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Phosphorylation of p130Cas initiates Rac activation and membrane ruffling.

Sharma A, Mayer BJ - BMC Cell Biol. (2008)

Bottom Line: 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.These 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences, 1260 Elm Street, Manchester, NH 03101, USA. alok.sharma@mcphs.edu

ABSTRACT

Background: 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. 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.

Results: We 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.

Conclusion: These 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.

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FIT as a tool to elucidate the role of kinases in a multi-molecular complex. FAK, Src and p130Cas are known to exist in multi-molecular complexes. Since both FAK and Src possess kinase activity, either one (or both) could phosphorylate p130Cas. The importance of Src or FAK as the kinase responsible for phosphorylating p130Cas can be easily studied by using kinase-dead (kd) versions of each. From our results, we conclude that the Src kinase activity is responsible for phosphorylating p130Cas.
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Figure 6: FIT as a tool to elucidate the role of kinases in a multi-molecular complex. FAK, Src and p130Cas are known to exist in multi-molecular complexes. Since both FAK and Src possess kinase activity, either one (or both) could phosphorylate p130Cas. The importance of Src or FAK as the kinase responsible for phosphorylating p130Cas can be easily studied by using kinase-dead (kd) versions of each. From our results, we conclude that the Src kinase activity is responsible for phosphorylating p130Cas.

Mentions: In order to obtain direct evidence that it is indeed Src kinase activity and not FAK activity that is responsible for p130Cas phosphorylation, we approached this seemingly complex problem with a very simple experiment utilizing FIT. 293T cells were transfected with Flag-tagged p130Cas, FIT-compatible Myc-tagged FAK, and FIT-compatible HA-tagged Src (Figure 6). Using FIT to force interaction of Src and FAK eliminates any need for kinase activity or phosphorylation to mediate this association. p130Cas was then immunoprecipitated by anti-Flag antibody, and blots probed by anti-pTyr antibody to monitor phosphorylation. As shown in Figure 7, tyrosine phosphorylation of p130Cas was seen only when FAK was present and when Src possessed kinase activity, but not when Src lacked kinase activity (Figure 7A). In contrast, p130Cas was still tyrosine phosphorylated when FAK lacked kinase activity (Figure 7B). Thus we conclude that the primary role of FAK in the complex is to serve as a scaffold to recruit Src and p130Cas to focal adhesions, promoting efficient phosphorylation of p130Cas by Src.


Phosphorylation of p130Cas initiates Rac activation and membrane ruffling.

Sharma A, Mayer BJ - BMC Cell Biol. (2008)

FIT as a tool to elucidate the role of kinases in a multi-molecular complex. FAK, Src and p130Cas are known to exist in multi-molecular complexes. Since both FAK and Src possess kinase activity, either one (or both) could phosphorylate p130Cas. The importance of Src or FAK as the kinase responsible for phosphorylating p130Cas can be easily studied by using kinase-dead (kd) versions of each. From our results, we conclude that the Src kinase activity is responsible for phosphorylating p130Cas.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553404&req=5

Figure 6: FIT as a tool to elucidate the role of kinases in a multi-molecular complex. FAK, Src and p130Cas are known to exist in multi-molecular complexes. Since both FAK and Src possess kinase activity, either one (or both) could phosphorylate p130Cas. The importance of Src or FAK as the kinase responsible for phosphorylating p130Cas can be easily studied by using kinase-dead (kd) versions of each. From our results, we conclude that the Src kinase activity is responsible for phosphorylating p130Cas.
Mentions: In order to obtain direct evidence that it is indeed Src kinase activity and not FAK activity that is responsible for p130Cas phosphorylation, we approached this seemingly complex problem with a very simple experiment utilizing FIT. 293T cells were transfected with Flag-tagged p130Cas, FIT-compatible Myc-tagged FAK, and FIT-compatible HA-tagged Src (Figure 6). Using FIT to force interaction of Src and FAK eliminates any need for kinase activity or phosphorylation to mediate this association. p130Cas was then immunoprecipitated by anti-Flag antibody, and blots probed by anti-pTyr antibody to monitor phosphorylation. As shown in Figure 7, tyrosine phosphorylation of p130Cas was seen only when FAK was present and when Src possessed kinase activity, but not when Src lacked kinase activity (Figure 7A). In contrast, p130Cas was still tyrosine phosphorylated when FAK lacked kinase activity (Figure 7B). Thus we conclude that the primary role of FAK in the complex is to serve as a scaffold to recruit Src and p130Cas to focal adhesions, promoting efficient phosphorylation of p130Cas by Src.

Bottom Line: 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.These 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences, 1260 Elm Street, Manchester, NH 03101, USA. alok.sharma@mcphs.edu

ABSTRACT

Background: 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. 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.

Results: We 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.

Conclusion: These 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.

Show MeSH
Related in: MedlinePlus