Actopaxin is a 42kDa focal adhesion protein that facilitates integrin signaling and interactions with the actin cytoskeleton. Phosphorylation of the actopaxin amino-terminus promotes lamellipodia formation and stimulates cell migration. Actopaxin also serves as a molecular scaffold for key regulators of cytoskeletal dynamics including ILK, paxillin, and TESK1. This proposal will focus on the importance of recently identified interactions between actopaxin and the Cdc42/Rac GEF PIX, CdGAP and the endocytic protein b2-Adaptin in the regulation of Rho family GTPase signaling and cell motility.
Aim 1 will examine how phosphorylation of actopaxin regulates lamellipodia formation and cell migration via the PIX-p21-activated kinase, PAK axis using transient transfection of actopaxin, PIX and PAK mutants combined with fluorescence microscopy, scrape wound, time-lapse and Boyden chamber migration assays. Rho family GTPase activity will be evaluated in cells using """"""""Raichu"""""""" FRET probes. Phospho-epitope specific antibodies will be generated to probe the spatio-temporal regulation of actopaxin phosphorylation.
In Aim 2 the role of CdGAP in integrin signaling and the importance of actopaxin binding in regulating cell migration will be examined as in Aim 1. GST-pulldown and co precipitation assays using mutants of CdGAP and actopaxin will delineate the respective binding domains. The importance of these domains for subcellular localization of CdGAP and activity will be assessed by epi- and Total Internal Reflection- (TIR-) fluorescence microscopy and in vitro GAP assays respectively.
Aim 3 will examine a role for the actopaxin-b2-Adaptin interaction in cell migration through the stimulation of cell polarity and focal adhesion disassembly, potentially through endocytic events. The binding site on actopaxin will be defined using GST-fusion proteins and co-precipitation. Effects on subcellular localization will be evaluated. Binding mutants will be introduced into fibroblasts to assess effect on cell polarity and focal adhesion disassembly utilizing potassium depletion and nocodazole washout assays respectively. These studies will provide further insight into how cell interactions with the extracellular matrix signals to the cytoskeleton to promote cell migration, and thus is of importance to understanding the dynamic processes of embryogenesis, tissue maintenance and repair as well as cardiovascular and musculoskeletal defects and metastatic transformation.
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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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