Research supported by this grant over the past two decades, has focused on using the oncogenic protein tyrosine kinase, Src, as a molecular guide to identify tyrosine phosphorylated proteins that play key roles in cellular signaling. Characterization of so-called """"""""Src targets"""""""" has led to the identification of a number of cellular proteins, such as Focal Adhesion Kinase (FAK), p130Cas and paxillin, proteins that function within the context of specific actin-based cytoskeletal structures, e.g., focal adhesions, as well as Cortactin, a protein that localizes predominantly to cortical actin, a site of dynamic actin remodeling. The long range goal of the studies outlined in this proposal is to understand how proteins such as Cortactin, function in the context of the cortical actin cytoskeleton to integrate cell surface receptor mediated cell signaling in a """"""""temporal and spacial"""""""" context. Information garnered over the past granting period shows that the Src-substrate Cortactin is a multifunctional adapter protein that interacts on the one hand with the """"""""cortical actin network"""""""" present in filopodia and lamellipodia by binding the Arp2/3 complex and links via its carboxyl-terminal SH3 domain to a number of functionally interesting and potentially important signaling and scaffolding proteins. This revised application again outlines four specific aims to address the function of Cortactin:
In Aim 1 we will use molecular genetic, cellular and biochemical approaches to study the factors that regulate the interaction of Cortactin with the Arp2/3 complex of proteins.
In Aim 2, we will extend and further develop our studies aimed at identifying and characterizing proteins that interact with the Cortactin SH3 domain. We will continue to characterize proteins that function as binding partners for Cortactin, including two recently identified SH3 effectors, WIP, Wasp Interacting Protein, a regulator of Arp2/3-dependent actin polymerization and dynamin, a GTPase implicated in the regulation of endocytic and secretory pathways. We will also extend and further develop studies to identify possible interactions of cellular proteins with the """"""""helical-proline-ser-rich"""""""" region of Cortactin, here-to-fore unstudied interaction domain.
In Aim 3 we will examine how tyrosine phosphorylation influences the interactions of Cortactin with known cellular binding partners. Finally in Aim 4, we will examine whether expression of full length Cortactin or predicted dominant inhibitory forms of Cortactin function to alter the dynamics of EGF-induced lamellipodia formation or cell migration. We will also determine whether Cortactin or Cortactin variants influence the dynamics of growth factor receptor signaling to downstream effectors, with particular attention to Ras signaling and EGF receptor internalization. Finally we propose to generate mice bearing a genetically altered allele of Cortactin, allowing for Cre-mediated excision and replacement of the Cortactin SH3 domain. Such mice, bearing a mutated form of Cortactin will allow one to directly test the role of Cortactin in mediating specific cellular functions in a cell type specific fashion.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA029243-23
Application #
6754369
Study Section
Special Emphasis Panel (ZRG1-CDF (01))
Program Officer
Ault, Grace S
Project Start
1981-01-01
Project End
2006-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
23
Fiscal Year
2004
Total Cost
$293,040
Indirect Cost
Name
University of Virginia
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Parsons, J Thomas; Slack-Davis, Jill; Tilghman, Robert et al. (2008) Focal adhesion kinase: targeting adhesion signaling pathways for therapeutic intervention. Clin Cancer Res 14:627-32
Iwanicki, Marcin P; Vomastek, Tomas; Tilghman, Robert W et al. (2008) FAK, PDZ-RhoGEF and ROCKII cooperate to regulate adhesion movement and trailing-edge retraction in fibroblasts. J Cell Sci 121:895-905
Owen, Katherine A; Pixley, Fiona J; Thomas, Keena S et al. (2007) Regulation of lamellipodial persistence, adhesion turnover, and motility in macrophages by focal adhesion kinase. J Cell Biol 179:1275-87
Vomastek, Tomas; Iwanicki, Marcin P; Schaeffer, Hans-Joerg et al. (2007) RACK1 targets the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to link integrin engagement with focal adhesion disassembly and cell motility. Mol Cell Biol 27:8296-305
Martin, Karen H; Jeffery, Erin D; Grigera, Pablo R et al. (2006) Cortactin phosphorylation sites mapped by mass spectrometry. J Cell Sci 119:2851-3
Liu, Yunhao; Yerushalmi, Gil M; Grigera, Pablo R et al. (2005) Mislocalization or reduced expression of Arf GTPase-activating protein ASAP1 inhibits cell spreading and migration by influencing Arf1 GTPase cycling. J Biol Chem 280:8884-92
Slack-Davis, Jill K; Eblen, Scott T; Zecevic, Maja et al. (2003) PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation. J Cell Biol 162:281-91
Kinley, Andrew W; Weed, Scott A; Weaver, Alissa M et al. (2003) Cortactin interacts with WIP in regulating Arp2/3 activation and membrane protrusion. Curr Biol 13:384-93
Head, Julie A; Jiang, Dongyan; Li, Min et al. (2003) Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton. Mol Biol Cell 14:3216-29
Parsons, J Thomas (2003) Focal adhesion kinase: the first ten years. J Cell Sci 116:1409-16

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