Abstract
BackgroundNon-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously. This makes it difficult to attribute a particular biological effect to the phosphorylation of a particular substrate. We developed the Functional Interaction Trap (FIT) method to phosphorylate specifically a single substrate of choice in living cells, thereby allowing the biological effect(s) of that phosphorylation to be assessed. In this study we have used FIT to investigate the effects of specific phosphorylation of p130Cas, a protein implicated in cell migration. We have also used this approach to address a controversy regarding whether it is Src family kinases or focal adhesion kinase (FAK) that phosphorylates p130Cas in the trimolecular Src-FAK-p130Cas complex.ResultsWe show here that SYF cells (mouse fibroblasts lacking the NTKs Src, Yes and Fyn) exhibit a low level of basal tyrosine phosphorylation at focal adhesions. FIT-mediated tyrosine phosphorylation of NTK substrates p130Cas, paxillin and FAK and cortactin was observed at focal adhesions, while FIT-mediated phosphorylation of cortactin was also seen at the cell periphery. Phosphorylation of p130Cas in SYF cells led to activation of Rac1 and increased membrane ruffling and lamellipodium formation, events associated with cell migration. We also found that the kinase activity of Src and not FAK is essential for phosphorylation of p130Cas when the three proteins exist as a complex in focal adhesions.ConclusionThese results demonstrate that tyrosine phosphorylation of p130Cas is sufficient for its localization to focal adhesions and for activation of downstream signaling events associated with cell migration. FIT provides a valuable tool to evaluate the contribution of individual components of the response to signals with multiple outputs, such as activation of NTKs.
Highlights
Non-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously
SYF cells are mouse embryonic fibroblasts (MEFs) that lack the three Src family kinases detectably expressed in fibroblastic cells (Src, Yes and Fyn)
These cells exhibit defects in cell migration [8], and could serve as an excellent system to determine the biological consequences that result from Functional Interaction Trap (FIT)-mediated tyrosine phosphorylation
Summary
Non-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously. This makes it difficult to attribute a particular biological effect to the phosphorylation of a particular substrate. Integrin receptor activation following adhesion to extracellular matrix is believed to be the primary stimulus to activate the signaling cascades mediated by NTKs. Following integrin receptor activation, autophosphorylation of FAK at tyrosine 397 [13,14,15,16] recruits Src [17] and p130Cas [18] leading to the activation of two separate pathways working in conjunction with each other. Rac activation promotes membrane ruffling, lamellipodium formation and actin reorganization [31] by acting on the WASP/WAVE family of Arp2/3 complex activators to stimulate actin polymerization [5,32,33]
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