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A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-crk signaling.

Kirsch K, Kensinger M, Hanafusa H, August A - BMC Cell Biol. (2002)

Bottom Line: A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling.We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation.This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Oncology, The Rockefeller University, NY, NY 10021, USA. kirschk@bu.edu

ABSTRACT

Background: The adaptor protein p130Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals.

Results: We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk.

Conclusion: Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.

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Tyrosine phosphorylated substrate domain of Cas blocks v-crk mediated transformation. a) NIH3T3 cells or those transformed with v-crk were either transfected with vector alone or with vector carrying the Src*/Cas(SD) chimera. Whole cell extracts (WCL) from cells were probed with anti-phosphotyrosine (top panel) or with anti-HA antibody (bottom panel). b) NIH3T3 cells stably transfected with vector alone (left panel), with v-crk (middle panel) or v-crk and the Src*/Cas(SD) chimera were assayed for growth in soft agar. Representative sections are shown. Colony numbers per view field were as follows: 26.1 (+/- 4.2) for v-crk alone vs. 2.9 (+/- 1.4), statistically significant (p = 7.76 × 10-8, t-test).
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Figure 5: Tyrosine phosphorylated substrate domain of Cas blocks v-crk mediated transformation. a) NIH3T3 cells or those transformed with v-crk were either transfected with vector alone or with vector carrying the Src*/Cas(SD) chimera. Whole cell extracts (WCL) from cells were probed with anti-phosphotyrosine (top panel) or with anti-HA antibody (bottom panel). b) NIH3T3 cells stably transfected with vector alone (left panel), with v-crk (middle panel) or v-crk and the Src*/Cas(SD) chimera were assayed for growth in soft agar. Representative sections are shown. Colony numbers per view field were as follows: 26.1 (+/- 4.2) for v-crk alone vs. 2.9 (+/- 1.4), statistically significant (p = 7.76 × 10-8, t-test).

Mentions: As our tyrosine phosphorylated Cas SD chimera can interact with v-crk, we decided to find out if the chimera would affect cell growth and/or transformation in normal cells and those transformed with v-crk. NIH3T3 cells or those transformed with v-crk were stably transfected with the Cas SD chimera (Fig. 5). Several independent clones were obtained for each set of transfections suggesting that expression of the tyrosine phosphorylated Cas SD domain did not negatively affect cell growth (data not shown). Figure 5a demonstrates that stable expression of the Src*/Cas(SD) chimera resulted in constitutive tyrosine phosphorylation of this protein in NIH3T3 cells. Analysis of the effect of this chimera in non-transformed NIH3T3 cells indicates that these cells were not transformed and had normal morphology, although they grew slightly slower than vector-transfected cells (data not shown). Analysis of the growth of either non-transformed or v-crk transformed NIH3T3 cells in soft agar confirmed that expression of tyrosine phosphorylated Cas SD chimera did not transform NIH3T3 cells as no colonies of these cells grew in soft agar (data not shown). Instead, expression of tyrosine phosphorylated Cas SD chimera resulted in significant reduction in the number of v-crk/NIH3T3 soft agar colonies as well as their size (Fig. 5b), suggesting that the Src*/Cas(SD) chimera blocked the ability of v-crk to transform these cells.


A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-crk signaling.

Kirsch K, Kensinger M, Hanafusa H, August A - BMC Cell Biol. (2002)

Tyrosine phosphorylated substrate domain of Cas blocks v-crk mediated transformation. a) NIH3T3 cells or those transformed with v-crk were either transfected with vector alone or with vector carrying the Src*/Cas(SD) chimera. Whole cell extracts (WCL) from cells were probed with anti-phosphotyrosine (top panel) or with anti-HA antibody (bottom panel). b) NIH3T3 cells stably transfected with vector alone (left panel), with v-crk (middle panel) or v-crk and the Src*/Cas(SD) chimera were assayed for growth in soft agar. Representative sections are shown. Colony numbers per view field were as follows: 26.1 (+/- 4.2) for v-crk alone vs. 2.9 (+/- 1.4), statistically significant (p = 7.76 × 10-8, t-test).
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Related In: Results  -  Collection

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Figure 5: Tyrosine phosphorylated substrate domain of Cas blocks v-crk mediated transformation. a) NIH3T3 cells or those transformed with v-crk were either transfected with vector alone or with vector carrying the Src*/Cas(SD) chimera. Whole cell extracts (WCL) from cells were probed with anti-phosphotyrosine (top panel) or with anti-HA antibody (bottom panel). b) NIH3T3 cells stably transfected with vector alone (left panel), with v-crk (middle panel) or v-crk and the Src*/Cas(SD) chimera were assayed for growth in soft agar. Representative sections are shown. Colony numbers per view field were as follows: 26.1 (+/- 4.2) for v-crk alone vs. 2.9 (+/- 1.4), statistically significant (p = 7.76 × 10-8, t-test).
Mentions: As our tyrosine phosphorylated Cas SD chimera can interact with v-crk, we decided to find out if the chimera would affect cell growth and/or transformation in normal cells and those transformed with v-crk. NIH3T3 cells or those transformed with v-crk were stably transfected with the Cas SD chimera (Fig. 5). Several independent clones were obtained for each set of transfections suggesting that expression of the tyrosine phosphorylated Cas SD domain did not negatively affect cell growth (data not shown). Figure 5a demonstrates that stable expression of the Src*/Cas(SD) chimera resulted in constitutive tyrosine phosphorylation of this protein in NIH3T3 cells. Analysis of the effect of this chimera in non-transformed NIH3T3 cells indicates that these cells were not transformed and had normal morphology, although they grew slightly slower than vector-transfected cells (data not shown). Analysis of the growth of either non-transformed or v-crk transformed NIH3T3 cells in soft agar confirmed that expression of tyrosine phosphorylated Cas SD chimera did not transform NIH3T3 cells as no colonies of these cells grew in soft agar (data not shown). Instead, expression of tyrosine phosphorylated Cas SD chimera resulted in significant reduction in the number of v-crk/NIH3T3 soft agar colonies as well as their size (Fig. 5b), suggesting that the Src*/Cas(SD) chimera blocked the ability of v-crk to transform these cells.

Bottom Line: A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling.We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation.This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Oncology, The Rockefeller University, NY, NY 10021, USA. kirschk@bu.edu

ABSTRACT

Background: The adaptor protein p130Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals.

Results: We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk.

Conclusion: Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.

Show MeSH
Related in: MedlinePlus