The overall objective of this renewal application remains an analysis of the signal transduction events generated by transmembrane integrin binding to the extracellular matrix protein fibronectin (FN) and how these signals act to regulate anchorage-dependent cell growth and cell migration. Since integrins do not possess intrinsic catalytic activity, intracellular signaling by these receptors must be mediated by proteins associated with the alpha or beta integrin subunits. During the previous funding cycle, this proposal addressed the role of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) in promoting FN-stimulated cell motility through alpha5beta1 integrin activation. Our current proposal is focused on integrin connections to the FAK-related PTK, Pyk2. There remain significant gaps in our knowledge of how the activity and specificity of Pyk2 function is regulated.
Aim -1 of this proposal will extend preliminary results showing that alpha4beta1integrin promotes Pyk2 recruitment to focal contact sites, Pyk2 activation, and the rescue of FN-stimulated FAK-/- cell motility to the CS-1 alpha4beta1 binding site) but not 9- 11 (alpha5beta1 site) fragment of FN. The adenoviral-mediated expression of various mutants in FAK-/- and T cells will be used to test the hypothesis that allpha4-chain specific connections to Pyk2 facilitate spatially appropriate activation of downstream signaling associated with focal contact remodeling and enhanced cell motility.
Aim -2 will extend preliminary results showing the association and phosphorylation of Pyk2 by a 135 kDa serine/threonine kinase. This connection was mediated by the N-terminal FERM domain of Pyk2 and we will test the hypothesis that Pyk2 is negatively regulated by serine phosphorylation and that this linkage represents a modulatory pathway for both Pyk2 and integrin activity.
Aim -3 will extend preliminary results and test the hypothesis that elevated Pyk2 expression in LNCaP human prostate carcinoma cells acts to promote FN integrin survival signals in vitro and enhanced solid tumor growth and angiogenesis in vivo. Dominant-inhibitory, antisense, and adenoviral-mediated Pyk2 overexpression experiments will be used to elucidate the signaling connections regulating gene expression changes in vitro and in vivo. Once completed, these studies will provide important fundamental insights into the molecular mechanisms of Pyk2 function as it plays an important signaling role in both normal physiology and in diverse pathologies such as cancer and inflammation.
| Vomaske, Jennifer; Melnychuk, Ryan M; Smith, Patricia P et al. (2009) Differential ligand binding to a human cytomegalovirus chemokine receptor determines cell type-specific motility. PLoS Pathog 5:e1000304 |
| Barbero, Simone; Mielgo, Ainhoa; Torres, Vicente et al. (2009) Caspase-8 association with the focal adhesion complex promotes tumor cell migration and metastasis. Cancer Res 69:3755-63 |
| Lim, Ssang-Taek; Chen, Xiao Lei; Lim, Yangmi et al. (2008) Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation. Mol Cell 29:9-22 |
| Wu, L; Bernard-Trifilo, J A; Lim, Y et al. (2008) Distinct FAK-Src activation events promote alpha5beta1 and alpha4beta1 integrin-stimulated neuroblastoma cell motility. Oncogene 27:1439-48 |
