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Alnuaimi AR, Nair VA, Malhab LJB, Abu-Gharbieh E, Ranade AV, Pintus G, Hamad M, Busch H, Kirfel J, Hamoudi R, Abdel-Rahman WM. Emerging role of caldesmon in cancer: A potential biomarker for colorectal cancer and other cancers. World J Gastrointest Oncol 2022; 14:1637-1653. [PMID: 36187394 PMCID: PMC9516648 DOI: 10.4251/wjgo.v14.i9.1637] [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: 03/16/2022] [Revised: 05/05/2022] [Accepted: 07/26/2022] [Indexed: 02/05/2023] Open
Abstract
Colorectal cancer (CRC) is a devastating disease, mainly because of metastasis. As a result, there is a need to better understand the molecular basis of invasion and metastasis and to identify new biomarkers and therapeutic targets to aid in managing these tumors. The actin cytoskeleton and actin-binding proteins are known to play an important role in the process of cancer metastasis because they control and execute essential steps in cell motility and contractility as well as cell division. Caldesmon (CaD) is an actin-binding protein encoded by the CALD1 gene as multiple transcripts that mainly encode two protein isoforms: High-molecular-weight CaD, expressed in smooth muscle, and low-molecular weight CaD (l-CaD), expressed in nonsmooth muscle cells. According to our comprehensive review of the literature, CaD, particularly l-CaD, plays a key role in the development, metastasis, and resistance to chemoradiotherapy in colorectal, breast, and urinary bladder cancers and gliomas, among other malignancies. CaD is involved in many aspects of the carcinogenic hallmarks, including epithelial mesenchymal transition via transforming growth factor-beta signaling, angiogenesis, resistance to hormonal therapy, and immune evasion. Recent data show that CaD is expressed in tumor cells as well as in stromal cells, such as cancer-associated fibroblasts, where it modulates the tumor microenvironment to favor the tumor. Interestingly, CaD undergoes selective tumor-specific splicing, and the resulting isoforms are generally not expressed in normal tissues, making these transcripts ideal targets for drug design. In this review, we will analyze these features of CaD with a focus on CRC and show how the currently available data qualify CaD as a potential candidate for targeted therapy in addition to its role in the diagnosis and prognosis of cancer.
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Affiliation(s)
- Alya R Alnuaimi
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
- College of Medicine, University of Sharjah, Sharjah 27272, United Arab Emirates
| | - Vidhya A Nair
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
| | - Lara J Bou Malhab
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
| | - Eman Abu-Gharbieh
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
- Department of Clinical Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
| | - Anu Vinod Ranade
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
- Department of Basic Medical Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
| | - Gianfranco Pintus
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
- Department of Medical Laboratory Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
- Department of Biomedical Sciences, University of Sassari, Sassari 07100, Italy
| | - Mohamad Hamad
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
- Department of Medical Laboratory Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
| | - Hauke Busch
- University Cancer Center Schleswig-Holstein and Luebeck Institute for Experimental Dermatology, University of Luebeck, Luebeck 23560, Germany
| | - Jutta Kirfel
- Institute of Pathology, University Hospital Schleswig-Holstein, Luebeck 23560, Germany
| | - Rifat Hamoudi
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
- Department of Clinical Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
- Division of Surgery and Interventional Science, University College London, London WC1E 6BT, United Kingdom
| | - Wael M Abdel-Rahman
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab Emirates
- Department of Medical Laboratory Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
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Yao YB, Xiao CF, Lu JG, Wang C. Caldesmon: Biochemical and Clinical Implications in Cancer. Front Cell Dev Biol 2021; 9:634759. [PMID: 33681215 PMCID: PMC7930484 DOI: 10.3389/fcell.2021.634759] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2020] [Accepted: 01/18/2021] [Indexed: 12/19/2022] Open
Abstract
Caldesmon, an actin-binding protein, can inhibit myosin binding to actin and regulate smooth muscle contraction and relaxation. However, caldesmon has recently attracted attention due to its importance in cancer. The upregulation of caldesmon in several solid cancer tissues has been reported. Caldesmon, as well as its two isoforms, is considered as a biomarker for cancer and a potent suppressor of cancer cell invasion by regulating podosome/invadopodium formation. Therefore, caldesmon may be a promising therapeutic target for diseases such as cancer. Here, we review new studies on the gene transcription, isoform structure, expression, and phosphorylation regulation of caldesmon and discuss its clinical implications in cancer.
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Affiliation(s)
- Yi-Bo Yao
- Department of Anorectal Surgery, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Chang-Fang Xiao
- Department of Anorectal Surgery, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Jin-Gen Lu
- Longhua Hospital, Institute of Chinese Traditional Surgery, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Chen Wang
- Department of Anorectal Surgery, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
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Michel JB. Phylogenic Determinants of Cardiovascular Frailty, Focus on Hemodynamics and Arterial Smooth Muscle Cells. Physiol Rev 2020; 100:1779-1837. [DOI: 10.1152/physrev.00022.2019] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The evolution of the circulatory system from invertebrates to mammals has involved the passage from an open system to a closed in-parallel system via a closed in-series system, accompanying the increasing complexity and efficiency of life’s biological functions. The archaic heart enables pulsatile motion waves of hemolymph in invertebrates, and the in-series circulation in fish occurs with only an endothelium, whereas mural smooth muscle cells appear later. The present review focuses on evolution of the circulatory system. In particular, we address how and why this evolution took place from a closed, flowing, longitudinal conductance at low pressure to a flowing, highly pressurized and bifurcating arterial compartment. However, although arterial pressure was the latest acquired hemodynamic variable, the general teleonomy of the evolution of species is the differentiation of individual organ function, supported by specific fueling allowing and favoring partial metabolic autonomy. This was achieved via the establishment of an active contractile tone in resistance arteries, which permitted the regulation of blood supply to specific organ activities via its localized function-dependent inhibition (active vasodilation). The global resistance to viscous blood flow is the peripheral increase in frictional forces caused by the tonic change in arterial and arteriolar radius, which backscatter as systemic arterial blood pressure. Consequently, the arterial pressure gradient from circulating blood to the adventitial interstitium generates the unidirectional outward radial advective conductance of plasma solutes across the wall of conductance arteries. This hemodynamic evolution was accompanied by important changes in arterial wall structure, supported by smooth muscle cell functional plasticity, including contractility, matrix synthesis and proliferation, endocytosis and phagocytosis, etc. These adaptive phenotypic shifts are due to epigenetic regulation, mainly related to mechanotransduction. These paradigms actively participate in cardio-arterial pathologies such as atheroma, valve disease, heart failure, aneurysms, hypertension, and physiological aging.
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Chan CL, Chen JY, Shih MC, Wang CLA, Liou YM. L-caldesmon alters cell spreading and adhesion force in RANKL-induced osteoclasts. J Biomed Sci 2019; 26:12. [PMID: 30678675 PMCID: PMC6345023 DOI: 10.1186/s12929-019-0505-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2018] [Accepted: 01/18/2019] [Indexed: 01/21/2023] Open
Abstract
Background Osteoclasts (OCs) are motile multinucleated cells derived from differentiation and fusion of hematopoietic progenitors of the monocyte-macrophage lineage that undergo a multistep process called osteoclastogenesis. The biological function of OCs is to resorb bone matrix for controlling bone strength and integrity, which is essential for bone development. The bone resorption function is based on the remodelling of the actin cytoskeleton into an F-actin-rich structure known as the sealing zone for bone anchoring and matrix degradation. Non-muscle caldesmon (l-CaD) is known to participate in the regulation of actin cytoskeletal remodeling, but its function in osteoclastogenesis remains unclear. Methods/results In this study, gain and loss of the l-CaD level in RAW264.7 murine macrophages followed by RANKL induction was used as an experimental approach to examine the involvement of l-CaD in the control of cell fusion into multinucleated OCs in osteoclastogenesis. In comparison with controls, l-CaD overexpression significantly increased TRAP activity, actin ring structure and mineral substrate resorption in RANKL-induced cells. In contrast, gene silencing against l-CaD decreased the potential for RANKL-induced osteoclastogenesis and mineral substrate resorption. In addition, OC precursor cells with l-CaD overexpression and gene silencing followed by RANKL induction caused 13% increase and 24% decrease, respectively, in cell fusion index. To further understand the mechanistic action of l-CaD in the modulation of OC fusion, atomic force microscopy was used to resolve the mechanical changes of cell spreading and adhesion force in RANKL-induced cells with and without l-CaD overexpression or gene silencing. Conclusions l-CaD plays a key role in the regulation of actin cytoskeletal remodeling for the formation of actin ring structure at the cell periphery, which may in turn alter the mechanical property of cell-spreading and cell surface adhesion force, thereby facilitating cell-cell fusion into multinucleated OCs during osteoclastogenesis. Electronic supplementary material The online version of this article (10.1186/s12929-019-0505-1) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Chu-Lung Chan
- Department of Life Sciences, National Chung-Hsing University, Taichung, 40227, Taiwan
| | - Jiann-Yeu Chen
- Research Center for Sustainable Energy and Nanotechnology, National Chung-Hsing University, Taichung, 40227, Taiwan
| | - Ming-Chih Shih
- Department of Physics, National Chung-Hsing University, Taichung, 40227, Taiwan
| | | | - Ying-Ming Liou
- Department of Life Sciences, National Chung-Hsing University, Taichung, 40227, Taiwan. .,The iEGG and Animal Biotechnology Center, and Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung, 40227, Taiwan.
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Liou YM, Chan CL, Huang R, Wang CLA. Effect of l-caldesmon on osteoclastogenesis in RANKL-induced RAW264.7 cells. J Cell Physiol 2018; 233:6888-6901. [PMID: 29377122 DOI: 10.1002/jcp.26452] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2017] [Accepted: 01/05/2018] [Indexed: 01/08/2023]
Abstract
Non-muscle caldesmon (l-CaD) is involved in the regulation of actin cytoskeletal remodeling in the podosome formation, but its function in osteoclastogenesis remains to be determined. In this study, RANKL-induced differentiation of RAW264.7 murine macrophages to osteoclast-like cells (OCs) was used as a model to determine the physiological role of l-CaD and its phosphorylation in osteoclastogenesis. Upon RANKL treatment, RAW264.7 cells undergo cell-cell fusion into multinucleate, and TRAP-positive large OCs with a concomitant increase of l-CaD expression. Using gain- and loss-of-function in OC precursor cells followed by RANKL induction, we showed that the expression of l-CaD in response to RANKL activation is an important event for osteoclastogenesis, and bone resorption. To determine the effect of l-CaD phosphorylation in osteoclastogenesis, three decoy peptides of l-CaD were used with, respectively, Ser-to-Ala mutations at the Erk- and Pak1-mediated phosphorylation sites, and Ser-to-Asp mutation at the Erk-mediated phosphorylation sites. Both the former two peptides competed with the C-terminal segment of l-CaD for F-actin binding and accelerated formation of podosome-like structures in RANKL-induced OCs, while the third peptide did not significantly affect the F-actin binding of l-CaD, and decreased the formation of podosome-like structures in OCs. With the experiments using dephosphorylated and phosphorylated l-CaD mutants, we further showed that dephosphorylated l-CaD mutant facilitated RANKL-induced TRAP activity with an increased cell fusion index, whereas phosphorylated l-CaD decreased the TRAP activity and cell fusion. Our findings suggested that both the level of l-CaD expression and the extent of l-CaD phosphorylation play a role in RANKL-induced osteoclast differentiation.
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Affiliation(s)
- Ying-Ming Liou
- Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan.,Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung, Taiwan
| | - Chu-Lung Chan
- Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan
| | - Renjian Huang
- Boston Biomedical Research Institute, Watertown, Massachusetts
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Nalluri SM, O'Connor JW, Virgi GA, Stewart SE, Ye D, Gomez EW. TGFβ1-induced expression of caldesmon mediates epithelial-mesenchymal transition. Cytoskeleton (Hoboken) 2018; 75:201-212. [PMID: 29466836 DOI: 10.1002/cm.21437] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2017] [Revised: 02/13/2018] [Accepted: 02/19/2018] [Indexed: 12/15/2022]
Abstract
Epithelial-mesenchymal transition (EMT) is an important process that mediates organ development and wound healing, and in pathological contexts, it can contribute to the progression of fibrosis and cancer. During EMT, cells exhibit marked changes in cytoskeletal organization and increased expression of a variety of actin associated proteins. Here, we sought to determine the role of caldesmon in mediating EMT in response to transforming growth factor (TGF)-β1. We find that the expression level and phosphorylation state of caldesmon increase as a function of time following induction of EMT by TGFβ1 and these changes in caldesmon correlate with increased focal adhesion number and size and increased cell contractility. Knockdown and forced expression of caldesmon in epithelial cells reveals that caldesmon expression plays an important role in regulating the expression of the myofibroblast marker alpha smooth muscle actin. Results from these studies provide insight into the role of cytoskeletal associated proteins in the regulation of EMT and may suggest ways to target the cell cytoskeleton for regulating EMT processes.
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Affiliation(s)
- Sandeep M Nalluri
- Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802
| | - Joseph W O'Connor
- Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802
| | - Gage A Virgi
- Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802
| | - Samantha E Stewart
- Department of Biomedical Engineering, University of South Carolina, Columbia, South Carolina 29208
| | - Dan Ye
- Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802
| | - Esther W Gomez
- Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802.,Department of Biomedical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802
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Paterson EK, Courtneidge SA. Invadosomes are coming: new insights into function and disease relevance. FEBS J 2017; 285:8-27. [PMID: 28548369 DOI: 10.1111/febs.14123] [Citation(s) in RCA: 112] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2017] [Revised: 05/09/2017] [Accepted: 05/24/2017] [Indexed: 12/21/2022]
Abstract
Invadopodia and podosomes are discrete, actin-based molecular protrusions that form in cancer cells and normal cells, respectively, in response to diverse signaling pathways and extracellular matrix cues. Although they participate in a host of different cellular processes, they share a common functional theme of controlling pericellular proteolytic activity, which sets them apart from other structures that function in migration and adhesion, including focal adhesions, lamellipodia, and filopodia. In this review, we highlight research that explores the function of these complex structures, including roles for podosomes in embryonic and postnatal development, in angiogenesis and remodeling of the vasculature, in maturation of the postsynaptic membrane, in antigen sampling and recognition, and in cell-cell fusion mechanisms, as well as the involvement of invadopodia at multiple steps of the metastatic cascade, and how all of this may apply in the treatment of human disease states. Finally, we explore recent research that implicates a novel role for exosomes and microvesicles in invadopodia-dependent and invadopodia-independent mechanisms of invasion, respectively.
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Affiliation(s)
- Elyse K Paterson
- Department of Cell, Developmental and Cancer Biology, Oregon Health & Science University, Portland, OR, USA
| | - Sara A Courtneidge
- Department of Cell, Developmental and Cancer Biology, Oregon Health & Science University, Portland, OR, USA.,Department of Biomedical Engineering, Oregon Health & Science University, Portland, OR, USA.,Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA
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Araki K, Ebata T, Guo AK, Tobiume K, Wolf SJ, Kawauchi K. p53 regulates cytoskeleton remodeling to suppress tumor progression. Cell Mol Life Sci 2015; 72:4077-94. [PMID: 26206378 PMCID: PMC11114009 DOI: 10.1007/s00018-015-1989-9] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2014] [Revised: 07/06/2015] [Accepted: 07/09/2015] [Indexed: 02/07/2023]
Abstract
Cancer cells possess unique characteristics such as invasiveness, the ability to undergo epithelial-mesenchymal transition, and an inherent stemness. Cell morphology is altered during these processes and this is highly dependent on actin cytoskeleton remodeling. Regulation of the actin cytoskeleton is, therefore, important for determination of cell fate. Mutations within the TP53 (tumor suppressor p53) gene leading to loss or gain of function (GOF) of the protein are often observed in aggressive cancer cells. Here, we highlight the roles of p53 and its GOF mutants in cancer cell invasion from the perspective of the actin cytoskeleton; in particular its reorganization and regulation by cell adhesion molecules such as integrins and cadherins. We emphasize the multiple functions of p53 in the regulation of actin cytoskeleton remodeling in response to the extracellular microenvironment, and oncogene activation. Such an approach provides a new perspective in the consideration of novel targets for anti-cancer therapy.
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Affiliation(s)
- Keigo Araki
- Frontiers of Innovative Research in Science and Technology, Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan
- Department of Biomedical Chemistry, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo, 669-1337, Japan
| | - Takahiro Ebata
- Frontiers of Innovative Research in Science and Technology, Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan
| | - Alvin Kunyao Guo
- Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, 8 College Road, Singapore, 169857, Singapore
| | - Kei Tobiume
- Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, Hiroshima, 734-8553, Japan
| | - Steven John Wolf
- Mechanobiology Institute, National University of Singapore, T-Lab, 5A Engineering Drive 1, Singapore, 117411, Singapore
| | - Keiko Kawauchi
- Frontiers of Innovative Research in Science and Technology, Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan.
- Mechanobiology Institute, National University of Singapore, T-Lab, 5A Engineering Drive 1, Singapore, 117411, Singapore.
- Department of Molecular Oncology, Institute for Advanced Medical Sciences, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533, Japan.
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Bisht S, Nolting J, Schütte U, Haarmann J, Jain P, Shah D, Brossart P, Flaherty P, Feldmann G. Cyclin-Dependent Kinase 5 (CDK5) Controls Melanoma Cell Motility, Invasiveness, and Metastatic Spread-Identification of a Promising Novel therapeutic target. Transl Oncol 2015; 8:295-307. [PMID: 26310376 PMCID: PMC4562979 DOI: 10.1016/j.tranon.2015.06.002] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2015] [Revised: 06/16/2015] [Accepted: 06/23/2015] [Indexed: 01/03/2023] Open
Abstract
Despite considerable progress in recent years, the overall prognosis of metastatic malignant melanoma remains poor, and curative therapeutic options are lacking. Therefore, better understanding of molecular mechanisms underlying melanoma progression and metastasis, as well as identification of novel therapeutic targets that allow inhibition of metastatic spread, are urgently required. The current study provides evidence for aberrant cyclin-dependent kinase 5 (CDK5) activation in primary and metastatic melanoma lesions by overexpression of its activator protein CDK5R1/p35. Moreover, using melanoma in vitro model systems, shRNA-mediated inducible knockdown of CDK5 was found to cause marked inhibition of cell motility, invasiveness, and anchorage-independent growth, while at the same time net cell growth was not affected. In vivo, CDK5 knockdown inhibited growth of orthotopic xenografts as well as formation of lung and liver colonies in xenogenic injection models mimicking systemic metastases. Inhibition of lung metastasis was further validated in a syngenic murine melanoma model. CDK5 knockdown was accompanied by dephosphorylation and overexpression of caldesmon, and concomitant caldesmon knockdown rescued cell motility and proinvasive phenotype. Finally, it was found that pharmacological inhibition of CDK5 activity by means of roscovitine as well as by a novel small molecule CDK5-inhibitor, N-(5-isopropylthiazol-2-yl)-3-phenylpropanamide, similarly caused marked inhibition of invasion/migration, colony formation, and anchorage-independent growth of melanoma cells. Thus, experimental data presented here provide strong evidence for a crucial role of aberrantly activated CDK5 in melanoma progression and metastasis and establish CDK5 as promising target for therapeutic intervention.
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Affiliation(s)
- Savita Bisht
- Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Germany
| | - Jens Nolting
- Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Germany
| | - Ute Schütte
- Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Germany
| | - Jens Haarmann
- Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Germany
| | - Prashi Jain
- Mylan School of Pharmacy, Medicinal Chemistry, Duquesne University, Pittsburgh, PA
| | - Dhruv Shah
- Mylan School of Pharmacy, Medicinal Chemistry, Duquesne University, Pittsburgh, PA
| | - Peter Brossart
- Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Germany
| | - Patrick Flaherty
- Mylan School of Pharmacy, Medicinal Chemistry, Duquesne University, Pittsburgh, PA
| | - Georg Feldmann
- Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Germany.
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Abstract
Cell invasion of the extracellular matrix is prerequisite to cross tissue migration of tumor cells in cancer metastasis, and vascular smooth muscle cells in atherosclerosis. The tumor suppressor p53, better known for its roles in the regulation of cell cycle and apoptosis, has ignited much interest in its function as a suppressor of cell migration and invasion. How p53 and its gain-of-function mutants regulate cell invasion remains a puzzle and a challenge for future studies. In recent years, podosomes and invadopodia have also gained center stage status as veritable apparatus specialized in cell invasion. It is not clear, however, whether p53 regulates cell invasion through podosomes and invadopodia. In this review, evidence supporting a negative role of p53 in podosomes formation in vascular smooth muscle cells will be surveyed, and signaling nodes that may mediate this regulation in other cell types will be explored.
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Affiliation(s)
- Alan S Mak
- Department of Biomedical and Molecular Sciences; Queen's University; Kingston, ON Canada
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Payne LJ, Eves RL, Jia L, Mak AS. p53 Down regulates PDGF-induced formation of circular dorsal ruffles in rat aortic smooth muscle cells. PLoS One 2014; 9:e108257. [PMID: 25247424 PMCID: PMC4172730 DOI: 10.1371/journal.pone.0108257] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2014] [Accepted: 08/27/2014] [Indexed: 01/15/2023] Open
Abstract
The tumor suppressor, p53, negatively regulates cell migration and invasion in addition to its role in apoptosis, cell cycle regulation and senescence. Here, we study the roles of p53 in PDGF-induced circular dorsal ruffle (CDR) formation in rat aortic smooth muscle (RASM) cells. In primary and immortalized RASM cells, up-regulation of p53 expression or increase in activity with doxorubicin inhibits CDR formation. In contrast, shRNA-knockdown of p53 or inhibition of its activity with pifithrin α promotes CDR formation. p53 acts by up-regulating PTEN expression, which antagonizes Rac and Cdc42 activation. Both lipid and protein phosphatase activities of PTEN are required for maximal suppression of CDR, but the lipid activity clearly plays the dominant role. N-WASP, the downstream effector of Cdc42, is the major positive contributor to CDR formation in RASM, and is an indirect target of p53. The Rac effector, WAVE2, appears to also play a minor role, while WAVE1 has no significant effect in CDR formation. In sum, we propose that p53 suppresses PDGF-induced CDR formation in RASM cells by upregulating PTEN leading mainly to the inhibition of the Cdc42-N-WASP pathway.
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Affiliation(s)
- Laura J. Payne
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
| | - Robert L. Eves
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
| | - Lilly Jia
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
| | - Alan S. Mak
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
- * E-mail:
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Terra LF, Teixeira PC, Wailemann RAM, Zelanis A, Palmisano G, Cunha-Neto E, Kalil J, Larsen MR, Labriola L, Sogayar MC. Proteins differentially expressed in human beta-cells-enriched pancreatic islet cultures and human insulinomas. Mol Cell Endocrinol 2013; 381:16-25. [PMID: 23891624 DOI: 10.1016/j.mce.2013.07.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/15/2013] [Revised: 06/28/2013] [Accepted: 07/04/2013] [Indexed: 02/06/2023]
Abstract
In view of the great demand for human beta-cells for physiological and medical studies, we generated cell lines derived from human insulinomas which secrete insulin, C-peptide and express neuroendocrine and islet markers. In this study, we set out to characterize their proteomes, comparing them to those of primary beta-cells using DIGE followed by MS. The results were validated by Western blotting. An average of 1800 spots was detected with less than 1% exhibiting differential abundance. Proteins more abundant in human islets, such as Caldesmon, are involved in the regulation of cell contractility, adhesion dependent signaling, and cytoskeletal organization. In contrast, almost all proteins more abundant in insulinoma cells, such as MAGE2, were first described here and could be related to cell survival and resistance to chemotherapy. Our proteomic data provides, for the first time, a molecular snapshot of the orchestrated changes in expression of proteins involved in key processes which could be correlated with the altered phenotype of human beta-cells. Collectively our observations prompt research towards the establishment of bioengineered human beta-cells providing a new and needed source of cultured human beta-cells for beta-cell research, along with the development of new therapeutic strategies for detection, characterization and treatment of insulinomas.
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Affiliation(s)
- Letícia F Terra
- Instituto de Química, Departamento de Bioquímica, Universidade de São Paulo (USP), São Paulo, Brazil
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Deng M, Boopathi E, Hypolite JA, Raabe T, Chang S, Zderic S, Wein AJ, Chacko S. Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle. Am J Physiol Renal Physiol 2013; 305:F1455-65. [PMID: 23986516 DOI: 10.1152/ajprenal.00174.2013] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.
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Affiliation(s)
- Maoxian Deng
- Dept. of Surgery and Dept. of Pathobiology, Univ. of Pennsylvania, 500 South Ridgeway Ave., Glenolden, PA 19036.
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14
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Gu Z, Fonseca V, Hai CM. Nicotinic acetylcholine receptor mediates nicotine-induced actin cytoskeletal remodeling and extracellular matrix degradation by vascular smooth muscle cells. Vascul Pharmacol 2013; 58:87-97. [PMID: 22940282 PMCID: PMC3530635 DOI: 10.1016/j.vph.2012.08.003] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2012] [Revised: 08/13/2012] [Accepted: 08/16/2012] [Indexed: 01/21/2023]
Abstract
Cigarette smoking is a significant risk factor for atherosclerosis, which involves the invasion of vascular smooth muscle cells (VSMCs) from the media to intima. A hallmark of many invasive cells is actin cytoskeletal remodeling in the form of podosomes, accompanied by extracellular matrix (ECM) degradation. A7r5 VSMCs form podosomes in response to PKC activation. In this study, we found that cigarette smoke extract, nicotine, and the cholinergic agonist, carbachol, were similarly effective in inducing the formation of podosome rosettes in A7r5 VSMCs. α-Bungarotoxin and atropine experiments confirmed the involvement of nicotinic acetylcholine receptors (nAChRs). Western blotting and immunofluorescence experiments revealed the aggregation of nAChRs at podosome rosettes. Cycloheximide experiments and media exchange experiments suggested that autocrine factor(s) and intracellular phenotypic modulation are putative mechanisms. In situ zymography experiments indicated that, in response to PKC activation, nicotine-treated cells degraded ECM near podosome rosettes, and possibly endocytose ECM fragments to intracellular compartments. Invasion assay of human aortic smooth muscle cells indicated that nicotine and PKC activation individually and synergistically enhanced cell invasion through ECM. Results from this study suggest that nicotine enhances the ability of VSMCs to degrade and invade ECM. nAChR activation, actin cytoskeletal remodeling and phenotypic modulation are possible mechanisms.
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MESH Headings
- Actin Cytoskeleton/drug effects
- Actin Cytoskeleton/metabolism
- Animals
- Aorta/cytology
- Aorta/drug effects
- Aorta/metabolism
- Blotting, Western
- Carbachol/pharmacology
- Cells, Cultured
- Extracellular Matrix/drug effects
- Extracellular Matrix/metabolism
- Fluorescent Antibody Technique
- Humans
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/drug effects
- Muscle, Smooth, Vascular/metabolism
- Myocytes, Smooth Muscle/drug effects
- Myocytes, Smooth Muscle/metabolism
- Nicotine/toxicity
- Protein Kinase C/metabolism
- Rats
- Receptors, Nicotinic/metabolism
- Smoke/adverse effects
- Smoking/adverse effects
- Nicotiana/chemistry
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Affiliation(s)
- Zhizhan Gu
- Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
| | - Vera Fonseca
- Department of Molecular Pharmacology, Physiology & Biotechnology, Brown University, Providence, RI 02912, USA
| | - Chi-Ming Hai
- Department of Molecular Pharmacology, Physiology & Biotechnology, Brown University, Providence, RI 02912, USA
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15
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Jensen MH, Morris EJ, Huang R, Rebowski G, Dominguez R, Weitz DA, Moore JR, Wang CLA. The conformational state of actin filaments regulates branching by actin-related protein 2/3 (Arp2/3) complex. J Biol Chem 2012; 287:31447-53. [PMID: 22791711 DOI: 10.1074/jbc.m112.350421] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Actin is a highly ubiquitous protein in eukaryotic cells that plays a crucial role in cell mechanics and motility. Cell motility is driven by assembling actin as polymerizing actin drives cell protrusions in a process closely involving a host of other actin-binding proteins, notably the actin-related protein 2/3 (Arp2/3) complex, which nucleates actin and forms branched filamentous structures. The Arp2/3 complex preferentially binds specific actin networks at the cell leading edge and forms branched filamentous structures, which drive cell protrusions, but the exact regulatory mechanism behind this process is not well understood. Here we show using in vitro imaging and binding assays that a fragment of the actin-binding protein caldesmon added to polymerizing actin increases the Arp2/3-mediated branching activity, whereas it has no effect on branch formation when binding to aged actin filaments. Because this caldesmon effect is shown to be independent of nucleotide hydrolysis and phosphate release from actin, our results suggest a mechanism by which caldesmon maintains newly polymerized actin in a distinct state that has a higher affinity for the Arp2/3 complex. Our data show that this new state does not affect the level of cooperativity of binding by Arp2/3 complex or its distribution on actin. This presents a novel regulatory mechanism by which caldesmon, and potentially other actin-binding proteins, regulates the interactions of actin with its binding partners.
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Affiliation(s)
- Mikkel Herholdt Jensen
- Department of Physiology and Biophysics, Boston University, Boston, Massachusetts 02118, USA
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16
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Xia Y, Bhattacharyya A, Roszell EE, Sandig M, Mequanint K. The role of endothelial cell-bound Jagged1 in Notch3-induced human coronary artery smooth muscle cell differentiation. Biomaterials 2012; 33:2462-72. [DOI: 10.1016/j.biomaterials.2011.12.001] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2011] [Accepted: 12/01/2011] [Indexed: 01/02/2023]
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17
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McInroy L, Määttä A. Plectin regulates invasiveness of SW480 colon carcinoma cells and is targeted to podosome-like adhesions in an isoform-specific manner. Exp Cell Res 2011; 317:2468-78. [PMID: 21821021 DOI: 10.1016/j.yexcr.2011.07.013] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2011] [Revised: 06/29/2011] [Accepted: 07/16/2011] [Indexed: 11/25/2022]
Abstract
Co-ordination of cytoskeletal networks and their dynamics is an essential feature of cell migration and cancer cell invasion. Plectin is a large cytolinker protein that influences tissue integrity, organisation of actin and intermediate filaments, and cell migration. Alternatively spliced plectin isoforms are targeted to different subcellular locations. Here, we show that plectin ablation by siRNA impaired migration, invasion and adhesion of SW480 colon carcinoma cells. A previously less well characterised plectin isoform, plectin-1k, co-localised with epithelial integrins, N-WASP, cortactin, and dynamin in podosome-like adhesions in invasive SW480 colon carcinoma cells. Transfection of alternative plectin N-terminal constructs demonstrated that the first exons of isoforms 1k, 1 and 1d can target the actin-binding domain of plectin to podosome-like adhesions. Finally, Plectin-1k N-terminus rescued adhesion site formation in plectin knock-down cells. Thus, plectin participates in actin assembly and invasiveness in carcinoma cells in an isoform-specific manner.
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Affiliation(s)
- Lorna McInroy
- School of Biological and Biomedical Sciences, Durham University, DH1 3LE Durham, United Kingdom
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18
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Nie S, Kee Y, Bronner-Fraser M. Caldesmon regulates actin dynamics to influence cranial neural crest migration in Xenopus. Mol Biol Cell 2011; 22:3355-65. [PMID: 21795398 PMCID: PMC3172261 DOI: 10.1091/mbc.e11-02-0165] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
A nonmuscle caldesmon (CaD) is highly expressed in premigratory and migrating Xenopus cranial neural crest cells. A loss-of-function approach shows that CaD is critical for neural crest migration. The results further suggest that CaD influences cell morphology and motility by modulating actin dynamics in neural crest cells. Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells to migrate a significantly shorter distance, prevents their segregation into distinct migratory streams, and later results in severe defects in cartilage formation. Demonstrating specificity, these effects are rescued by adding back exogenous CaD. Interestingly, CaD proteins with mutations in the Ca2+-calmodulin–binding sites or ErK/Cdk1 phosphorylation sites fail to rescue the knockdown phenotypes, whereas mutation of the PAK phosphorylation site is able to rescue them. Analysis of neural crest explants reveals that CaD is required for the dynamic arrangements of actin and, thus, for cell shape changes and process formation. Taken together, these results suggest that the actin-modulating activity of CaD may underlie its critical function and is regulated by distinct signaling pathways during normal neural crest migration.
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Affiliation(s)
- Shuyi Nie
- Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA
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19
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Quintavalle M, Elia L, Price JH, Heynen-Genel S, Courtneidge SA. A cell-based high-content screening assay reveals activators and inhibitors of cancer cell invasion. Sci Signal 2011; 4:ra49. [PMID: 21791703 DOI: 10.1126/scisignal.2002032] [Citation(s) in RCA: 85] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Acquisition of invasive cell behavior underlies tumor progression and metastasis. To further define the molecular mechanisms underlying invasive behavior, we developed a high-throughput screening strategy to quantitate invadopodia, which are actin-rich membrane protrusions of cancer cells that contribute to tissue invasion and matrix remodeling. We tested the LOPAC 1280 collection of pharmacologically active agents in a high-content, image-based assay and identified compounds that inhibited invadopodium formation without overt toxicity, as well as compounds that increased invadopodia number. The chemotherapeutic agent paclitaxel increased both the number of invadopodia and the invasive behavior of various human cancer cell lines, effects that have potential clinical implications for its use before surgical removal of a primary tumor (neoadjuvant therapy) or in patients with chemoresistant tumors. Several compounds that inhibited invasion have been characterized as cyclin-dependent kinase (Cdk) inhibitors, and loss-of-function experiments determined that Cdk5 was the relevant target. We further determined that Cdk5 promoted both invadopodium formation and cancer cell invasion by phosphorylating and thus decreasing the abundance of the actin regulatory protein caldesmon.
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Affiliation(s)
- Manuela Quintavalle
- Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
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20
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Mak AS. p53 regulation of podosome formation and cellular invasion in vascular smooth muscle cells. Cell Adh Migr 2011; 5:144-9. [PMID: 21164280 DOI: 10.4161/cam.5.2.14375] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
The p53 transcription factor, discovered in 1979 ( 1;2) , is well known as a potent suppressor of tumor development by inhibiting cell cycle progression, and promoting senescence or apoptosis, when the genome is compromised or under oncogenic stress ( 3) . Accumulating evidence has pointed to an alternative role of p53 in the curtailment of tumor progression and colonization of secondary sites by negatively regulating tumor cell metastasis ( 4;5) . Recently, we have found that p53 suppresses Src-induced formation of podosomes and associated invasive phenotypes in fibroblasts and vascular smooth muscle cells (VSMC) ( 6;7) . In this review, I will focus on some recent studies that have identified p53 as a suppressor of cell migration and invasion in general, and VSMC podosome formation and ECM degradation in particular.
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Affiliation(s)
- Alan S Mak
- Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
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21
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Mayanagi T, Sobue K. Diversification of caldesmon-linked actin cytoskeleton in cell motility. Cell Adh Migr 2011; 5:150-9. [PMID: 21350330 DOI: 10.4161/cam.5.2.14398] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The actin cytoskeleton plays a key role in regulating cell motility. Caldesmon (CaD) is an actin-linked regulatory protein found in smooth muscle and non-muscle cells that is conserved among a variety of vertebrates. It binds and stabilizes actin filaments, as well as regulating actin-myosin interaction in a calcium (Ca2+)/calmodulin (CaM)- and/or phosphorylation-dependent manner. CaD function is regulated qualitatively by Ca2+/CaM and by its phosphorylation state and quantitatively at the mRNA level, by three different transcriptional regulation of the CALD1 gene. CaD has numerous functions in cell motility, such as migration, invasion, and proliferation, exerted via the reorganization of the actin cytoskeleton. Here we will outline recent findings regarding CaD's structural features and functions.
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Affiliation(s)
- Taira Mayanagi
- Department of Neuroscience, Osaka University Graduate School of Medicine, Osaka, Japan
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22
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Hamden SS, Schroeter MM, Chalovich JM. Phosphorylation of caldesmon at sites between residues 627 and 642 attenuates inhibitory activity and contributes to a reduction in Ca2+-calmodulin affinity. Biophys J 2011; 99:1861-8. [PMID: 20858431 DOI: 10.1016/j.bpj.2010.07.018] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2010] [Revised: 07/12/2010] [Accepted: 07/14/2010] [Indexed: 11/19/2022] Open
Abstract
Caldesmon is an actin- and myosin-binding protein found in smooth muscle that inhibits actin activation of myosin ATPase activity. The activity of caldesmon is controlled by phosphorylation and by binding to Ca(2+)-calmodulin. We investigated the effects of phosphorylation by p(21)-activated kinase 3 (PAK) and calmodulin on the 22 kDa C-terminal fragment of caldesmon (CaD22). We substituted the major PAK sites, Ser-672 and Ser-702, with either alanine or aspartic acid to mimic nonphosphorylated and constitutively phosphorylated states of caldesmon, respectively. The aspartic acid mutation of CaD22 weakened Ca(2+)-calmodulin binding but had no effect on inhibition of ATPase activity. Phosphorylation of the aspartic acid mutant with PAK resulted in the slow phosphorylation of Thr-627, Ser-631, Ser-635, and Ser-642. Phosphorylation at these sites weakened Ca(2+)-calmodulin binding further and reduced the inhibitory activity of CaD22 in the absence of Ca(2+)-calmodulin. Phosphorylation of these sites of the alanine mutant of CaD22 had no effect on Ca(2+)-calmodulin binding but did reduce inhibition of ATPase activity. Thus, the region between residues 627 and 642 may contribute to the overall regulation of caldesmon's activity.
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Affiliation(s)
- Svetlana S Hamden
- Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina, USA
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23
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The protein kinase C cascade regulates recruitment of matrix metalloprotease 9 to podosomes and its release and activation. Mol Cell Biol 2010; 30:5545-61. [PMID: 20937775 DOI: 10.1128/mcb.00382-10] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Podosomes are transient cell surface structures essential for degradation of extracellular matrix during cell invasion. Protein kinase C (PKC) is involved in the regulation of podosome formation; however, the roles of individual PKC isoforms in podosome formation and proteolytic function are largely unknown. Recently, we reported that PDBu, a PKC activator, induced podosome formation in normal human bronchial epithelial cells. Here, we demonstrate that phorbol-12,13-dibutyrate (PDBu)-induced podosome formation is mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes. Interestingly, although blocking atypical PKCζ did not affect PDBu-induced podosome formation, it significantly reduced matrix degradation at podosomes. Inhibition of PKCζ reduced recruitment of matrix metalloprotease 9 (MMP-9) to podosomes and its release and activation. Downregulation of MMP-9 by small interfering RNA (siRNA) or neutralization antibody also significantly reduced matrix degradation. The regulatory effects of PKCζ on matrix degradation and recruitment of MMP-9 to podosomes were PKCζ kinase activity dependent. PDBu-induced recruitment of PKCζ and MMP-9 to podosomes was blocked by inhibition of novel PKC with rottlerin or PKCδ siRNA. Our data suggest that multiple PKC isozymes form a signaling cascade that controls podosome formation and dynamics and MMP-9 recruitment, release, and activation in a coordinated fashion.
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24
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Abstract
We have recently shown that Src induces the formation of podosomes and cell invasion by suppressing endogenous p53, while enhanced p53 strongly represses the Src-induced invasive phenotype. However, the mechanism by which Src and p53 play antagonistic roles in cell invasion is unknown. Here we show that the Stat3 oncogene is a required downstream effector of Src in inducing podosome structures and related invasive phenotypes. Stat3 promotes Src phenotypes through the suppression of p53 and the p53-inducible protein caldesmon, a known podosome antagonist. In contrast, enhanced p53 attenuates Stat3 function and Src-induced podosome formation by upregulating the tumor suppressor PTEN. PTEN, through the inactivation of Src/Stat3 function, also stabilizes the podosome-antagonizing p53/caldesmon axis, thereby further enhancing the anti-invasive potential of the cell. Furthermore, the protein phosphatase activity of PTEN plays a major role in the negative regulation of the Src/Stat3 pathway and represses podosome formation. Our data suggest that cellular invasiveness is dependent on the balance between two opposing forces: the proinvasive oncogenes Src-Stat3 and the anti-invasive tumor suppressors p53-PTEN.
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25
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Analysis of the signaling pathways regulating Src-dependent remodeling of the actin cytoskeleton. Eur J Cell Biol 2010; 90:143-56. [PMID: 20719402 DOI: 10.1016/j.ejcb.2010.07.006] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2010] [Revised: 06/18/2010] [Accepted: 07/13/2010] [Indexed: 12/24/2022] Open
Abstract
Cell adhesion to the extracellular matrix is mediated by adhesion receptors, mainly integrins, which upon interaction with the extracellular matrix, bind to the actin cytoskeleton via their cytoplasmic domains. This association is mediated by a variety of scaffold and signaling proteins, which control the mechanical and signaling activities of the adhesion site. Upon transformation of fibroblasts with active forms of Src (e.g., v-Src), focal adhesions are disrupted, and transformed into dot-like contacts known as podosomes, and consisting of a central actin core surrounded by an adhesion ring. To clarify the mechanism underlying Src-dependent modulation of the adhesive phenotype, and its influence on podosome organization, we screened for the effect of siRNA-mediated knockdown of tyrosine kinases, MAP kinases and phosphatases on the reorganization of the adhesion-cytoskeleton complex, induced by a constitutively active Src mutant (SrcY527F). In this screen, we discovered several genes that are involved in Src-induced remodeling of the actin cytoskeleton. We further showed that knockdown of Src in osteoclasts abolishes the formation of the podosome-based rings and impairs cell spreading, without inducing stress fiber development. Our work points to several genes that are involved in this process, and sheds new light on the molecular plasticity of integrin adhesions.
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26
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JIANG QF, CAI SX, YAN XQ. The Effect of Caldesmon Phosphorylation on Metastatic Tumor Cell Mobility*. PROG BIOCHEM BIOPHYS 2010. [DOI: 10.3724/sp.j.1206.2009.00545] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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27
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Maślikowski BM, Néel BD, Wu Y, Wang L, Rodrigues NA, Gillet G, Bédard PA. Cellular processes of v-Src transformation revealed by gene profiling of primary cells--implications for human cancer. BMC Cancer 2010; 10:41. [PMID: 20152043 PMCID: PMC2837010 DOI: 10.1186/1471-2407-10-41] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2009] [Accepted: 02/12/2010] [Indexed: 01/05/2023] Open
Abstract
Background Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. In this study, we took advantage of several strains of the Rous sarcoma virus (RSV) to characterize the patterns of v-Src-dependent gene expression in two different primary cell types, namely chicken embryo fibroblasts (CEF) and chicken neuroretinal (CNR) cells. We identified a common set of v-Src regulated genes and assessed if their expression is associated with disease-free survival using several independent human tumor data sets. Methods CEF and CNR cells were infected with transforming, non-transforming, and temperature sensitive mutants of RSV to identify the patterns of gene expression in response to v-Src-transformation. Microarray analysis was used to measure changes in gene expression and to define a common set of v-Src regulated genes (CSR genes) in CEF and CNR cells. A clustering enrichment regime using the CSR genes and two independent breast tumor data-sets was used to identify a 42-gene aggressive tumor gene signature. The aggressive gene signature was tested for its prognostic value by conducting survival analyses on six additional tumor data sets. Results The analysis of CEF and CNR cells revealed that cell transformation by v-Src alters the expression of 6% of the protein coding genes of the genome. A common set of 175 v-Src regulated genes (CSR genes) was regulated in both CEF and CNR cells. Within the CSR gene set, a group of 42 v-Src inducible genes was associated with reduced disease- and metastasis-free survival in several independent patient cohorts with breast or lung cancer. Gene classes represented within this group include DNA replication, cell cycle, the DNA damage and stress responses, and blood vessel morphogenesis. Conclusion By studying the v-Src-dependent changes in gene expression in two types of primary cells, we identified a set of 42 inducible genes associated with poor prognosis in breast and lung cancer. The identification of these genes provides a set of biomarkers of aggressive tumor behavior and a framework for the study of cancer cells characterized by elevated Src kinase activity.
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Affiliation(s)
- Bart M Maślikowski
- Department of Biology, McMaster University, 1280 Main street West, Hamilton, ON, L8S 4K1, Canada
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28
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Jiang Q, Huang R, Cai S, Wang CLA. Caldesmon regulates the motility of vascular smooth muscle cells by modulating the actin cytoskeleton stability. J Biomed Sci 2010; 17:6. [PMID: 20128924 PMCID: PMC2846900 DOI: 10.1186/1423-0127-17-6] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2009] [Accepted: 02/03/2010] [Indexed: 02/16/2023] Open
Abstract
Background Migration of vascular smooth muscle cells (SMCs) from the media to intima constitutes a critical step in the development of proliferative vascular diseases. To elucidate the regulatory mechanism of vacular SMC motility, the roles of caldesmon (CaD) and its phosphorylation were investigated. Methods We have performed Transwell migration assays, immunofluorescence microscopy, traction microscopy and cell rounding assays using A7r5 cells transfected with EGFP (control), EGFP-wtCaD or phosphomimetic CaD mutants, including EGFP-A1A2 (the two PAK sites Ser452 and Ser482 converted to Ala), EGFP-A3A4 (the two Erk sites Ser497 and Ser527 converted to Ala), EGFP-A1234 (both PAK- and Erk-sites converted to Ala) and EGFP-D1234 (both PAK- and Erk-sites converted to Asp). Results We found that cells transfected with wtCaD, A1A2 or A3A4 mutants of CaD migrated at a rate approximately 50% more slowly than those EGFP-transfected cells. The migration activity for A1234 cells was only about 13% of control cells. Thus it seems both MAPK and PAK contribute to the motility of A7r5 cells and the effects are comparable and additive. The A1234 mutant also gave rise to highest strain energy and lowest rate of cell rounding. The migratory and contractile properties of these cells are consistent with stabilized actin cytoskeletal structures. Indeed, the A1234 mutant cells exhibited most robust stress fibers, whereas cells transfected with wtCaD or A3A4 (and A1A2) had moderately reinforced actin cytoskeleton. The control cells (transfected with EGFP alone) exhibited actin cytoskeleton that was similar to that in untransfected cells, and also migrated at about the same speed as the untransfected cells. Conclusions These results suggest that both the expression level and the level of MAPK- and/or PAK-mediated phosphorylation of CaD play key roles in regulating the cell motility by modulating the actin cytoskeleton stability in dedifferentiated vascular SMCs such as A7r5.
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Affiliation(s)
- Qifeng Jiang
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044, China
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29
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Wang CLA, Coluccio LM. New insights into the regulation of the actin cytoskeleton by tropomyosin. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2010; 281:91-128. [PMID: 20460184 DOI: 10.1016/s1937-6448(10)81003-2] [Citation(s) in RCA: 70] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The actin cytoskeleton is regulated by a variety of actin-binding proteins including those constituting the tropomyosin family. Tropomyosins are coiled-coil dimers that bind along the length of actin filaments. In muscles, tropomyosin regulates the interaction of actin-containing thin filaments with myosin-containing thick filaments to allow contraction. In nonmuscle cells where multiple tropomyosin isoforms are expressed, tropomyosins participate in a number of cellular events involving the cytoskeleton. This chapter reviews the current state of the literature regarding tropomyosin structure and function and discusses the evidence that tropomyosins play a role in regulating actin assembly.
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30
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Juchem G, Weiss DR, Gansera B, Kemkes BM, Mueller-Hoecker J, Nees S. Pericytes in the macrovascular intima: possible physiological and pathogenetic impact. Am J Physiol Heart Circ Physiol 2009; 298:H754-70. [PMID: 20023125 DOI: 10.1152/ajpheart.00343.2009] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
The frequently observed de-endothelialization of venous coronary bypass grafts prepared using standard methods exposes subendothelial prothrombotic cells to blood components, thus endangering patients by inducing acute thromboembolic infarction or long-term proliferative stenosis. Our aim was to gain deeper histological and physiological insight into these relations. An intricate network of subendothelial cells, characterized by histological features specific for true pericytes, was detected even in healthy vessels and forms, coupled to the luminal endothelium, a second leaflet of the macrovascular intima. These cells, and particularly those in the venous intima, express enormous concentrations of tissue factor and can recruit additional amounts of up to the 25-fold concentration within 1 h during preincubation with serum (intimal pericytes of venous origin activate 30.71 +/- 4.07 pmol coagulation factor x.min(-1).10(-6) cells; n = 15). Moreover, decoupled from the endothelium, they proliferate rapidly (generation time, 15 +/- 2.1 h, n = 8). Central regions of atherosclerotic plaques, as well as of those of restenosed areas of coronary vein grafts, consist almost completely of these cells. In stark contrast with the prothrombogenicity of the intimal pericytes, intact luminal endothelium recruits high concentrations of thrombomodulin (CD 141) specifically within its intercellular junctions, activates Protein C rapidly (42 +/- 5.1 pmol/min.10(6) venous endothelial cells at thrombin saturation; n = 15), can thus actively prevent coagulatory processes, and never expresses histologically detectable and functionally active tissue factor. Given this strongly prothrombotic potential of the intimal pericytes and their overshooting growth behavior in endothelium-denuded vascular regions, they may play important roles in the development of atherosclerosis, thrombosis, and saphenous vein graft disease.
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Affiliation(s)
- Gerd Juchem
- Departments of Cardiac Surgery, University of Munich, Germany
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31
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p53 suppresses Src-induced podosome and rosette formation and cellular invasiveness through the upregulation of caldesmon. Mol Cell Biol 2009; 29:3088-98. [PMID: 19349302 DOI: 10.1128/mcb.01816-08] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The tumor-suppressive role of p53 at the level of tumor initiation is well documented. It has also been shown previously that p53 acts against tumor progression/metastasis. However, its role in modulating cell migration and invasion leading to metastasis is poorly understood. In this study, using vascular smooth muscle cells and NIH 3T3 fibroblast cells, we have shown that p53 potently suppresses Src-induced podosome/rosette formation, extracellular matrix digestion, cell migration, and invasion. The overexpression of exogenous wild-type p53 or the activation of the endogenous p53 function suppresses, while the short hairpin RNA-mediated knockdown of p53 expression or the pageing of its function exacerbates, Src-induced migratory and invasive phenotypes. We have also found that p53 expression and function are downregulated in cells stably transformed with constitutively active Src that exhibit aggressive invasive properties. Lastly, p53 upregulates the expression of caldesmon, an actin-binding protein that has been shown to be an inhibitor of podosome/invadopodium formation. The ability of p53 to suppress Src phenotypes in transformed cells was largely abolished by knocking down caldesmon. This study reports a novel molecular mechanism (caldesmon), as well as a structural basis (podosomes/rosettes), to show how p53 can act as an anti-motility/invasion/metastasis agent.
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32
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Kim HR, Appel S, Vetterkind S, Gangopadhyay SS, Morgan KG. Smooth muscle signalling pathways in health and disease. J Cell Mol Med 2009. [PMID: 19120701 DOI: 10.1111/j.1582-4934.2008.00552] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Smooth muscle contractile activity is a major regulator of function of the vascular system, respiratory system, gastrointestinal system and the genitourinary systems. Malfunction of contractility in these systems leads to a host of clinical disorders, and yet, we still have major gaps in our understanding of the molecular mechanisms by which contractility of the differentiated smooth muscle cell is regulated. This review will summarize recent advances in the molecular understanding of the regulation of smooth muscle myosin activity via phosphorylation/dephosphorylation of myosin, the regulation of the accessibility of actin to myosin via the actin-binding proteins calponin and caldesmon, and the remodelling of the actin cytoskeleton. Understanding of the molecular 'players' should identify target molecules that could point the way to novel drug discovery programs for the treatment of smooth muscle disorders such as cardiovascular disease, asthma, functional bowel disease and pre-term labour.
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Affiliation(s)
- H R Kim
- Department of Health Sciences, Boston University, Boston, MA 02215, USA
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33
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Jin L, Hastings NE, Blackman BR, Somlyo AV. Mechanical properties of the extracellular matrix alter expression of smooth muscle protein LPP and its partner palladin; relationship to early atherosclerosis and vascular injury. J Muscle Res Cell Motil 2009; 30:41-55. [PMID: 19205907 DOI: 10.1007/s10974-009-9173-1] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2008] [Accepted: 01/08/2009] [Indexed: 12/12/2022]
Abstract
Lipoma preferred partner (LPP) localizes to focal adhesions/dense bodies, is selectively expressed in smooth muscle cells (SMC) and enhances cell migration. SMCs cultured on denatured collagen or on a rigid substrate, up regulated expression of LPP, its partner palladin, tenascin C (TN-C), phosphorylated focal adhesion kinase (pFAK) and exhibited robust stress fibers. In an endothelial (EC)/SMC hemodynamic flow system, shear stress waveforms mimicking atheroprone flow, applied to the EC layer, significantly decreased expression of SMC LPP and palladin. They were also down regulated with TN-C, in an ApoE murine model of atherosclerosis and with oxidative stress but up regulated in an arterial injury model in response to upstream sequential changes in pFAK, Prx1 and TN-C. In conclusion, expression of LPP and palladin are modulated by a mix of mechanical cues, oxidative stress and substrate composition which translate into their up or down regulation in vessel wall injury and early atherogenesis.
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Affiliation(s)
- Li Jin
- Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA
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34
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Xiao H, Eves R, Yeh C, Kan W, Xu F, Mak AS, Liu M. Phorbol ester-induced podosomes in normal human bronchial epithelial cells. J Cell Physiol 2009; 218:366-75. [DOI: 10.1002/jcp.21609] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
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35
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Lin JJ, Li Y, Eppinga RD, Wang Q, Jin J. Chapter 1 Roles of Caldesmon in Cell Motility and Actin Cytoskeleton Remodeling. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2009; 274:1-68. [DOI: 10.1016/s1937-6448(08)02001-7] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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36
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Kim HR, Appel S, Vetterkind S, Gangopadhyay SS, Morgan KG. Smooth muscle signalling pathways in health and disease. J Cell Mol Med 2008; 12:2165-80. [PMID: 19120701 PMCID: PMC2692531 DOI: 10.1111/j.1582-4934.2008.00552.x] [Citation(s) in RCA: 154] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2008] [Accepted: 10/08/2008] [Indexed: 12/24/2022] Open
Abstract
Smooth muscle contractile activity is a major regulator of function of the vascular system, respiratory system, gastrointestinal system and the genitourinary systems. Malfunction of contractility in these systems leads to a host of clinical disorders, and yet, we still have major gaps in our understanding of the molecular mechanisms by which contractility of the differentiated smooth muscle cell is regulated. This review will summarize recent advances in the molecular understanding of the regulation of smooth muscle myosin activity via phosphorylation/dephosphorylation of myosin, the regulation of the accessibility of actin to myosin via the actin-binding proteins calponin and caldesmon, and the remodelling of the actin cytoskeleton. Understanding of the molecular 'players' should identify target molecules that could point the way to novel drug discovery programs for the treatment of smooth muscle disorders such as cardiovascular disease, asthma, functional bowel disease and pre-term labour.
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Affiliation(s)
- H R Kim
- Department of Health Sciences, Boston UniversityBoston, MA, USA
| | - S Appel
- Department of Health Sciences, Boston UniversityBoston, MA, USA
| | - S Vetterkind
- Department of Health Sciences, Boston UniversityBoston, MA, USA
| | | | - K G Morgan
- Department of Health Sciences, Boston UniversityBoston, MA, USA
- Boston Biomedical Research InstituteWatertown, MA, USA
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37
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Kudryashova TV, Rutkevich PN, Shevelev AY, Vlasik TN, Vorotnikov AV. Caldesmon affects actin organization at the leading edge and inhibits cell migration. Biophysics (Nagoya-shi) 2008. [DOI: 10.1134/s0006350908060110] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
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Aga M, Bradley JM, Keller KE, Kelley MJ, Acott TS. Specialized podosome- or invadopodia-like structures (PILS) for focal trabecular meshwork extracellular matrix turnover. Invest Ophthalmol Vis Sci 2008; 49:5353-65. [PMID: 18641286 DOI: 10.1167/iovs.07-1666] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
PURPOSE There are distinctive areas of colocalization of matrix metalloproteinase (MMP)-2 and -14 on trabecular meshwork (TM) cells that resemble podosomes or invadopodia. Studies were conducted to determine whether TM cells exhibit podosome- or invadopodia-like structures (PILS) and whether they produce focal extracellular matrix (ECM) turnover. METHODS Porcine and human TM cells and perfused anterior segment organ cultures were studied. Localization of PILS components on TM cells and in sections from anterior segments was determined by immunohistochemistry and confocal microscopy. Cells were grown on type I collagen labeled with fluorescein isothiocyanate (FITC) for degradation analysis. Confocal time lapse images were taken of labeled TM cells on FITC-collagen. RESULTS Immunostaining for MMP-2, MMP-14, and the typical PILS components cortactin, caldesmon, alpha-actinin, N-WASP, Arp-3, and cdc42 colocalized on these distinctive structures. Integrin-alphaV and -beta1, fibronectin, and versican colocalized with PILS components. TM cells on FITC-conjugated collagen developed focal regions of degradation. Time-lapse imaging showed dramatic and controlled movement of TM cell processes during this ECM degradation and fragment internalization. MMP-2, MMP-14, and cortactin colocalized at regions that appear to be PILS on cells within the outflow pathway in sections of human anterior segments. CONCLUSIONS TM cells exhibit areas where PILS components colocalize with MMP-2 and -14. Similar structures are found in sections, suggesting that PILS occur in situ in the outflow pathway. The collagen degradation suggests that PILS may serve as focal sites for targeted ECM turnover, an event linked to modifications of aqueous outflow resistance and intraocular pressure homeostasis.
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Affiliation(s)
- Mini Aga
- Casey Eye Institute, Oregon Health & Science University, Portland, Oregon 97239-4197, USA
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39
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Dorfleutner A, Cho Y, Vincent D, Cunnick J, Lin H, Weed SA, Stehlik C, Flynn DC. Phosphorylation of AFAP-110 affects podosome lifespan in A7r5 cells. J Cell Sci 2008; 121:2394-405. [PMID: 18577577 DOI: 10.1242/jcs.026187] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
AFAP-110 is an actin-binding and -crosslinking protein that is enriched in Src and phorbol ester (PE)-induced podosomes. In vascular smooth muscle cells endogenous AFAP-110 localized to actin stress fibers and, in response to treatment with phorbol-12,13-dibutyrate (PDBu), to actin-rich podosomes. Since PEs can activate PKCalpha, AFAP-110 is a substrate of PKCalpha and PKCalpha-AFAP-110 interactions direct podosome formation, we sought to identify a PE-induced phosphorylation site in AFAP-110 and determine whether phosphorylation is linked to the formation of podosomes. Mutational analysis revealed Ser277 of AFAP-110 to be phosphorylated in PE-treated cells. The use of a newly generated, phospho-specific antibody directed against phosphorylated Ser277 revealed that PKCalpha activation is associated with PE-induced AFAP-110 phosphorylation. In PDBu-treated A7r5 rat vascular smooth muscle cells, immunolabeling using the phospho-specific antibody showed that phospho-AFAP-110 is primarily associated with actin in podosomes. Although mutation of Ser at position 277 to Ala (AFAP-110(S277A)) did not alter the ability of AFAP-110 to localize to podosomes, overexpression of AFAP-110(S277A) in treated and untreated A7r5 cells resulted in an increased number of cells that display podosomes. Video microscopy demonstrated that AFAP-110(S277A) expression correlates with an increased number of long-lived podosomes. Therefore, we hypothesize that AFAP-110 phosphorylation and/or dephosphorylation is involved in the regulation of podosome stability and lifespan.
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Affiliation(s)
- Andrea Dorfleutner
- The Mary Babb Randolph Cancer Center and Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA
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40
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Flynn DC, Cho Y, Vincent D, Cunnick JM. Podosomes and Invadopodia: Related structures with Common Protein Components that May Promote Breast Cancer Cellular Invasion. Breast Cancer (Auckl) 2008; 2:17-29. [PMID: 21655365 PMCID: PMC3085414 DOI: 10.4137/bcbcr.s789] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
A rate-limiting step in breast cancer progression is acquisition of the invasive phenotype, which can precede metastasis. Expression of cell-surface proteases at the leading edge of a migrating cell provides cells with a mechanism to cross tissue barriers. A newly appreciated mechanism that may be relevant for breast cancer cell invasion is the formation of invadopodia, well-defined structures that project from the ventral membrane and promote degradation of the extracellular matrix, allowing the cell to cross a tissue barrier. Recently, there has been some controversy and discussion as to whether invadopodia, which are associated with carcinoma cells, are related to a similar structure called podosomes, which are associated with normal cells. Invadopodia and podosomes share many common characteristics, including a similar size, shape, subcellular localization and an ability to promote invasion. These two structures also share many common protein components, which we outline herein. It has been speculated that podosomes may be precursors to invadopodia and by extension both structures may be relevant to cancer cell invasion. Here, we compare and contrast the protein components of invadopodia and podosomes and discuss a potential role for these proteins and the evidence that supports a role for invadopodia and podosomes in breast cancer invasion.
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Affiliation(s)
- Daniel C. Flynn
- Mary Babb Randolph Cancer Center
- Department of Microbiology, Immunology and Cell Biology and
| | - YoungJin Cho
- Mary Babb Randolph Cancer Center
- Department of Microbiology, Immunology and Cell Biology and
| | - Deanne Vincent
- Mary Babb Randolph Cancer Center
- Department of Microbiology, Immunology and Cell Biology and
| | - Jess M. Cunnick
- Mary Babb Randolph Cancer Center
- Department of Pathology, West Virginia University, Morgantown, WV 26506-9300
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41
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Wang CLA. Caldesmon and the regulation of cytoskeletal functions. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2008; 644:250-72. [PMID: 19209827 DOI: 10.1007/978-0-387-85766-4_19] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Caldesmon (CaD) is an extraordinary actin-binding protein, because in addition to actin, it also bindsmyosin, calmodulin and tropomyosin. As a component of the smoothmuscle and nonmuscle contractile apparatus CaD inhibits the actomyosin ATPase activity and its inhibitory action is modulated by both Ca2+ and phosphorylation. The multiplicity of binding partners and diverse biochemical properties suggest CaD is a potent and versatile regulatory protein both in contractility and cell motility. However, after decades ofinvestigation in numerous laboratories, hard evidence is still lacking to unequivocally identify its in vivo functions, although indirect evidence is mounting to support an important role in connection with the actin cytoskeleton. This chapter reviews the highlights of the past findings and summarizes the current views on this protein, with emphasis of its interaction with tropomyosin.
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Affiliation(s)
- C L Albert Wang
- Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA.
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42
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Yoshio T, Morita T, Kimura Y, Tsujii M, Hayashi N, Sobue K. Caldesmon suppresses cancer cell invasion by regulating podosome/invadopodium formation. FEBS Lett 2007; 581:3777-82. [PMID: 17631293 DOI: 10.1016/j.febslet.2007.06.073] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2007] [Accepted: 06/28/2007] [Indexed: 12/15/2022]
Abstract
The podosome and invadopodium are dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin-binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmon's involvement in podosome/invadopodium-mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.
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Affiliation(s)
- T Yoshio
- Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
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43
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Webb BA, Jia L, Eves R, Mak AS. Dissecting the functional domain requirements of cortactin in invadopodia formation. Eur J Cell Biol 2007; 86:189-206. [PMID: 17343955 DOI: 10.1016/j.ejcb.2007.01.003] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2006] [Revised: 01/19/2007] [Accepted: 01/22/2007] [Indexed: 12/28/2022] Open
Abstract
Cells degrade extracellular matrix (ECM) barriers at focal locations by the formation of membrane protrusions called invadopodia. Polymerization of the actin cytoskeleton is critical to the extension of these processes into the ECM. We used a short interference RNA/rescue strategy to investigate the role of cortactin in the formation of Src-induced invadopodia in 3T3 fibroblasts, and subsequent degradation of the ECM. Cortactin-depleted cells did not form invadopodia or degrade the ECM. Functional invadopodia were restored in cortactin-depleted cells by expression of full-length cortactin, and fragments that contained the intact actin-binding repeats. Mutation of the three Src-targeted Tyr sites to Phe caused a loss in its rescuing ability, while mutation of the Erk phosphorylation sites had little effect on invadopodia formation. Interestingly, knock-down of cortactin did not affect the formation of lamellipodia and only slightly attenuated random cell motility. Our data shows that formation of functional invadopodia requires interaction between cortactin and filamentous actin, while interaction with SH3- and NTA-binding partners plays a less significant role. Furthermore, phosphorylation of cortactin by Src, but not by Erk, is essential for functional invadopodia formation. These results also suggest that cortactin plays a different role in invadopodia-dependent ECM degradation and lamellipodia formation in cell movement.
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Affiliation(s)
- Bradley A Webb
- Department of Biochemistry and Protein Function Discovery Program, Queen's University, Room 616 Botterell Hall, Kingston, Ont., Canada K7L 3N6
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44
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Morita T, Mayanagi T, Yoshio T, Sobue K. Changes in the Balance between Caldesmon Regulated by p21-activated Kinases and the Arp2/3 Complex Govern Podosome Formation. J Biol Chem 2007; 282:8454-63. [PMID: 17224451 DOI: 10.1074/jbc.m609983200] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.
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Affiliation(s)
- Tsuyoshi Morita
- Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita, Osaka 565-0871, Japan
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45
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The matrix corroded: podosomes and invadopodia in extracellular matrix degradation. Trends Cell Biol 2007; 17:107-17. [PMID: 17275303 DOI: 10.1016/j.tcb.2007.01.002] [Citation(s) in RCA: 482] [Impact Index Per Article: 26.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2006] [Revised: 12/20/2006] [Accepted: 01/17/2007] [Indexed: 01/09/2023]
Abstract
Podosomes and invadopodia are unique actin-rich adhesions that establish close contact to the substratum but can also degrade components of the extracellular matrix. Accordingly, matrix degradation localized at podosomes or invadopodia is thought to contribute to cellular invasiveness in physiological and pathological situations. Cell types that form podosomes include monocytic, endothelial and smooth muscle cells, whereas invadopodia have been mostly observed in carcinoma cells. This review highlights important new developments in the field, discusses the common and divergent features of podosomes and invadopodia and summarizes current knowledge about matrix-degrading proteinases at these structures.
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Gu Z, Kordowska J, Williams GL, Wang CLA, Hai CM. Erk1/2 MAPK and caldesmon differentially regulate podosome dynamics in A7r5 vascular smooth muscle cells. Exp Cell Res 2006; 313:849-66. [PMID: 17239373 PMCID: PMC2040298 DOI: 10.1016/j.yexcr.2006.12.005] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2006] [Revised: 12/05/2006] [Accepted: 12/06/2006] [Indexed: 01/27/2023]
Abstract
We tested the hypothesis that the MEK/Erk/caldesmon phosphorylation cascade regulates PKC-mediated podosome dynamics in A7r5 cells. We observed the phosphorylation of MEK, Erk and caldesmon, and their translocation to the podosomes upon phorbol dibutyrate (PDBu) stimulation, together with the nuclear translocation of phospho-MEK and phospho-Erk. After MEK inhibition by U0126, Erk translocated to the interconnected actin-rich columns but failed to translocate to the nucleus, suggesting that podosomes served as a site for Erk phosphorylation. The interconnected actin-rich columns in U0126-treated, PDBu-stimulated cells contained alpha-actinin, caldesmon, vinculin, and metalloproteinase-2. Caldesmon and vinculin became integrated with F-actin at the columns, in contrast to their typical location at the ring of podosomes. Live-imaging experiments suggested the growth of these columns from podosomes that were slow to disassemble. The observed modulation of podosome size and life time in A7r5 cells overexpressing wild-type and phosphorylation-deficient caldesmon-GFP mutants in comparison to untransfected cells suggests that caldesmon and caldesmon phosphorylation modulate podosome dynamics in A7r5 cells. These results suggest that Erk1/2 and caldesmon differentially modulate PKC-mediated formation and/or dynamics of podosomes in A7r5 vascular smooth muscle cells.
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Affiliation(s)
- Zhizhan Gu
- Department of Molecular Pharmacology, Physiology & Biotechnology, Brown University, Providence, RI 02912
| | | | - Geoffrey L. Williams
- Department of Molecular Biology, Cell Biology & Biochemistry, Brown University, Providence, RI 02912
| | | | - Chi-Ming Hai
- Department of Molecular Pharmacology, Physiology & Biotechnology, Brown University, Providence, RI 02912
- *Correspondence: Chi-Ming Hai, Ph.D., Department of Molecular Pharmacology, Physiology & Biotechnology, Box G-B3, Brown University, Providence, RI 02912, Tel. (401) 863-3288, Fax. (401) 863-1222, Email.
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Webb BA, Zhou S, Eves R, Shen L, Jia L, Mak AS. Phosphorylation of cortactin by p21-activated kinase. Arch Biochem Biophys 2006; 456:183-93. [PMID: 16854367 DOI: 10.1016/j.abb.2006.06.011] [Citation(s) in RCA: 71] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2006] [Revised: 06/06/2006] [Accepted: 06/12/2006] [Indexed: 12/25/2022]
Abstract
Cortactin is an F-actin binding protein that is enriched in dynamic cytoskeletal organelles such as podosomes, membrane ruffles, and lamellipodia. We have shown previously that Src-phosphorylation of cortactin is not required for its translocation to phorbol-ester induced podosomes in A7r5 aortic smooth muscle cells, but may be important for stability and turnover of podosomes. However, little is known of the role of Ser/Thr kinases in the regulation of cortactin. Here, we report that p21-associated kinase (PAK), which plays a crucial role in the formation of podosome and membrane ruffles, is able to phosphorylate cortactin in vitro. The predominant phosphorylation site is located at Ser113 in the first actin-binding repeat. Phosphorylation by PAK is not required for the translocation of cortactin to podosomes, lamellipodia, or membrane ruffles in A7r5 smooth muscle cells. However, binding of cortactin to F-actin is significantly reduced by PAK-phosphorylation. Taken together, these results suggest a role for PAK-phosphorylation of cortactin in the regulation of the dynamics of branched actin filaments in dynamic cytoskeletal organelles.
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Affiliation(s)
- Bradley A Webb
- Department of Biochemistry and Protein Function Discovery Program, Queen's University, Kingston, Ont., Canada K7L 3N6
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