Integrin-mediated interaction of cells with the extracellular matrix control a diverse set of physiologic processes including cell migration during embryogenesis and metastasis, cell proliferation, tissue maintenance, differentiation and repair. Protein complexes associated with the integrin cytoplasmic domains facilitate integrin signaling and interactions with the actin cytoskeleton. This proposal will focus on the importance of recently identified interactions between the integrin-linked kinase ILK, the actin binding protein actopaxin and the molecular adapter protein paxillin in the regulation of cell adhesion, motility, proliferation and differentiation.
In Aim 1 epitope-tagged mutant cDNAs of ILK and actopaxin will be transfected into fibroblasts followed by co-precipitation experiments to further characterize the ILK and actopaxin binding domains. The importance of these domains for subcellular localization will be assessed by immunofluorescence microscopy. We have recently demonstrated that actopaxin mutants perturb cell adhesion/spreading on collagen. Experiments proposed in Aim 2 will identify the molecular basis for this defect by assaying for changes in integrin function as well as effect on downstream signaling events. The role of ILK and actopaxin in mediating cell motility will be addressed using time-lapse microscopy, Boyden chamber and wound assays.
In Aim 3 we will investigate the importance of ILK-actopaxin associations in regulating cell proliferation and differentiation. Sites of actopaxin phosphorylation resulting from associated cyclinB/cdc2 kinase activity will be delineated. The role of phosphorylation of these sites in regulating protein-protein interactions and cytoskeleton changes associated with transition through mitosis will be examined by co-precipitation analysis and immunofluorescence microscopy. Potential effects on proliferation and cell survival will be examined. The involvement of ILK and actopaxin interactions in regulating cell differentiation will be examined in the context of skeletal muscle myoblast differentiation. Together these experiments will address evolutionarily conserved mechanisms of protein linkages between the extracellular matrix and the actin cytoskeleton that are of importance to the understanding of cardiovascular and musculoskeletal defects and metastatic transformation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL070244-01
Application #
6468278
Study Section
Pathobiochemistry Study Section (PBC)
Program Officer
Pearson, Gail D
Project Start
2002-04-01
Project End
2006-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
1
Fiscal Year
2002
Total Cost
$367,000
Indirect Cost
Name
Upstate Medical University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
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Wormer, Duncan; Deakin, Nicholas O; Turner, Christopher E (2012) CdGAP regulates cell migration and adhesion dynamics in two-and three-dimensional matrix environments. Cytoskeleton (Hoboken) 69:644-58
Deakin, Nicholas O; Ballestrem, Christoph; Turner, Christopher E (2012) Paxillin and Hic-5 interaction with vinculin is differentially regulated by Rac1 and RhoA. PLoS One 7:e37990
Pignatelli, Jeanine; Tumbarello, David A; Schmidt, Ronald P et al. (2012) Hic-5 promotes invadopodia formation and invasion during TGF-ýý-induced epithelial-mesenchymal transition. J Cell Biol 197:421-37
Pignatelli, Jeanine; LaLonde, Sara E; LaLonde, David P et al. (2012) Actopaxin (?-parvin) phosphorylation is required for matrix degradation and cancer cell invasion. J Biol Chem 287:37309-20
Devallière, Julie; Chatelais, Mathias; Fitau, Juliette et al. (2012) LNK (SH2B3) is a key regulator of integrin signaling in endothelial cells and targets ?-parvin to control cell adhesion and migration. FASEB J 26:2592-606
Yu, Jianxin A; Deakin, Nicholas O; Turner, Christopher E (2010) Emerging role of paxillin-PKL in regulation of cell adhesion, polarity and migration. Cell Adh Migr 4:342-7
Yu, Jianxin A; Deakin, Nicholas O; Turner, Christopher E (2009) Paxillin-kinase-linker tyrosine phosphorylation regulates directional cell migration. Mol Biol Cell 20:4706-19
Deakin, Nicholas O; Turner, Christopher E (2008) Paxillin comes of age. J Cell Sci 121:2435-44
LaLonde, David P; Grubinger, Markus; Lamarche-Vane, Nathalie et al. (2006) CdGAP associates with actopaxin to regulate integrin-dependent changes in cell morphology and motility. Curr Biol 16:1375-85

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