 518055 China
- Shenzhen Key Laboratory of Functional Proteomics, Guangming Advanced Research Institute, Southern University of Science and Technology Shenzhen 518055 China
- State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics Beijing 102206 China
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4
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Wilson SE. Two-phase mechanism in the treatment of corneal stromal fibrosis with topical losartan. Exp Eye Res 2024; 242:109884. [PMID: 38570181 DOI: 10.1016/j.exer.2024.109884] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2024] [Revised: 03/12/2024] [Accepted: 03/31/2024] [Indexed: 04/05/2024]
Abstract
Recent studies in rabbits and case reports in humans have demonstrated the efficacy of topical losartan in the treatment of corneal scarring fibrosis after a wide range of injuries, including chemical burns, infections, surgical complications, and some diseases. It is hypothesized that the effect of losartan on the fibrotic corneal stroma occurs through a two-phase process in which losartan first triggers the elimination of myofibroblasts by directing their apoptosis via inhibition of extracellular signal-regulated kinase (ERK)-mediated signal transduction, and possibly through signaling effects on the viability and development of corneal fibroblast and fibrocyte myofibroblast precursor cells. This first step likely occurs within a week or two in most corneas with fibrosis treated with topical losartan, but the medication must be continued for much longer until the epithelial basement membrane (EBM) is fully regenerated or new myofibroblasts will develop from precursor cells. Once the myofibroblasts are eliminated from the fibrotic stroma, corneal fibroblasts can migrate into the fibrotic tissue and reabsorb/reorganize the disordered extracellular matrix (ECM) previously produced by the myofibroblasts. This second stage is longer and more variable in different eyes of rabbits and humans, and accounts for most of the variability in the time it takes for the stromal opacity to be markedly reduced by topical losartan treatment. Eventually, keratocytes reemerge in the previously fibrotic stromal tissue to fine-tune the collagens and other ECM components and maintain the normal structure of the corneal stroma. The efficacy of losartan in the prevention and treatment of corneal fibrosis suggests that it acts as a surrogate for the EBM, by suppressing TGF beta-directed scarring of the wounded corneal stroma, until control over TGF beta action is re-established by a healed EBM, while also supporting regeneration of the EBM by allowing corneal fibroblasts to occupy the subepithelial stroma in the place of myofibroblasts.
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Affiliation(s)
- Steven E Wilson
- Cole Eye Institute, The Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH, USA.
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5
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Andrews SS, Wiley HS, Sauro HM. Design patterns of biological cells. Bioessays 2024; 46:e2300188. [PMID: 38247191 PMCID: PMC10922931 DOI: 10.1002/bies.202300188] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2023] [Revised: 12/03/2023] [Accepted: 12/14/2023] [Indexed: 01/23/2024]
Abstract
Design patterns are generalized solutions to frequently recurring problems. They were initially developed by architects and computer scientists to create a higher level of abstraction for their designs. Here, we extend these concepts to cell biology to lend a new perspective on the evolved designs of cells' underlying reaction networks. We present a catalog of 21 design patterns divided into three categories: creational patterns describe processes that build the cell, structural patterns describe the layouts of reaction networks, and behavioral patterns describe reaction network function. Applying this pattern language to the E. coli central metabolic reaction network, the yeast pheromone response signaling network, and other examples lends new insights into these systems.
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Affiliation(s)
- Steven S. Andrews
- Department of Bioengineering, University of Washington, Seattle, WA, USA
| | - H. Steven Wiley
- Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA
| | - Herbert M. Sauro
- Department of Bioengineering, University of Washington, Seattle, WA, USA
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6
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Raji L, Tetteh A, Amin ARMR. Role of c-Src in Carcinogenesis and Drug Resistance. Cancers (Basel) 2023; 16:32. [PMID: 38201459 PMCID: PMC10778207 DOI: 10.3390/cancers16010032] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2023] [Revised: 12/12/2023] [Accepted: 12/15/2023] [Indexed: 01/12/2024] Open
Abstract
The aberrant transformation of normal cells into cancer cells, known as carcinogenesis, is a complex process involving numerous genetic and molecular alterations in response to innate and environmental stimuli. The Src family kinases (SFK) are key components of signaling pathways implicated in carcinogenesis, with c-Src and its oncogenic counterpart v-Src often playing a significant role. The discovery of c-Src represents a compelling narrative highlighting groundbreaking discoveries and valuable insights into the molecular mechanisms underlying carcinogenesis. Upon oncogenic activation, c-Src activates multiple downstream signaling pathways, including the PI3K-AKT pathway, the Ras-MAPK pathway, the JAK-STAT3 pathway, and the FAK/Paxillin pathway, which are important for cell proliferation, survival, migration, invasion, metastasis, and drug resistance. In this review, we delve into the discovery of c-Src and v-Src, the structure of c-Src, and the molecular mechanisms that activate c-Src. We also focus on the various signaling pathways that c-Src employs to promote oncogenesis and resistance to chemotherapy drugs as well as molecularly targeted agents.
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Affiliation(s)
| | | | - A. R. M. Ruhul Amin
- Department of Pharmaceutical Sciences, Marshall University School of Pharmacy, Huntington, WV 25755, USA; (L.R.); (A.T.)
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7
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Powell AM, Edwards NA, Hunter H, Kiser P, Watson AJ, Cumming RC, Betts DH. Deletion of p66Shc Dysregulates ERK and STAT3 Activity in Mouse Embryonic Stem Cells, Enhancing Their Naive-Like Self-Renewal in the Presence of Leukemia Inhibitory Factor. Stem Cells Dev 2023; 32:434-449. [PMID: 37183401 DOI: 10.1089/scd.2022.0283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/16/2023] Open
Abstract
The ShcA adapter protein is necessary for early embryonic development. The role of ShcA in development is primarily attributed to its 52 and 46 kDa isoforms that transduce receptor tyrosine kinase signaling through the extracellular signal regulated kinase (ERK). During embryogenesis, ERK acts as the primary signaling effector, driving fate acquisition and germ layer specification. P66Shc, the largest of the ShcA isoforms, has been observed to antagonize ERK in several contexts; however, its role during embryonic development remains poorly understood. We hypothesized that p66Shc could act as a negative regulator of ERK activity during embryonic development, antagonizing early lineage commitment. To explore the role of p66Shc in stem cell self-renewal and differentiation, we created a p66Shc knockout murine embryonic stem cell (mESC) line. Deletion of p66Shc enhanced basal ERK activity, but surprisingly, instead of inducing mESC differentiation, loss of p66Shc enhanced the expression of core and naive pluripotency markers. Using pharmacologic inhibitors to interrogate potential signaling mechanisms, we discovered that p66Shc deletion permits the self-renewal of naive mESCs in the absence of conventional growth factors, by increasing their responsiveness to leukemia inhibitory factor (LIF). We discovered that loss of p66Shc enhanced not only increased ERK phosphorylation but also increased phosphorylation of Signal transducer and activator of transcription in mESCs, which may be acting to stabilize their naive-like identity, desensitizing them to ERK-mediated differentiation cues. These findings identify p66Shc as a regulator of both LIF-mediated ESC pluripotency and of signaling cascades that initiate postimplantation embryonic development and ESC commitment.
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Affiliation(s)
- Andrew M Powell
- Department of Biology, The University of Western Ontario, London, Canada
| | - Nicole A Edwards
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada
| | - Hailey Hunter
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada
| | - Patti Kiser
- Department of Pathology and Laboratory Medicine, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada
| | - Andrew J Watson
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada
- Department of Obstetrics and Gynaecology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada
- Genetics and Development Division, The Children's Health Research Institute, Lawson Health Research Institute, London, Canada
| | - Robert C Cumming
- Department of Biology, The University of Western Ontario, London, Canada
- Genetics and Development Division, The Children's Health Research Institute, Lawson Health Research Institute, London, Canada
| | - Dean H Betts
- Department of Biology, The University of Western Ontario, London, Canada
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada
- Department of Obstetrics and Gynaecology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada
- Genetics and Development Division, The Children's Health Research Institute, Lawson Health Research Institute, London, Canada
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8
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Barros P, Matos AM, Matos P, Jordan P. YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1. Biomolecules 2023; 13:949. [PMID: 37371529 DOI: 10.3390/biom13060949] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2023] [Revised: 05/26/2023] [Accepted: 06/01/2023] [Indexed: 06/29/2023] Open
Abstract
Recent developments in CFTR modulator drugs have had a significant transformational effect on the treatment of individuals with Cystic Fibrosis (CF) who carry the most frequent F508del-CFTR mutation in at least one allele. However, the clinical effects of these revolutionary drugs remain limited by their inability to fully restore the plasma membrane (PM) stability of the rescued mutant channels. Here, we shed new light on the molecular mechanisms behind the reduced half-life of rescued F508del-CFTR at the PM of airway cells. We describe that YES1 protein kinase is enriched in F508del-CFTR protein PM complexes, and that its interaction with rescued channels is mediated and dependent on the adaptor protein YAP1. Moreover, we show that interference with this complex, either by depletion of one of these components or inhibiting YES1 activity, is sufficient to significantly improve the abundance and stability of modulator-rescued F508del-CFTR at the surface of airway cells. In addition, we found that this effect was mediated by a decreased phosphorylation of the scaffold protein SHC1, a key regulator of MAPK pathway activity. In fact, we showed that depletion of SHC1 or inhibition of MAPK pathway signaling was sufficient to improve rescued F508del-CFTR surface levels, whereas an ectopic increase in pathway activation downstream of SHC1, through the use of a constitutively active H-RAS protein, abrogated the stabilizing effect of YES1 inhibition on rescued F508del-CFTR. Taken together, our findings not only provide new mechanistic insights into the regulation of modulator-rescued F508del-CFTR membrane stability, but also open exciting new avenues to be further explored in CF research and treatment.
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Affiliation(s)
- Patrícia Barros
- Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge, 1649-016 Lisboa, Portugal
- BioISI-Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, 1749-016 Lisboa, Portugal
| | - Ana M Matos
- Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge, 1649-016 Lisboa, Portugal
- BioISI-Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, 1749-016 Lisboa, Portugal
| | - Paulo Matos
- Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge, 1649-016 Lisboa, Portugal
- BioISI-Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, 1749-016 Lisboa, Portugal
| | - Peter Jordan
- Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge, 1649-016 Lisboa, Portugal
- BioISI-Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, 1749-016 Lisboa, Portugal
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9
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Hayes B, van der Geer P. STS-1 and STS-2, Multi-Enzyme Proteins Equipped to Mediate Protein-Protein Interactions. Int J Mol Sci 2023; 24:ijms24119214. [PMID: 37298164 DOI: 10.3390/ijms24119214] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2023] [Revised: 05/10/2023] [Accepted: 05/12/2023] [Indexed: 06/12/2023] Open
Abstract
STS-1 and STS-2 form a small family of proteins that are involved in the regulation of signal transduction by protein-tyrosine kinases. Both proteins are composed of a UBA domain, an esterase domain, an SH3 domain, and a PGM domain. They use their UBA and SH3 domains to modify or rearrange protein-protein interactions and their PGM domain to catalyze protein-tyrosine dephosphorylation. In this manuscript, we discuss the various proteins that have been found to interact with STS-1 or STS-2 and describe the experiments used to uncover their interactions.
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Affiliation(s)
- Barbara Hayes
- Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92105, USA
| | - Peter van der Geer
- Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego, CA 92105, USA
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10
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Jacobs NR, Norton PA. Role of chromosome 1q copy number variation in hepatocellular carcinoma. World J Hepatol 2021; 13:662-672. [PMID: 34239701 PMCID: PMC8239492 DOI: 10.4254/wjh.v13.i6.662] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Revised: 05/13/2021] [Accepted: 06/04/2021] [Indexed: 02/06/2023] Open
Abstract
Chromosome 1q often has been observed to be amplified in hepatocellular carcinoma. This review summarizes literature reports of multiple genes that have been proposed as possible 1q amplification drivers. These largely fall within 1q21-1q23. In addition, publicly available copy number alteration data from The Cancer Genome Atlas project were used to identify additional candidate genes involved in carcinogenesis. The most frequent location for gene amplification was 1q22, consistent with the results of the literature search. The genes TPM3 and NUF2 were found to be candidates whose amplification and/or mRNA up-regulation was most highly associated with poorer hepatocellular carcinoma outcomes.
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Affiliation(s)
- Nathan R Jacobs
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA 19102, United States
| | - Pamela A Norton
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA 19102, United States
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11
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Elumalai S, Karunakaran U, Moon JS, Won KC. NADPH Oxidase (NOX) Targeting in Diabetes: A Special Emphasis on Pancreatic β-Cell Dysfunction. Cells 2021; 10:cells10071573. [PMID: 34206537 PMCID: PMC8307876 DOI: 10.3390/cells10071573] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2021] [Revised: 06/12/2021] [Accepted: 06/16/2021] [Indexed: 12/24/2022] Open
Abstract
In type 2 diabetes, metabolic stress has a negative impact on pancreatic β-cell function and survival (T2D). Although the pathogenesis of metabolic stress is complex, an imbalance in redox homeostasis causes abnormal tissue damage and β-cell death due to low endogenous antioxidant expression levels in β-cells. Under diabetogenic conditions, the susceptibility of β-cells to oxidative damage by NADPH oxidase has been related to contributing to β-cell dysfunction. Here, we consider recent insights into how the redox response becomes deregulated under diabetic conditions by NADPH oxidase, as well as the therapeutic benefits of NOX inhibitors, which may provide clues for understanding the pathomechanisms and developing strategies aimed at the treatment or prevention of metabolic stress associated with β-cell failure.
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Affiliation(s)
- Suma Elumalai
- Innovative Center for Aging Research, Yeungnam University Medical Center, Daegu 42415, Korea; (S.E.); (U.K.)
| | - Udayakumar Karunakaran
- Innovative Center for Aging Research, Yeungnam University Medical Center, Daegu 42415, Korea; (S.E.); (U.K.)
| | - Jun-Sung Moon
- Innovative Center for Aging Research, Yeungnam University Medical Center, Daegu 42415, Korea; (S.E.); (U.K.)
- Department of Internal Medicine, Yeungnam Universtiy College of Medicine, Daegu 42415, Korea
- Correspondence: (J.-S.M.); (K.-C.W.); Tel.: +82-53-620-3825 (J.-S.W.); +82-53-620-3846 (K.-C.W.)
| | - Kyu-Chang Won
- Innovative Center for Aging Research, Yeungnam University Medical Center, Daegu 42415, Korea; (S.E.); (U.K.)
- Department of Internal Medicine, Yeungnam Universtiy College of Medicine, Daegu 42415, Korea
- Correspondence: (J.-S.M.); (K.-C.W.); Tel.: +82-53-620-3825 (J.-S.W.); +82-53-620-3846 (K.-C.W.)
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12
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Yoshizawa R, Umeki N, Yamamoto A, Murata M, Sako Y. Biphasic spatiotemporal regulation of GRB2 dynamics by p52SHC for transient RAS activation. Biophys Physicobiol 2021; 18:1-12. [PMID: 33665062 PMCID: PMC7902154 DOI: 10.2142/biophysico.bppb-v18.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Accepted: 01/05/2021] [Indexed: 12/14/2022] Open
Abstract
RTK-RAS-MAPK systems are major signaling pathways for cell fate decisions. Among the several RTK species, it is known that the transient activation of ERK (MAPK) stimulates cell proliferation, whereas its sustained activation induces cell differentiation. In both instances however, RAS activation is transient, suggesting that the strict temporal regulation of its activity is critical in normal cells. RAS on the cytoplasmic side of the plasma membrane is activated by SOS through the recruitment of GRB2/SOS complex to the RTKs that are phosphorylated after stimulation with growth factors. The adaptor protein GRB2 recognizes phospho-RTKs both directly and indirectly via another adaptor protein, SHC. We here studied the regulation of GRB2 recruitment under the SHC pathway using single-molecule imaging and fluorescence correlation spectroscopy in living cells. We stimulated MCF7 cells with a differentiation factor, heregulin, and observed the translocation, complex formation, and phosphorylation of cell signaling molecules including GRB2 and SHC. Our results suggest a biphasic regulation of the GRB2/SOS-RAS pathway by SHC: At the early stage (<10 min) of stimulation, SHC increased the amplitude of RAS activity by increasing the association sites for the GRB2/SOS complex on the plasma membrane. At the later stage however, SHC suppressed RAS activation and sequestered GRB2 molecules from the membrane through the complex formation in the cytoplasm. The latter mechanism functions additively to other mechanisms of negative feedback regulation of RAS from MEK and/or ERK to complete the transient activation dynamics of RAS.
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Affiliation(s)
- Ryo Yoshizawa
- Cellular Informatics Lab, RIKEN, Wako, Saitama 351-0198, Japan.,Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
| | - Nobuhisa Umeki
- Cellular Informatics Lab, RIKEN, Wako, Saitama 351-0198, Japan
| | | | - Masayuki Murata
- Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
| | - Yasushi Sako
- Cellular Informatics Lab, RIKEN, Wako, Saitama 351-0198, Japan
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13
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Buday L, Vas V. Novel regulation of Ras proteins by direct tyrosine phosphorylation and dephosphorylation. Cancer Metastasis Rev 2020; 39:1067-1073. [PMID: 32936431 PMCID: PMC7680326 DOI: 10.1007/s10555-020-09918-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2020] [Accepted: 06/19/2020] [Indexed: 12/01/2022]
Abstract
Somatic mutations in the RAS genes are frequent in human tumors, especially in pancreatic, colorectal, and non-small-cell lung cancers. Such mutations generally decrease the ability of Ras to hydrolyze GTP, maintaining the protein in a constitutively active GTP-bound form that drives uncontrolled cell proliferation. Efforts to develop drugs that target Ras oncoproteins have been unsuccessful. Recent emerging data suggest that Ras regulation is more complex than the scientific community has believed for decades. In this review, we summarize advances in the "textbook" view of Ras activation. We also discuss a novel type of Ras regulation that involves direct phosphorylation and dephosphorylation of Ras tyrosine residues. The discovery that pharmacological inhibition of the tyrosine phosphoprotein phosphatase SHP2 maintains mutant Ras in an inactive state suggests that SHP2 could be a novel drug target for the treatment of Ras-driven human cancers.
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Affiliation(s)
- László Buday
- Institute of Enzymology, Research Centre for Natural Sciences, Budapest, 1117, Hungary.
- Department of Medical Chemistry, Semmelweis University Medical School, Budapest, 1094, Hungary.
| | - Virág Vas
- Institute of Enzymology, Research Centre for Natural Sciences, Budapest, 1117, Hungary
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14
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Parker MI, Nikonova AS, Sun D, Golemis EA. Proliferative signaling by ERBB proteins and RAF/MEK/ERK effectors in polycystic kidney disease. Cell Signal 2020; 67:109497. [PMID: 31830556 PMCID: PMC6957738 DOI: 10.1016/j.cellsig.2019.109497] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2019] [Revised: 12/05/2019] [Accepted: 12/06/2019] [Indexed: 12/24/2022]
Abstract
A primary pathological feature of polycystic kidney disease (PKD) is the hyperproliferation of epithelial cells in renal tubules, resulting in formation of fluid-filled cysts. The proliferative aspects of the two major forms of PKD-autosomal dominant PKD (ADPKD), which arises from mutations in the polycystins PKD1 and PKD2, and autosomal recessive PKD (ARPKD), which arises from mutations in PKHD1-has encouraged investigation into protein components of the core cell proliferative machinery as potential drivers of PKD pathogenesis. In this review, we examine the role of signaling by ERBB proteins and their effectors, with a primary focus on ADPKD. The ERBB family of receptor tyrosine kinases (EGFR/ERBB1, HER2/ERBB2, ERBB3, and ERBB4) are activated by extracellular ligands, inducing multiple pro-growth signaling cascades; among these, activation of signaling through the RAS GTPase, and the RAF, MEK1/2, and ERK1/2 kinases enhance cell proliferation and restrict apoptosis during renal tubuloepithelial cyst formation. Characteristics of PKD include overexpression and mislocalization of the ERBB receptors and ligands, leading to enhanced activation and increased activity of downstream signaling proteins. The altered regulation of ERBBs and their effectors in PKD is influenced by enhanced activity of SRC kinase, which is promoted by the loss of cytoplasmic Ca2+ and an increase in cAMP-dependent PKA kinase activity that stimulates CFTR, driving the secretory phenotype of ADPKD. We discuss the interplay between ERBB/SRC signaling, and polycystins and their depending signaling, with emphasis on thes changes that affect cell proliferation in cyst expansion, as well as the inflammation-associated fibrogenesis, which characterizes progressive disease. We summarize the current progress of preclinical and clinical trials directed at inhibiting this signaling axis, and discuss potential future strategies that may be productive for controlling PKD.
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Affiliation(s)
- Mitchell I Parker
- Program in Molecular Therapeutics, Fox Chase Cancer Center, 19111, USA; Molecular & Cell Biology & Genetics (MCBG) Program, Drexel University College of Medicine, 19102, USA
| | - Anna S Nikonova
- Program in Molecular Therapeutics, Fox Chase Cancer Center, 19111, USA
| | - Danlin Sun
- Program in Molecular Therapeutics, Fox Chase Cancer Center, 19111, USA; Institute of Life Science, Jiangsu University, Jingkou District, Zhenjiang, Jiangsu 212013, China
| | - Erica A Golemis
- Program in Molecular Therapeutics, Fox Chase Cancer Center, 19111, USA.
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15
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Lin CC, Suen KM, Stainthorp A, Wieteska L, Biggs GS, Leitão A, Montanari CA, Ladbury JE. Targeting the Shc-EGFR interaction with indomethacin inhibits MAP kinase pathway signalling. Cancer Lett 2019; 457:86-97. [PMID: 31100409 PMCID: PMC6584941 DOI: 10.1016/j.canlet.2019.05.008] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2019] [Revised: 05/09/2019] [Accepted: 05/10/2019] [Indexed: 12/27/2022]
Abstract
Receptor tyrosine kinase (RTK)-mediated hyperactivation of the MAPK/Erk pathway is responsible for a large number of pathogenic outcomes including many cancers. Considerable effort has been directed at targeting this pathway with varying degrees of long term therapeutic success. Under non-stimulated conditions Erk is bound to the adaptor protein Shc preventing aberrant signalling by sequestering Erk from activation by Mek. Activated RTK recruits Shc, via its phosphotyrosine binding (PTB) domain (ShcPTB), precipitating the release of Erk to engage in a signalling response. Here we describe a novel approach to inhibition of MAP kinase signal transduction through attempting to preserve the Shc-Erk complex under conditions of activated receptor. A library of existing drug molecules was computationally screened for hits that would bind to the ShcPTB and block its interaction with the RTKs EGFR and ErbB2. The primary hit from the screen was indomethacin, a non-steroidal anti-inflammatory drug. Validation of this molecule in vitro and in cellular efficacy studies in cancer cells provides proof of principle of the approach to pathway down-regulation and a potential optimizable lead compound.
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Affiliation(s)
- Chi-Chuan Lin
- School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Kin Man Suen
- School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK; Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK
| | - Amy Stainthorp
- School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Lukasz Wieteska
- School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - George S Biggs
- Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EQ, UK
| | - Andrei Leitão
- Medicinal Chemistry Group (NEQUIMED), São Carlos Institute of Chemistry, University of São Paulo (IQSC-USP), 13566-590, São Carlos, SP, Brazil
| | - Carlos A Montanari
- Medicinal Chemistry Group (NEQUIMED), São Carlos Institute of Chemistry, University of São Paulo (IQSC-USP), 13566-590, São Carlos, SP, Brazil
| | - John E Ladbury
- School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK; Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India.
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16
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Zeglinski MR, Moghadam AR, Ande SR, Sheikholeslami K, Mokarram P, Sepehri Z, Rokni H, Mohtaram NK, Poorebrahim M, Masoom A, Toback M, Sareen N, Saravanan S, Jassal DS, Hashemi M, Marzban H, Schaafsma D, Singal P, Wigle JT, Czubryt MP, Akbari M, Dixon IM, Ghavami S, Gordon JW, Dhingra S. Myocardial Cell Signaling During the Transition to Heart Failure. Compr Physiol 2018; 9:75-125. [DOI: 10.1002/cphy.c170053] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
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17
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Mugabo Y, Lim GE. Scaffold Proteins: From Coordinating Signaling Pathways to Metabolic Regulation. Endocrinology 2018; 159:3615-3630. [PMID: 30204866 PMCID: PMC6180900 DOI: 10.1210/en.2018-00705] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/06/2018] [Accepted: 09/05/2018] [Indexed: 01/13/2023]
Abstract
Among their pleiotropic functions, scaffold proteins are required for the accurate coordination of signaling pathways. It has only been within the past 10 years that their roles in glucose homeostasis and metabolism have emerged. It is well appreciated that changes in the expression or function of signaling effectors, such as receptors or kinases, can influence the development of chronic diseases such as diabetes and obesity. However, little is known regarding whether scaffolds have similar roles in the pathogenesis of metabolic diseases. In general, scaffolds are often underappreciated in the context of metabolism or metabolic diseases. In the present review, we discuss various scaffold proteins and their involvement in signaling pathways related to metabolism and metabolic diseases. The aims of the present review were to highlight the importance of scaffold proteins and to raise awareness of their physiological contributions. A thorough understanding of how scaffolds influence metabolism could aid in the discovery of novel therapeutic approaches to treat chronic conditions, such as diabetes, obesity, and cardiovascular disease, for which the incidence of all continue to increase at alarming rates.
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Affiliation(s)
- Yves Mugabo
- Cardiometabolic Axis, Centre de Recherche de Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada
- Montréal Diabetes Research Centre, Montreal, Quebec, Canada
- Department of Medicine, Université de Montréal, Montreal, Quebec, Canada
| | - Gareth E Lim
- Cardiometabolic Axis, Centre de Recherche de Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada
- Montréal Diabetes Research Centre, Montreal, Quebec, Canada
- Department of Medicine, Université de Montréal, Montreal, Quebec, Canada
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18
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Shan J, Dudenhausen E, Kilberg MS. Induction of early growth response gene 1 (EGR1) by endoplasmic reticulum stress is mediated by the extracellular regulated kinase (ERK) arm of the MAPK pathways. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2018; 1866:371-381. [PMID: 30290239 DOI: 10.1016/j.bbamcr.2018.09.009] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/07/2018] [Revised: 09/13/2018] [Accepted: 09/23/2018] [Indexed: 12/11/2022]
Abstract
Endoplasmic reticulum (ER) stress activates three principal signaling pathways, collectively known as the unfolded protein response, leading to translational and transcriptional control mechanisms that dictate the cell's response as adaptive or apoptotic. The present study illustrates that for HepG2 human hepatocellular carcinoma cells the signaling pathways triggered by ER stress extend beyond the three principal pathways to include mitogen-activated protein kinase (MAPK) signaling, leading to activation of transcription from the early growth response 1 (EGR1) gene. Analysis provided evidence for a SRC-RAS-RAF-MEK-ERK cascade mechanism that leads to enhanced phosphorylation of the transcription factor ELK1. ELK1 and serum response factor (SRF) are constitutively bound to the EGR1 promoter and are phosphorylated by nuclear localized ERK. The promoter abundance of both phospho-SRF and phopsho-ELK1 was increased by ER stress, but the SRF phosphorylation was transient. Knockdown of ELK1 had little effect on the basal EGR1 mRNA content, but completely blocked the increase in response to ER stress. Conversely, knockdown of SRF suppressed basal EGR1 mRNA content, but had only a small effect on the induction by ER stress. This research highlights the importance of MAPK signaling in response to ER stress and identifies ELK1 as a transcriptional mediator and the EGR1 gene as a target.
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Affiliation(s)
- Jixiu Shan
- Department of Biochemistry and Molecular Biology, Genetics Institute, Shands Cancer Center and Center for Nutritional Sciences, University of Florida College of Medicine, Gainesville 32610, FL, United States of America
| | - Elizabeth Dudenhausen
- Department of Biochemistry and Molecular Biology, Genetics Institute, Shands Cancer Center and Center for Nutritional Sciences, University of Florida College of Medicine, Gainesville 32610, FL, United States of America
| | - Michael S Kilberg
- Department of Biochemistry and Molecular Biology, Genetics Institute, Shands Cancer Center and Center for Nutritional Sciences, University of Florida College of Medicine, Gainesville 32610, FL, United States of America.
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19
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Kakizawa S. [Functional roles of phosphotyrosine adaptor Shc in the brain]. Nihon Yakurigaku Zasshi 2018; 152:84-89. [PMID: 30101865 DOI: 10.1254/fpj.152.84] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Abstract
Adaptor molecules (adaptor proteins) have indispensable roles in cellular signaling, essential for cellular proliferation, development and metabolism. Shc (Src homology and collagen homology)-family molecule is a group of adaptor molecules, and indicated to be involved in intracellular phosphotyrosine signaling. Shc family has 4 subtypes, ShcA-ShcD, and there are long and short isoforms in ShcA and ShcC whereas ShcB and ShcD have short isoform only. There are three domains conserved in all Shc-family isoforms: phosphotyrosine-binding (PTB) domain, collagen-homology 1 (CH1) domain and Src-homology 2 (SH2) domain, from the N-terminal to C-terminal. PTB and SH2 domains recognize and bind to phosphotyrosine in other molecules, and CH1 domain is recognized and bind to SH2 domain in Grb2, an adaptor molecule, when the tyrosine residues in the domain are phosphorylated. Expression of ShcA is observed in all tissues except for brain in adult animals, although ShcA mRNA is detected in brain during embryonic days. On the other hand, in adult brain, expressions of ShcB, ShcC, and ShcD are observed. Analysis of single knockout mice (ShcA (neuron specific), ShcB, ShcC) and double knockout mice for ShcB and C indicated essential roles of Shc-family molecules in proliferation and survival of cells in various brain regions as well as synaptic plasticity and higher brain functions such as learning and memory. Studies on multiple-knockout mice of Shc-family molecules may further clarify possible involvements of Shc family in physiological and pathophysiological functions in brain.
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Affiliation(s)
- Sho Kakizawa
- Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University
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20
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Tsutsui Y, Hays FA. A Link Between Alzheimer's and Type II Diabetes Mellitus? Ca +2 -Mediated Signal Control and Protein Localization. Bioessays 2018; 40:e1700219. [PMID: 29694668 PMCID: PMC6166406 DOI: 10.1002/bies.201700219] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2017] [Revised: 03/16/2018] [Indexed: 01/28/2023]
Abstract
We propose protein localization dependent signal activation (PLDSA) as a model to describe pre-existing protein partitioning between the cytosol, and membrane surface, as a means to modulate signal activation, specificity, and robustness. We apply PLDSA to explain possible molecular links between type II diabetes mellitus (T2DM) and Alzheimer's disease (AD) by describing Ca+2 -mediated interactions between the Src non-receptor tyrosine kinase and p52Shc adaptor protein. We suggest that these interactions may serve as a contributing factor to disease development and progression. In particular, we propose that signaling response is regulated, in part, by Ca+2 -mediated partitioning of lipid-bound and soluble forms of Src and p52shc. Thus, protein-protein interactions that drive signaling in response to extracellular ligand binding are also mediated by partitioning of signaling proteins between membrane-bound and soluble populations. We propose that PLDSA effects may explain, in part, the evolutionary basis of promiscuous protein interaction domains and their importance in cellular function.
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Affiliation(s)
- Yuko Tsutsui
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States
| | - Franklin A. Hays
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States
- Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, 73104, United States
- Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, 73104, United States
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21
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Boopathy GTK, Lynn JLS, Wee S, Gunaratne J, Hong W. Phosphorylation of Mig6 negatively regulates the ubiquitination and degradation of EGFR mutants in lung adenocarcinoma cell lines. Cell Signal 2017; 43:21-31. [PMID: 29196224 DOI: 10.1016/j.cellsig.2017.11.006] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2017] [Revised: 11/16/2017] [Accepted: 11/27/2017] [Indexed: 12/29/2022]
Abstract
Activating mutations in the kinase domain of epidermal growth factor receptor (EGFR) leads to the constitutively active kinase, improves the EGFR stability and promotes malignant transformation in lung adenocarcinoma. Despite the clinical significance, the mechanism by which the increased kinase activity stabilizes the receptor is not completely understood. Using SILAC phosphoproteomic approach, we identify that Mig6 is highly phosphorylated at S256 in EGFR mutants (19del and L858R). Loss of Mig6 contributes to the efficient degradation of EGFR wildtype and mutants in lung cancer cells. Mig6 regulates the recruitment of c-Cbl to EGFR as the ablation of Mig6 enables efficient ubiquitination of the EGFR mutants through elevated recruitment of c-Cbl. We show that the cells with activating mutants of EGFR inactivate Mig6 through phosphorylation at S256. Inactivated Mig6 causes inefficient ubiquitination of EGFR, leading to defective degradation of the receptor thus contributing to the increased stability of the receptor. Taken together, we show a novel function of Mig6 in regulating the ubiquitination of EGFR.
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Affiliation(s)
- Gandhi T K Boopathy
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, 138673, Singapore.
| | - Julia Lim Sze Lynn
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, 138673, Singapore
| | - Sheena Wee
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, 138673, Singapore
| | - Jayantha Gunaratne
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, 138673, Singapore
| | - Wanjin Hong
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, 138673, Singapore.
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22
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Wright KD, Staruschenko A, Sorokin A. Role of adaptor protein p66Shc in renal pathologies. Am J Physiol Renal Physiol 2017; 314:F143-F153. [PMID: 28978535 DOI: 10.1152/ajprenal.00414.2017] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
p66Shc is one of the three adaptor proteins encoded by the Shc1 gene, which are expressed in many organs, including the kidney. Recent studies shed new light on several key questions concerning the signaling mechanisms mediated by p66Shc. The central goal of this review article is to summarize recent findings on p66Shc and the role it plays in kidney physiology and pathology. This article provides a review of the various mechanisms whereby p66Shc has been shown to function within the kidney through a wide range of actions. The mitochondrial and cytoplasmic signaling of p66Shc, as it relates to production of reactive oxygen species (ROS) and renal pathologies, is further discussed.
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Affiliation(s)
- Kevin D Wright
- Cardiovascular Center, Medical College of Wisconsin , Milwaukee, Wisconsin.,Department of Medicine, Medical College of Wisconsin , Milwaukee, Wisconsin
| | - Alexander Staruschenko
- Cardiovascular Center, Medical College of Wisconsin , Milwaukee, Wisconsin.,Department of Physiology, Medical College of Wisconsin , Milwaukee, Wisconsin
| | - Andrey Sorokin
- Cardiovascular Center, Medical College of Wisconsin , Milwaukee, Wisconsin.,Department of Medicine, Medical College of Wisconsin , Milwaukee, Wisconsin
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23
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Engin HB, Carlin D, Pratt D, Carter H. Modeling of RAS complexes supports roles in cancer for less studied partners. BMC BIOPHYSICS 2017; 10:5. [PMID: 28815022 PMCID: PMC5558186 DOI: 10.1186/s13628-017-0037-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Background RAS protein interactions have predominantly been studied in the context of the RAF and PI3kinase oncogenic pathways. Structural modeling and X-ray crystallography have demonstrated that RAS isoforms bind to canonical downstream effector proteins in these pathways using the highly conserved switch I and II regions. Other non-canonical RAS protein interactions have been experimentally identified, however it is not clear whether these proteins also interact with RAS via the switch regions. Results To address this question we constructed a RAS isoform-specific protein-protein interaction network and predicted 3D complexes involving RAS isoforms and interaction partners to identify the most probable interaction interfaces. The resulting models correctly captured the binding interfaces for well-studied effectors, and additionally implicated residues in the allosteric and hyper-variable regions of RAS proteins as the predominant binding site for non-canonical effectors. Several partners binding to this new interface (SRC, LGALS1, RABGEF1, CALM and RARRES3) have been implicated as important regulators of oncogenic RAS signaling. We further used these models to investigate competitive binding and multi-protein complexes compatible with RAS surface occupancy and the putative effects of somatic mutations on RAS protein interactions. Conclusions We discuss our findings in the context of RAS localization to the plasma membrane versus within the cytoplasm and provide a list of RAS protein interactions with possible cancer-related consequences, which could help guide future therapeutic strategies to target RAS proteins. Electronic supplementary material The online version of this article (doi:10.1186/s13628-017-0037-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- H Billur Engin
- Division of Medical Genetics, Department of Medicine, Universsity of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093 USA
| | - Daniel Carlin
- Division of Medical Genetics, Department of Medicine, Universsity of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093 USA
| | - Dexter Pratt
- Division of Medical Genetics, Department of Medicine, Universsity of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093 USA
| | - Hannah Carter
- Division of Medical Genetics, Department of Medicine, Universsity of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093 USA
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24
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Wills MKB, Lau HR, Jones N. The ShcD phosphotyrosine adaptor subverts canonical EGF receptor trafficking. J Cell Sci 2017; 130:2808-2820. [PMID: 28724758 DOI: 10.1242/jcs.198903] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2016] [Accepted: 07/09/2017] [Indexed: 12/15/2022] Open
Abstract
Shc family signalling adaptors connect activated transmembrane receptors to proximal effectors, and most also contain a sequence involved in clathrin-mediated receptor endocytosis. Notably, this AP2 adaptin-binding motif (AD) is absent from the ShcD (also known as Shc4) homolog, which also uniquely promotes ligand-independent phosphorylation of the epidermal growth factor receptor (EGFR). We now report that cultured cells expressing ShcD exhibit reduced EGF uptake, commensurate with a decrease in EGFR surface presentation. Under basal conditions, ShcD colocalises with the EGFR and facilitates its phosphorylation, ubiquitylation and accumulation in juxtanuclear vesicles identified as Rab11-positive endocytic recycling compartments. Accordingly, ShcD also functions as a constitutive binding partner for the E3 ubiquitin ligase Cbl. EGFR phosphorylation and focal accumulation likewise occur upon ShcD co-expression in U87 glioma cells. Loss of ShcD phosphotyrosine-binding function or insertion of the ShcA AD sequence each restore ligand acquisition through distinct mechanisms. The AD region also contains a nuclear export signal, indicating its multifunctionality. Overall, ShcD appears to possess several molecular permutations that actively govern the EGFR, which may have implications in development and disease.
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Affiliation(s)
- Melanie K B Wills
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada
| | - Hayley R Lau
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada
| | - Nina Jones
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada
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25
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van der Meulen T, Swarts S, Fischer W, van der Geer P. Identification of STS-1 as a novel ShcA-binding protein. Biochem Biophys Res Commun 2017; 490:1334-1339. [PMID: 28690151 DOI: 10.1016/j.bbrc.2017.07.024] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2017] [Accepted: 07/05/2017] [Indexed: 11/19/2022]
Abstract
ShcA is a cytoplasmic signaling protein that supports signal transduction by receptor protein-tyrosine kinases by providing auxiliary tyrosine phosphorylation sites that engage additional signaling proteins. The principal binding partner for tyrosine phosphorylation sites on ShcA is Grb2. In the current study, we have used phosphotyrosine-containing peptides to isolate and identify STS-1 as a novel ShcA-binding protein. Our results further show that the interaction between STS-1 and ShcA is regulated in response to EGF receptor activation.
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Affiliation(s)
- Talitha van der Meulen
- Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-1030, USA
| | - Spencer Swarts
- Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-1030, USA
| | - Wolfgang Fischer
- Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Rd, La Jolla, CA 92037, USA
| | - Peter van der Geer
- Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-1030, USA.
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26
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The Shc1 adaptor simultaneously balances Stat1 and Stat3 activity to promote breast cancer immune suppression. Nat Commun 2017; 8:14638. [PMID: 28276425 PMCID: PMC5347092 DOI: 10.1038/ncomms14638] [Citation(s) in RCA: 49] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2016] [Accepted: 01/18/2017] [Indexed: 02/07/2023] Open
Abstract
Tyrosine kinase signalling within cancer cells is central to the establishment of an immunosuppressive microenvironment. Although tyrosine kinase inhibitors act, in part, to augment adaptive immunity, the increased heterogeneity and functional redundancy of the tyrosine kinome is a hurdle to achieving durable responses to immunotherapies. We previously identified the Shc1 (ShcA) scaffold, a central regulator of tyrosine kinase signalling, as essential for promoting breast cancer immune suppression. Herein we show that the ShcA pathway simultaneously activates STAT3 immunosuppressive signals and impairs STAT1-driven immune surveillance in breast cancer cells. Impaired Y239/Y240-ShcA phosphorylation selectively reduces STAT3 activation in breast tumours, profoundly sensitizing them to immune checkpoint inhibitors and tumour vaccines. Finally, the ability of diminished tyrosine kinase signalling to initiate STAT1-driven immune surveillance can be overcome by compensatory STAT3 hyperactivation in breast tumours. Our data indicate that inhibition of pY239/240-ShcA-dependent STAT3 signalling may represent an attractive therapeutic strategy to sensitize breast tumours to multiple immunotherapies. Tyrosine kinase signalling in cancer cells promotes immune evasion. Here, the authors show that tyrosine kinases engage scaffold protein Shc1 to promote immunosuppression in breast cancer by simultaneously activating STAT3 immunosuppressive signals and impairing STAT1-driven anti-tumour immune responses.
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27
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The Roles of Insulin-Like Growth Factors in Mesenchymal Stem Cell Niche. Stem Cells Int 2017; 2017:9453108. [PMID: 28298931 PMCID: PMC5337393 DOI: 10.1155/2017/9453108] [Citation(s) in RCA: 50] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Revised: 12/22/2016] [Accepted: 01/18/2017] [Indexed: 12/12/2022] Open
Abstract
Many tissues contain adult mesenchymal stem cells (MSCs), which may be used in tissue regeneration therapies. However, the MSC availability in most tissues is limited which demands expansion in vitro following isolation. Like many developing cells, the state of MSCs is affected by the surrounding microenvironment, and mimicking this natural microenvironment that supports multipotent or differentiated state in vivo is essential to understand for the successful use of MSC in regenerative therapies. Many researchers are, therefore, optimizing cell culture conditions in vitro by altering growth factors, extracellular matrices, chemicals, oxygen tension, and surrounding pH to enhance stem cells self-renewal or differentiation. Insulin-like growth factors (IGFs) system has been demonstrated to play an important role in stem cell biology to either promote proliferation and self-renewal or enhance differentiation onset and outcome, depending on the cell culture conditions. In this review, we will describe the importance of IGFs, IGF-1 and IGF-2, in development and in the MSC niche and how they affect the pluripotency or differentiation towards multiple lineages of the three germ layers.
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A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling. J Virol 2017; 91:JVI.01774-16. [PMID: 27852846 DOI: 10.1128/jvi.01774-16] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2016] [Accepted: 10/29/2016] [Indexed: 02/06/2023] Open
Abstract
Middle T antigen (MT), the principal oncoprotein of murine polyomavirus, transforms by association with cellular proteins. Protein phosphatase 2A (PP2A), YAP, Src family tyrosine kinases, Shc, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ1) have all been implicated in MT transformation. Mutant dl1015, with deletion of residues 338 to 347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets Src homology domain 3 (SH3) domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation defective. Extensive analysis of the E349K mutant is described here. Similar to wild-type MT, the E349K mutant associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase, and PLCγ1. The E349K mutant was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by coimmunoprecipitation, showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of the E349K mutant with PI3K, cells expressing the mutant failed to elevate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in mutant-expressing cells. These results indicate a novel unsuspected aspect to PI3K control. IMPORTANCE The gene coding for middle T antigen (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like the wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like the wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect.
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Kano Y, Cook JD, Lee JE, Ohh M. New structural and functional insight into the regulation of Ras. Semin Cell Dev Biol 2016; 58:70-8. [DOI: 10.1016/j.semcdb.2016.06.006] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2016] [Revised: 06/06/2016] [Accepted: 06/07/2016] [Indexed: 10/21/2022]
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30
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Huang H, Svoboda RA, Lazenby AJ, Saowapa J, Chaika N, Ding K, Wheelock MJ, Johnson KR. Up-regulation of N-cadherin by Collagen I-activated Discoidin Domain Receptor 1 in Pancreatic Cancer Requires the Adaptor Molecule Shc1. J Biol Chem 2016; 291:23208-23223. [PMID: 27605668 DOI: 10.1074/jbc.m116.740605] [Citation(s) in RCA: 48] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2016] [Indexed: 12/13/2022] Open
Abstract
Pancreatic ductal adenocarcinomas are highly malignant cancers characterized by extensive invasion into surrounding tissues, metastasis to distant organs, and a limited response to therapy. A main feature of pancreatic ductal adenocarcinomas is desmoplasia, which leads to extensive deposition of collagen I. We have demonstrated that collagen I can induce epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. A hallmark of EMT is an increase in the expression of the mesenchymal cadherin N-cadherin. Previously we showed up-regulation of N-cadherin promotes tumor cell invasion and that collagen I-induced EMT is mediated by two collagen receptors, α2β1-integrin and discoidin domain receptor 1 (DDR1). DDR1 is a receptor-tyrosine kinase widely expressed during embryonic development and in many adult tissues and is also highly expressed in many different cancers. In the signaling pathway initiated by collagen, we have shown proline-rich tyrosine kinase 2 (Pyk2) is downstream of DDR1. In this study we found isoform b of DDR1 is responsible for collagen I-induced up-regulation of N-cadherin and tyrosine 513 of DDR1b is necessary. Knocking down Shc1, which binds to tyrosine 513 of DDR1b via its PTB (phosphotyrosine binding) domain, eliminates the up-regulation of N-cadherin. The signaling does not require a functional SH2 domain or the tyrosine residues commonly phosphorylated in Shc1 but is mediated by the interaction between a short segment of the central domain of Shc1 and the proline-rich region of Pyk2. Taken together, these data illustrate DDR1b, but not DDR1a, mediates collagen I-induced N-cadherin up-regulation, and Shc1 is involved in this process by coupling to both DDR1 and Pyk2.
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Affiliation(s)
- Huocong Huang
- From the Department of Biochemistry and Molecular Biology, College of Medicine
| | | | - Audrey J Lazenby
- Department of Pathology and Microbiology, College of Medicine, and
| | | | - Nina Chaika
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha Nebraska 68198
| | - Ke Ding
- State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, #190 Kaiyuan Avenue, Guangzhou 510530, China, and
| | - Margaret J Wheelock
- From the Department of Biochemistry and Molecular Biology, College of Medicine.,Department of Oral Biology, College of Dentistry.,Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha Nebraska 68198
| | - Keith R Johnson
- From the Department of Biochemistry and Molecular Biology, College of Medicine, .,Department of Oral Biology, College of Dentistry.,Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha Nebraska 68198.,Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198
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31
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Ha JR, Siegel PM, Ursini-Siegel J. The Tyrosine Kinome Dictates Breast Cancer Heterogeneity and Therapeutic Responsiveness. J Cell Biochem 2016; 117:1971-90. [PMID: 27392311 DOI: 10.1002/jcb.25561] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2016] [Accepted: 03/24/2016] [Indexed: 12/13/2022]
Abstract
Phospho-tyrosine signaling networks control numerous biological processes including cellular differentiation, cell growth and survival, motility, and invasion. Aberrant regulation of the tyrosine kinome is a hallmark of malignancy and influences all stages of breast cancer progression, from initiation to the development of metastatic disease. The success of specific tyrosine kinase inhibitors strongly validates the clinical relevance of tyrosine phosphorylation networks in breast cancer pathology. However, a significant degree of redundancy exists within the tyrosine kinome. Numerous receptor and cytoplasmic tyrosine kinases converge on a core set of signaling regulators, including adaptor proteins and tyrosine phosphatases, to amplify pro-tumorigenic signal transduction pathways. Mutational activation, amplification, or overexpression of one or more components of the tyrosine kinome represents key contributing events responsible for the tumor heterogeneity that is observed in breast cancers. It is this molecular heterogeneity that has become the most significant barrier to durable clinical responses due to the development of therapeutic resistance. This review focuses on recent literature that supports a prominent role for specific components of the tyrosine kinome in the emergence of unique breast cancer subtypes and in shaping breast cancer plasticity, sensitivity to targeted therapies, and the eventual emergence of acquired resistance. J. Cell. Biochem. 117: 1971-1990, 2016. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Jacqueline R Ha
- Lady Davis Institute for Medical Research, Montreal, Quebec, Canada.,Department of Medicine, McGill University, Montreal, Quebec, Canada.,Department of Oncology, McGill University, Montreal, Quebec, Canada
| | - Peter M Siegel
- Department of Medicine, McGill University, Montreal, Quebec, Canada.,Department of Oncology, McGill University, Montreal, Quebec, Canada.,Goodman Cancer Research Centre, McGill University, Montreal, Quebec, Canada.,Department of Biochemistry, McGill University, Montreal, Quebec, Canada.,Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada
| | - Josie Ursini-Siegel
- Lady Davis Institute for Medical Research, Montreal, Quebec, Canada.,Department of Medicine, McGill University, Montreal, Quebec, Canada.,Department of Oncology, McGill University, Montreal, Quebec, Canada
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32
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EGF-receptor specificity for phosphotyrosine-primed substrates provides signal integration with Src. Nat Struct Mol Biol 2015; 22:983-90. [PMID: 26551075 PMCID: PMC4824005 DOI: 10.1038/nsmb.3117] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2013] [Accepted: 09/30/2015] [Indexed: 01/07/2023]
Abstract
Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK pathway and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Using peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, thus promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly than singly phosphorylated peptide to the Ras activator Grb2; this binding is a key step in activating the Ras-MAPK pathway. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras.
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33
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AKT inactivation causes persistent drug tolerance to EGFR inhibitors. Pharmacol Res 2015; 102:132-7. [PMID: 26453958 DOI: 10.1016/j.phrs.2015.09.022] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/29/2015] [Accepted: 09/30/2015] [Indexed: 12/31/2022]
Abstract
Drug resistance is a major obstacle to the success of EGFR-targeted therapy. We recently studied the mechanism by which a small subset of EGFR mutant lung cancer cells remains viable after EGFR inhibition. We found that this drug-tolerant subpopulation develops because EGFR inhibition prevents AKT activity and thus inactivates Ets-1 function. In this article, we discuss how changes in intrinsic cell signaling after EGFR inhibition open a new avenue to drug resistance in NSCLCs, and comment on combined TKI and MEK inhibitor treatment to reduce the probability of emergent resistance to EGFR TKIs.
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Tang N, Lyu D, Liu T, Chen F, Jing S, Hao T, Liu S. Different Effects of p52SHC1 and p52SHC3 on the Cell Cycle of Neurons and Neural Stem Cells. J Cell Physiol 2015; 231:172-80. [DOI: 10.1002/jcp.25069] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2014] [Revised: 04/27/2015] [Accepted: 06/05/2015] [Indexed: 12/27/2022]
Affiliation(s)
- Ning Tang
- State Key Laboratory of Proteomics and; Department of Neurobiology; Institute of Basic Medical Sciences; Beijing P. R. China
- Reproductive Medicine Center; Jinan Military General Hospital; Jinan P. R. China
| | - Dan Lyu
- State Key Laboratory of Proteomics and; Department of Neurobiology; Institute of Basic Medical Sciences; Beijing P. R. China
- Dan Lyu is currently working in Department of Pain Management; Tianjin First Center Hospital; Tianjin P. R. China
| | - Tao Liu
- State Key Laboratory of Proteomics and; Department of Neurobiology; Institute of Basic Medical Sciences; Beijing P. R. China
| | - Fangjin Chen
- State Key Laboratory of Proteomics and; Department of Neurobiology; Institute of Basic Medical Sciences; Beijing P. R. China
| | - Shuqian Jing
- State Key Laboratory of Proteomics and; Department of Neurobiology; Institute of Basic Medical Sciences; Beijing P. R. China
| | - Tianyu Hao
- Reproductive Medicine Center; Jinan Military General Hospital; Jinan P. R. China
| | - Shaojun Liu
- State Key Laboratory of Proteomics and; Department of Neurobiology; Institute of Basic Medical Sciences; Beijing P. R. China
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EGFR inhibition evokes innate drug resistance in lung cancer cells by preventing Akt activity and thus inactivating Ets-1 function. Proc Natl Acad Sci U S A 2015; 112:E3855-63. [PMID: 26150526 DOI: 10.1073/pnas.1510733112] [Citation(s) in RCA: 79] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. About 14% of NSCLCs harbor mutations in epidermal growth factor receptor (EGFR). Despite remarkable progress in treatment with tyrosine kinase inhibitors (TKIs), only 5% of patients achieve tumor reduction >90%. The limited primary responses are attributed partly to drug resistance inherent in the tumor cells before therapy begins. Recent reports showed that activation of receptor tyrosine kinases (RTKs) is an important determinant of this innate drug resistance. In contrast, we demonstrate that EGFR inhibition promotes innate drug resistance despite blockade of RTK activity in NSCLC cells. EGFR TKIs decrease both the mitogen-activated protein kinase (MAPK) and Akt protein kinase pathways for a short time, after which the Ras/MAPK pathway becomes reactivated. Akt inhibition selectively blocks the transcriptional activation of Ets-1, which inhibits its target gene, dual specificity phosphatase 6 (DUSP6), a negative regulator specific for ERK1/2. As a result, ERK1/2 is activated. Furthermore, elevated c-Src stimulates Ras GTP-loading and activates Raf and MEK kinases. These observations suggest that not only ERK1/2 but also Akt activity is essential to maintain Ets-1 in an active state. Therefore, despite high levels of ERK1/2, Ets-1 target genes including DUSP6 and cyclins D1, D3, and E2 remain suppressed by Akt inhibition. Reduction of DUSP6 in combination with elevated c-Src renews activation of the Ras/MAPK pathway, which enhances cell survival by accelerating Bim protein turnover. Thus, EGFR TKIs evoke innate drug resistance by preventing Akt activity and inactivating Ets-1 function in NSCLC cells.
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SHC1 sensitizes cancer cells to the 8-Cl-cAMP treatment. Biochem Biophys Res Commun 2015; 463:673-8. [PMID: 26043699 DOI: 10.1016/j.bbrc.2015.05.123] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2015] [Accepted: 05/30/2015] [Indexed: 02/01/2023]
Abstract
8-Chloro-cyclic AMP (8-Cl-cAMP) is a cyclic AMP analog that induces growth inhibition and apoptosis in a broad spectrum of cancer cells. Previously, we found that 8-Cl-cAMP-induced growth inhibition is mediated by AMP-activated protein kinase (AMPK) as well as p38 mitogen-activated protein kinase (p38 MAPK). To identify downstream mediators of the 8-Cl-cAMP signaling, we performed co-immunoprecipitation combined with mass spectrometry using the anti-AMPK or p38 MAPK antibodies. Through this approach, SHC1 was identified as one of the binding partners of p38 MAPK. SHC1 phosphorylation was suppressed by 8-Cl-cAMP in HeLa and MCF7 cancer cells, which was mediated by its metabolites, 8-Cl-adenosine and 8-Cl-ATP; however, 8-Cl-cAMP showed no effect on SHC1 phosphorylation in normal human fibroblasts. SHC1 siRNA induced AMPK and p38 MAPK phosphorylation and growth inhibition in cancer cells, and SHC1 overexpression re-sensitized human foreskin fibroblasts to the 8-Cl-cAMP treatment. SHC1 phosphorylation was unaffected by Compound C (an AMPK inhibitor) and SB203580 (a p38 MAPK inhibitor), which suggests that SHC1 is upstream of AMPK and p38 MAPK in the 8-Cl-cAMP-stimulated signaling cascade. On the basis of these findings, we conclude that SHC1 functions as a sensor during the 8-Cl-cAMP-induced growth inhibition in SHC1-overexpressing cancer cells.
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37
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Tsutsui Y, Johnson JM, Demeler B, Kinter MT, Hays FA. Conformation-Dependent Human p52Shc Phosphorylation by Human c-Src. Biochemistry 2015; 54:3469-82. [PMID: 25961473 DOI: 10.1021/acs.biochem.5b00122] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Phosphorylation of the human p52Shc adaptor protein is a key determinant in modulating signaling complex assembly in response to tyrosine kinase signaling cascade activation. The underlying mechanisms that govern p52Shc phosphorylation status are unknown. In this study, p52Shc phosphorylation by human c-Src was investigated using purified proteins to define mechanisms that affect the p52Shc phosphorylation state. We conducted biophysical characterizations of both human p52Shc and human c-Src in solution as well as membrane-mimetic environments using the acidic lipid phosphatidylinositol 4-phosphate or a novel amphipathic detergent (2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside). We then identified p52Shc phosphorylation sites under various solution conditions, and the amount of phosphorylation at each identified site was quantified using mass spectrometry. These data demonstrate that the p52Shc phosphorylation level is altered by the solution environment without affecting the fraction of active c-Src. Mass spectrometry analysis of phosphorylated p52Shc implies functional linkage among phosphorylation sites. This linkage may drive preferential coupling to protein binding partners during signaling complex formation, such as during initial binding interactions with the Grb2 adaptor protein leading to activation of the Ras/MAPK signaling cascade. Remarkably, tyrosine residues involved in Grb2 binding were heavily phosphorylated in a membrane-mimetic environment. The increased phosphorylation level in Grb2 binding residues was also correlated with a decrease in the thermal stability of purified human p52Shc. A schematic for the phosphorylation-dependent interaction between p52Shc and Grb2 is proposed. The results of this study suggest another possible therapeutic strategy for altering protein phosphorylation to regulate signaling cascade activation.
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Affiliation(s)
- Yuko Tsutsui
- †Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States
| | - Jennifer M Johnson
- †Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States
| | - Borries Demeler
- ‡Department of Biochemistry, The University of Texas Health Sciences Center at San Antonio, 7750 Floyd Curl Drive, San Antonio, Texas 78229-3900, United States
| | - Michael T Kinter
- ∥Department of Geriatric Medicine, Reynolds Oklahoma Center on Aging, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States
| | - Franklin A Hays
- †Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.,⊥Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.,∇Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States
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38
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Lei Q, Chen J, Huang W, Wu D, Lin H, Lai Y. Proteomic analysis of the effect of extracellular calcium ions on human mesenchymal stem cells: Implications for bone tissue engineering. Chem Biol Interact 2015; 233:139-46. [PMID: 25824407 DOI: 10.1016/j.cbi.2015.03.021] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2014] [Revised: 01/29/2015] [Accepted: 03/19/2015] [Indexed: 12/16/2022]
Abstract
Human mesenchymal stem cells-bone marrow (BM-hMSCs) are considered as the most suitable seed cells for bone tissue engineering. Calcium ions (Ca(2+)) forms an important component of a number of commercial bone substitutes and support materials. For efficient bone tissue engineering, it is crucial to explore the effect of extracellular Ca(2+) on the growth and differentiation of BM-hMSCs, and to understand their molecular mechanisms. Therefore, in the present study, BM-hMSCs were cultivated in serum free growth medium or serum free growth medium with additional 4 or 6mM Ca(2+) for 3weeks, following which, the proliferation and osteoblastic differentiation of these cells were evaluated. Differentially expressed proteins were established using iTRAQ labeling coupled with nano-LC-MS/MS. Our data revealed that Ca(2+) significantly promoted the proliferation of BM-hMSCs in the early stage. Furthermore, Ca(2+) showed osteoinduction properties. MAPKs signaling pathway might participate in the osteogenic differentiation of BM-hMSCs caused by Ca(2+). Certain newly found proteins could be potentially important for the osteogenic differentiation of BM-hMSCs and may be associated with osteogenesis.
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Affiliation(s)
- Qun Lei
- Department of Oral Implantology, Affiliated Stomatological Hospital of Fujian Medical University, Fuzhou, Fujian 350002, China
| | - Jiang Chen
- Department of Oral Implantology, Affiliated Stomatological Hospital of Fujian Medical University, Fuzhou, Fujian 350002, China
| | - Wenxiu Huang
- Department of Oral Implantology, Affiliated Stomatological Hospital of Fujian Medical University, Fuzhou, Fujian 350002, China
| | - Dong Wu
- Department of Oral Implantology, Affiliated Stomatological Hospital of Fujian Medical University, Fuzhou, Fujian 350002, China.
| | - Hengzhang Lin
- Department of Stomatology, Affiliated Provincial Governmental Hospital of Fujian Health College, Fuzhou, Fujian 350002, China
| | - Yingzhen Lai
- Department of Stomatology, Xiamen Medical College, Xiamen, Fujian 361008, China
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39
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Bardita C, Predescu DN, Sha F, Patel M, Balaji G, Predescu SA. Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5. J Cell Sci 2015; 128:1528-41. [PMID: 25720380 PMCID: PMC4406123 DOI: 10.1242/jcs.163030] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2014] [Accepted: 02/17/2015] [Indexed: 12/11/2022] Open
Abstract
Recently, we demonstrated in cultured endothelial cells and in vivo that deficiency of an isoform of intersectin-1, ITSN-1s, impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e. enlarged endocytic structures, membranous rings or tubules) that are normally underrepresented. We now show that these endocytic structures internalize the broadly expressed transforming growth factor β receptor I (TGFβ-RI or TGFBR1), also known as Alk5, leading to its ubiquitylation and degradation. Moreover, the apoptotic or activated vascular cells of the ITSN-1s-knockdown mice release Alk5-bearing microparticles to the systemic circulation. These interact with and transfer Alk5 to endocytosis-deficient endothelial cells, resulting in lung endothelial cell survival and phenotypic alteration towards proliferation through activation of Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively). We also show that non-productive assembly of the Alk5–Smad–SARA (Smad anchor for receptor activation, also known as ZFYVE9) signaling complex and preferential formation of the Alk5–mSos–Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary endothelial cells. Taken together, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 by microparticles and endothelial cell survival and proliferation, and define a novel molecular mechanism for TGFβ and Alk5-dependent Erk1/2MAPK signaling that is significant for proliferative signaling and abnormal growth.
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Affiliation(s)
- Cristina Bardita
- Department of Pharmacology, Rush University, Chicago, IL 60612, USA
| | - Dan N Predescu
- Department of Pharmacology, Rush University, Chicago, IL 60612, USA Pulmonary and Critical Care Medicine, Rush University Medical Center, Chicago, IL 60612, USA
| | - Fei Sha
- Department of Pharmacology, Rush University, Chicago, IL 60612, USA
| | - Monal Patel
- Department of Pharmacology, Rush University, Chicago, IL 60612, USA
| | - Ganesh Balaji
- Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Sanda A Predescu
- Department of Pharmacology, Rush University, Chicago, IL 60612, USA Pulmonary and Critical Care Medicine, Rush University Medical Center, Chicago, IL 60612, USA
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40
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Mlih M, Host L, Martin S, Niederhoffer N, Monassier L, Terrand J, Messaddeq N, Radke M, Gotthardt M, Bruban V, Kober F, Bernard M, Canet-Soulas E, Abt-Jijon F, Boucher P, Matz RL. The Src homology and collagen A (ShcA) adaptor protein is required for the spatial organization of the costamere/Z-disk network during heart development. J Biol Chem 2014; 290:2419-30. [PMID: 25488665 DOI: 10.1074/jbc.m114.597377] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.
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Affiliation(s)
- Mohamed Mlih
- From the CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch, France
| | - Lionel Host
- From the CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch, France
| | - Sophie Martin
- From the CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch, France
| | - Nathalie Niederhoffer
- the Laboratory of Neurobiology and Cardiovascular Pharmacology Department, EA 7296, Federation of Translational Medicine, University of Strasbourg, 67000 Strasbourg, France
| | - Laurent Monassier
- the Laboratory of Neurobiology and Cardiovascular Pharmacology Department, EA 7296, Federation of Translational Medicine, University of Strasbourg, 67000 Strasbourg, France
| | - Jérôme Terrand
- From the CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch, France
| | - Nadia Messaddeq
- the IGBMC, INSERM U964 CNRS UMR 7104, University of Strasbourg, 67401 Illkirch, France
| | - Michael Radke
- the Neuromuscular and Cardiovascular Cell Biology, Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany, the DZHK, German Centre for Cardiovascular Research, partner site, 13347 Berlin, Germany
| | - Michael Gotthardt
- the Neuromuscular and Cardiovascular Cell Biology, Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany, the DZHK, German Centre for Cardiovascular Research, partner site, 13347 Berlin, Germany
| | - Véronique Bruban
- From the CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch, France
| | - Frank Kober
- the CRMBM, CNRS, UMR 7339, University of Aix-Marseille, 13385 Marseille, France, and
| | - Monique Bernard
- the CRMBM, CNRS, UMR 7339, University of Aix-Marseille, 13385 Marseille, France, and
| | - Emmanuelle Canet-Soulas
- the CREATIS-LRMN, CNRS, UMR 5220, U630 INSERM, 69621 Villeurbanne, Lyon-1 University, Lyon, France
| | | | - Philippe Boucher
- From the CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch, France,
| | - Rachel L Matz
- From the CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch, France,
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41
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Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation. Proc Natl Acad Sci U S A 2014; 111:E3785-94. [PMID: 25157176 DOI: 10.1073/pnas.1406559111] [Citation(s) in RCA: 81] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Mutations in Ras GTPase and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers and also have been identified in several familial developmental syndromes. Over the past few decades it has become clear that the activity or the oncogenic potential of Ras is dependent on the nonreceptor tyrosine kinase Src to promote the Ras/Raf/MAPK pathway essential for proliferation, differentiation, and survival of eukaryotic cells. However, no direct relationship between Ras and Src has been established. We show here that Src binds to and phosphorylates GTP-, but not GDP-, loaded Ras on a conserved Y32 residue within the switch I region in vitro and that in vivo, Ras-Y32 phosphorylation markedly reduces the binding to effector Raf and concomitantly increases binding to GTPase-activating proteins and the rate of GTP hydrolysis. These results suggest that, in the context of predetermined crystallographic structures, Ras-Y32 serves as an Src-dependent keystone regulatory residue that modulates Ras GTPase activity and ensures unidirectionality to the Ras GTPase cycle.
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42
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Klammer M, Dybowski JN, Hoffmann D, Schaab C. Identification of significant features by the Global Mean Rank test. PLoS One 2014; 9:e104504. [PMID: 25119995 PMCID: PMC4132091 DOI: 10.1371/journal.pone.0104504] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2014] [Accepted: 07/14/2014] [Indexed: 12/24/2022] Open
Abstract
With the introduction of omics-technologies such as transcriptomics and proteomics, numerous methods for the reliable identification of significantly regulated features (genes, proteins, etc.) have been developed. Experimental practice requires these tests to successfully deal with conditions such as small numbers of replicates, missing values, non-normally distributed expression levels, and non-identical distributions of features. With the MeanRank test we aimed at developing a test that performs robustly under these conditions, while favorably scaling with the number of replicates. The test proposed here is a global one-sample location test, which is based on the mean ranks across replicates, and internally estimates and controls the false discovery rate. Furthermore, missing data is accounted for without the need of imputation. In extensive simulations comparing MeanRank to other frequently used methods, we found that it performs well with small and large numbers of replicates, feature dependent variance between replicates, and variable regulation across features on simulation data and a recent two-color microarray spike-in dataset. The tests were then used to identify significant changes in the phosphoproteomes of cancer cells induced by the kinase inhibitors erlotinib and 3-MB-PP1 in two independently published mass spectrometry-based studies. MeanRank outperformed the other global rank-based methods applied in this study. Compared to the popular Significance Analysis of Microarrays and Linear Models for Microarray methods, MeanRank performed similar or better. Furthermore, MeanRank exhibits more consistent behavior regarding the degree of regulation and is robust against the choice of preprocessing methods. MeanRank does not require any imputation of missing values, is easy to understand, and yields results that are easy to interpret. The software implementing the algorithm is freely available for academic and commercial use.
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Affiliation(s)
- Martin Klammer
- Dept. of Bioinformatics, Evotec (München) GmbH, Martinsried, Germany
| | | | - Daniel Hoffmann
- Center for Medical Biotechnology, University of Duisburg-Essen, Essen, Germany
| | - Christoph Schaab
- Dept. of Bioinformatics, Evotec (München) GmbH, Martinsried, Germany
- Dept. Proteomics and Signal Transduction, Max-Plack Institute for Biochemistry, Martinsried, Germany
- * E-mail:
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43
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Im YK, La Selva R, Gandin V, Ha JR, Sabourin V, Sonenberg N, Pawson T, Topisirovic I, Ursini-Siegel J. The ShcA adaptor activates AKT signaling to potentiate breast tumor angiogenesis by stimulating VEGF mRNA translation in a 4E-BP-dependent manner. Oncogene 2014; 34:1729-35. [PMID: 24837366 DOI: 10.1038/onc.2014.110] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2013] [Revised: 03/10/2014] [Accepted: 03/12/2014] [Indexed: 12/12/2022]
Abstract
The ShcA adaptor protein is engaged by numerous receptor tyrosine kinases (RTKs) in breast cancer cells. Once activated, RTKs phosphorylate three key tyrosine phosphorylation sites (Y239, Y240 and Y317) within ShcA that creates a docking site for Grb2/SOS and Grb2/Gab-containing complexes to activate the MAPK and AKT signaling pathways, respectively. We previously demonstrated that a tyrosine to phenylalanine substitution of the ShcA tyrosine phosphorylation sites (Shc3F-Y239/240/313F) significantly impairs breast tumor growth and angiogenesis in transgenic mouse models, in part, through the regulation of vascular endothelial growth factor (VEGF) production. Despite this fact, the underlying molecular mechanisms by which ShcA transduces pro-tumorigenic signals in breast cancer cells remain poorly defined. In this study, we demonstrate that ShcA-dependent activation of AKT, but not the RAS/MAPK pathway, induces VEGF production by bolstering VEGF mRNA translation. Accordingly, ShcA drives breast tumor growth and angiogenesis in vivo in a 4E-BP-dependent manner. These findings establish ShcA as a biological bridge that links AKT activation downstream of RTKs to cap-dependent VEGF mRNA translation in order to promote mammary tumorigenesis.
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Affiliation(s)
- Y K Im
- Department of Oncology, Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada
| | - R La Selva
- Department of Oncology, Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada
| | - V Gandin
- Department of Oncology, Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada
| | - J R Ha
- Department of Oncology, Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada
| | - V Sabourin
- Department of Oncology, Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada
| | - N Sonenberg
- Goodman Cancer Research Centre, Montreal, Quebec, Canada
| | - T Pawson
- Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada
| | - I Topisirovic
- Department of Oncology, Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada
| | - J Ursini-Siegel
- Department of Oncology, Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada
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Schaller-Schönitz M, Barzan D, Williamson AJK, Griffiths JR, Dallmann I, Battmer K, Ganser A, Whetton AD, Scherr M, Eder M. BCR-ABL affects STAT5A and STAT5B differentially. PLoS One 2014; 9:e97243. [PMID: 24836440 PMCID: PMC4023949 DOI: 10.1371/journal.pone.0097243] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2013] [Accepted: 04/16/2014] [Indexed: 11/21/2022] Open
Abstract
Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors linking extracellular signals to target gene transcription. Hematopoietic cells express two highly conserved STAT5-isoforms (STAT5A/STAT5B), and STAT5 is directly activated by JAK2 downstream of several cytokine receptors and the oncogenic BCR-ABL tyrosine kinase. Using an IL-3-dependent cell line with inducible BCR-ABL-expression we compared STAT5-activation by IL-3 and BCR-ABL in a STAT5-isoform specific manner. RNAi targeting of STAT5B strongly inhibits BCR-ABL-dependent cell proliferation, and STAT5B but not STAT5A is essential for BCL-XL-expression in the presence of BCR-ABL. Although BCR-ABL induces STAT5-tyrosine phosphorylation independent of JAK2-kinase activity, BCR-ABL is less efficient in inducing active STAT5A:STAT5B-heterodimerization than IL-3, leaving constitutive STAT5A and STAT5B-homodimerization unaffected. In comparison to IL-3, nuclear accumulation of a STAT5A-eGFP fusion protein is reduced by BCR-ABL, and BCR-ABL tyrosine kinase activity induces STAT5A-eGFP translocation to the cell membrane and co-localization with the IL-3 receptor. Furthermore, BCR-ABL-dependent phosphorylation of Y682 in STAT5A was detected by mass-spectrometry. Finally, RNAi targeting STAT5B but not STAT5A sensitizes human BCR-ABL-positive cell lines to imatinib-treatment. These data demonstrate differences between IL-3 and BCR-ABL-mediated STAT5-activation and isoform-specific effects, indicating therapeutic options for isoform-specific STAT5-inhibition in BCR-ABL-positive leukemia.
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Affiliation(s)
- Michael Schaller-Schönitz
- Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany
| | - David Barzan
- Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany
| | - Andrew J. K. Williamson
- Faculty of Medical and Human Sciences, Manchester Academic Health Science Centre, University of Manchester, Wolfson Molecular Imaging Centre, Manchester, United Kingdom
| | - John R. Griffiths
- Faculty of Medical and Human Sciences, Manchester Academic Health Science Centre, University of Manchester, Wolfson Molecular Imaging Centre, Manchester, United Kingdom
| | - Iris Dallmann
- Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany
| | - Karin Battmer
- Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany
| | - Arnold Ganser
- Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany
| | - Anthony D. Whetton
- Faculty of Medical and Human Sciences, Manchester Academic Health Science Centre, University of Manchester, Wolfson Molecular Imaging Centre, Manchester, United Kingdom
| | - Michaela Scherr
- Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany
| | - Matthias Eder
- Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany
- * E-mail:
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45
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Wills MKB, Tong J, Tremblay SL, Moran MF, Jones N. The ShcD signaling adaptor facilitates ligand-independent phosphorylation of the EGF receptor. Mol Biol Cell 2014; 25:739-52. [PMID: 24430869 PMCID: PMC3952845 DOI: 10.1091/mbc.e13-08-0434] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2013] [Revised: 12/06/2013] [Accepted: 01/08/2014] [Indexed: 11/12/2022] Open
Abstract
Proto-oncogenic Src homology and collagen (Shc) proteins have been considered archetypal adaptors of epidermal growth factor receptor (EGFR)-mediated signaling. We report that in addition to its role as an EGFR-binding partner and Grb2 platform, ShcD acts noncanonically to promote phosphorylation of select EGFR residues. Unexpectedly, Y1068, Y1148, and Y1173 are subject to ShcD-induced, cell-autonomous hyperphosphorylation in the absence of external stimuli. This response is not elicited by other Shc proteins and requires the intrinsic EGFR kinase, as well as the ShcD phosphotyrosine-binding (PTB) domain. Assessments of Erk, Akt, phospholipase C 1γ, and FAK pathways reveal no apparent distal signaling targets of ShcD. Nevertheless, the capacity of cultured cells to repopulate a wounded monolayer is markedly accelerated by ShcD in an EGFR kinase-dependent manner. Furthermore, detection of overexpressed ShcD coincident with EGFR phosphorylation in human gliomas suggests a clinical application for these findings. We thus demonstrate unique and relevant synergy between ShcD and EGFR that is unprecedented among signaling adaptors.
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Affiliation(s)
- Melanie K. B. Wills
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada
| | - Jiefei Tong
- Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
| | - Sylvie L. Tremblay
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada
| | - Michael F. Moran
- Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
- Department of Molecular Genetics and Banting and Best Department of Medical Research, University of Toronto, Toronto, ON M5G 1L6, Canada
| | - Nina Jones
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada
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Abstract
Proteins generally act by binding to other molecules, including proteins. When proteins bind to other proteins, we speak of protein-protein interactions. It has become apparent that protein-protein interactions are critically important to many processes that take place in the cell, including signal transduction, regulation of gene expression, vesicular transport, nuclear import and export, and cell migration (Pawson and Nash, 2003). This has led to the recognition of protein-protein interactions as targets for drug development and to an increased interest in the identification of novel protein-protein interactions (Fry and Vassilev, 2005; Fry, 2006; Tord et al., 2007). Coimmunoprecipitation is a technique that is used to confirm novel protein-protein interactions in the context of a living cell or organism. In addition, coimmunoprecipitation is also used to study the dynamics of protein-protein interactions in response to intra- or extracellular stimuli, or can be used to study the effect of mutations on the ability of a protein to engage its binding partner. In a coimmunoprecipitation experiment, a protein of interest is isolated by immunoprecipitation. Subsequently, the presence of binding partners can be assessed by immunoblotting (see Western Blotting using Chemiluminescent Substrates).
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Affiliation(s)
- Peter van der Geer
- Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA, USA.
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47
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Mierke CT. The role of focal adhesion kinase in the regulation of cellular mechanical properties. Phys Biol 2013; 10:065005. [PMID: 24304934 DOI: 10.1088/1478-3975/10/6/065005] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
The regulation of mechanical properties is necessary for cell invasion into connective tissue or intra- and extravasation through the endothelium of blood or lymph vessels. Cell invasion is important for the regulation of many healthy processes such as immune response reactions and wound healing. In addition, cell invasion plays a role in disease-related processes such as tumor metastasis and autoimmune responses. Until now the role of focal adhesion kinase (FAK) in regulating mechanical properties of cells and its impact on cell invasion efficiency is still not well known. Thus, this review focuses on mechanical properties regulated by FAK in comparison to the mechano-regulating protein vinculin. Moreover, it points out the connection between cancer cell invasion and metastasis and FAK by showing that FAK regulates cellular mechanical properties required for cellular motility. Furthermore, it sheds light on the indirect interaction of FAK with vinculin by binding to paxillin, which then impairs the binding of paxillin to vinculin. In addition, this review emphasizes whether FAK fulfills regulatory functions similar to vinculin. In particular, it discusses the differences and the similarities between FAK and vinculin in regulating the biomechanical properties of cells. Finally, this paper highlights that both focal adhesion proteins, vinculin and FAK, synergize their functions to regulate the mechanical properties of cells such as stiffness and contractile forces. Subsequently, these mechanical properties determine cellular invasiveness into tissues and provide a source sink for future drug developments to inhibit excessive cell invasion and hence, metastases formation.
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Affiliation(s)
- Claudia Tanja Mierke
- Faculty of Physics and Earth Science, Institute of Experimental Physics I, Biological Physics Division, University of Leipzig, Linnéstr. 5, D-04103 Leipzig, Germany
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48
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Molecular mechanisms of SH2- and PTB-domain-containing proteins in receptor tyrosine kinase signaling. Cold Spring Harb Perspect Biol 2013; 5:a008987. [PMID: 24296166 DOI: 10.1101/cshperspect.a008987] [Citation(s) in RCA: 106] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.
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49
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Bardita C, Predescu D, Justice MJ, Petrache I, Predescu S. In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs. Apoptosis 2013; 18:57-76. [PMID: 23054079 PMCID: PMC3543613 DOI: 10.1007/s10495-012-0762-x] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Intersectin-1s (ITSN-1s) is a general endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, via MEK/Erk1/2MAPK signaling. To investigate the in vivo effects of ITSN-1s deficiency and the resulting ECs apoptosis on pulmonary vasculature and lung homeostasis, we used an ITSN-1s knocked-down (KDITSN) mouse generated by repeated delivery of a specific siRNA targeting ITSN-1 gene (siRNAITSN). Biochemical and histological analyses as well as electron microscopy (EM) revealed that acute KDITSN [3-days (3d) post-siRNAITSN treatment] inhibited Erk1/2MAPK pro-survival signaling, causing significant ECs apoptosis and lung injury; at 10d of KDITSN, caspase-3 activation was at peak, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive ECs showed 3.4-fold increase, the mean linear intercept (MLI) showed 48 % augment and pulmonary microvessel density as revealed by aquaporin-1 staining (AQP-1) decreased by 30 %, all compared to controls; pulmonary function was altered. Concomitantly, expression of several growth factors known to activate Erk1/2MAPK and suppress Bad pro-apoptotic activity increased. KDITSN altered Smads activity, downstream of the transforming growth factor beta-receptor-1 (TβR1), as shown by subcellular fractionation and immunoblot analyses. Moreover, 24d post-siRNAITSN, surviving ECs became hyper-proliferative and apoptotic-resistant against ITSN-1s deficiency, as demonstrated by EM imaging, 5-bromo-deoxyuridine (BrdU) incorporation and Bad-Ser112/155 phosphorylation, respectively, leading to increased microvessel density and repair of the injured lungs, as well as matrix deposition. In sum, ECs endocytic dysfunction and apoptotic death caused by KDITSN contribute to the initial lung injury and microvascular loss, followed by endothelial phenotypic changes and microvascular remodeling in the remaining murine pulmonary microvascular bed.
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Affiliation(s)
- Cristina Bardita
- Department of Pharmacology, Rush University, 1735 W. Harrison St., Chicago, IL 60612, USA
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50
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Linossi EM, Chandrashekaran IR, Kolesnik TB, Murphy JM, Webb AI, Willson TA, Kedzierski L, Bullock AN, Babon JJ, Norton RS, Nicola NA, Nicholson SE. Suppressor of Cytokine Signaling (SOCS) 5 utilises distinct domains for regulation of JAK1 and interaction with the adaptor protein Shc-1. PLoS One 2013; 8:e70536. [PMID: 23990909 PMCID: PMC3749136 DOI: 10.1371/journal.pone.0070536] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2013] [Accepted: 06/20/2013] [Indexed: 12/02/2022] Open
Abstract
Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.
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Affiliation(s)
- Edmond M. Linossi
- Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- The University of Melbourne, Parkville, Victoria, Australia
| | - Indu R. Chandrashekaran
- Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
| | - Tatiana B. Kolesnik
- Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- The University of Melbourne, Parkville, Victoria, Australia
| | - James M. Murphy
- Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- The University of Melbourne, Parkville, Victoria, Australia
| | - Andrew I. Webb
- Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- The University of Melbourne, Parkville, Victoria, Australia
| | - Tracy A. Willson
- Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- The University of Melbourne, Parkville, Victoria, Australia
| | - Lukasz Kedzierski
- Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- The University of Melbourne, Parkville, Victoria, Australia
| | - Alex N. Bullock
- Structural Genomics Consortium, University of Oxford, Oxford, United Kingdom
| | - Jeffrey J. Babon
- Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- The University of Melbourne, Parkville, Victoria, Australia
| | - Raymond S. Norton
- Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
| | - Nicos A. Nicola
- Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- The University of Melbourne, Parkville, Victoria, Australia
| | - Sandra E. Nicholson
- Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- The University of Melbourne, Parkville, Victoria, Australia
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