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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 Cas substrate domain blocks activation of JNK. a) NIH3T3 cells stably transfected with vector alone, the Src*/Cas(SD) chimera alone, v-crk alone or v-crk plus the Src*/Cas(SD) chimera were assayed for total cellular tyrosine phosphorylation (top panel). JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (middle and bottom panels). Lanes 1, 2 and 3, NIH3T3 vector transfected clones; lane 4 NIH3T3 transfected with the Src*/Cas(SD) chimera; lanes 5 and 6, NIH3T3 transfected with v-crk; lane 7, NIH3T3/v-crk cells transfected with the Src*/Cas(SD) chimera. Arrows are pointing to the Src*/Cas(SD) chimera (top panel), gst-c-jun (middle panel) and JNK (bottom panel). b) Cos-7 cells were transiently transfected with gst-JNK alone, or along with v-crk, or v-crk plus the Src*/Cas(SD) chimera. gst-JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (first panel). The expression levels of the gst-JNK, v-crk and the Src*/Cas(SD) chimera were determined as indicated. Arrows are pointing to the gst-c-jun substrate (first panel), gst-JNK (second panel), v-crk (third panel) and the Src*/Cas(SD) chimera (fourth panel).
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Figure 6: Tyrosine phosphorylated Cas substrate domain blocks activation of JNK. a) NIH3T3 cells stably transfected with vector alone, the Src*/Cas(SD) chimera alone, v-crk alone or v-crk plus the Src*/Cas(SD) chimera were assayed for total cellular tyrosine phosphorylation (top panel). JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (middle and bottom panels). Lanes 1, 2 and 3, NIH3T3 vector transfected clones; lane 4 NIH3T3 transfected with the Src*/Cas(SD) chimera; lanes 5 and 6, NIH3T3 transfected with v-crk; lane 7, NIH3T3/v-crk cells transfected with the Src*/Cas(SD) chimera. Arrows are pointing to the Src*/Cas(SD) chimera (top panel), gst-c-jun (middle panel) and JNK (bottom panel). b) Cos-7 cells were transiently transfected with gst-JNK alone, or along with v-crk, or v-crk plus the Src*/Cas(SD) chimera. gst-JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (first panel). The expression levels of the gst-JNK, v-crk and the Src*/Cas(SD) chimera were determined as indicated. Arrows are pointing to the gst-c-jun substrate (first panel), gst-JNK (second panel), v-crk (third panel) and the Src*/Cas(SD) chimera (fourth panel).

Mentions: The JNK pathway is regulated by interaction of c- or v-crk with Cas via the substrate domain [38]. In determining which pathways are affected in our stable cell lines generated, we determined if JNK was activated in the v-crk transformed cells, and if the tyrosine phosphorylated Cas chimera was affecting this activation. JNK was immunoprecipitated and tested for activity from control NIH3T3 cells, those expressing the Cas chimera and those expressing the Cas chimera and v-crk (Fig. 6a). We found that while JNK activity was elevated in v-crk transformed NIH3T3 cells, expression of the tyrosine phosphorylated Cas chimera resulted in reduced activity of JNK (Fig. 6a, cf. lanes 1, 5 and 7). Interestingly, JNK activity was also lower in the NIH3T3 cells expressing the tyrosine phosphorylated Cas chimera than the vector transfected cells, suggesting that this chimera also affected JNK activity in those cells (Fig. 6a, cf. lanes 1 and 4). These data suggest that the tyrosine phosphorylated Cas chimera perturbs signaling via v-crk and probably via endogenous c-crk to affect the activation of the JNK pathway.


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 Cas substrate domain blocks activation of JNK. a) NIH3T3 cells stably transfected with vector alone, the Src*/Cas(SD) chimera alone, v-crk alone or v-crk plus the Src*/Cas(SD) chimera were assayed for total cellular tyrosine phosphorylation (top panel). JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (middle and bottom panels). Lanes 1, 2 and 3, NIH3T3 vector transfected clones; lane 4 NIH3T3 transfected with the Src*/Cas(SD) chimera; lanes 5 and 6, NIH3T3 transfected with v-crk; lane 7, NIH3T3/v-crk cells transfected with the Src*/Cas(SD) chimera. Arrows are pointing to the Src*/Cas(SD) chimera (top panel), gst-c-jun (middle panel) and JNK (bottom panel). b) Cos-7 cells were transiently transfected with gst-JNK alone, or along with v-crk, or v-crk plus the Src*/Cas(SD) chimera. gst-JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (first panel). The expression levels of the gst-JNK, v-crk and the Src*/Cas(SD) chimera were determined as indicated. Arrows are pointing to the gst-c-jun substrate (first panel), gst-JNK (second panel), v-crk (third panel) and the Src*/Cas(SD) chimera (fourth panel).
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Figure 6: Tyrosine phosphorylated Cas substrate domain blocks activation of JNK. a) NIH3T3 cells stably transfected with vector alone, the Src*/Cas(SD) chimera alone, v-crk alone or v-crk plus the Src*/Cas(SD) chimera were assayed for total cellular tyrosine phosphorylation (top panel). JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (middle and bottom panels). Lanes 1, 2 and 3, NIH3T3 vector transfected clones; lane 4 NIH3T3 transfected with the Src*/Cas(SD) chimera; lanes 5 and 6, NIH3T3 transfected with v-crk; lane 7, NIH3T3/v-crk cells transfected with the Src*/Cas(SD) chimera. Arrows are pointing to the Src*/Cas(SD) chimera (top panel), gst-c-jun (middle panel) and JNK (bottom panel). b) Cos-7 cells were transiently transfected with gst-JNK alone, or along with v-crk, or v-crk plus the Src*/Cas(SD) chimera. gst-JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (first panel). The expression levels of the gst-JNK, v-crk and the Src*/Cas(SD) chimera were determined as indicated. Arrows are pointing to the gst-c-jun substrate (first panel), gst-JNK (second panel), v-crk (third panel) and the Src*/Cas(SD) chimera (fourth panel).
Mentions: The JNK pathway is regulated by interaction of c- or v-crk with Cas via the substrate domain [38]. In determining which pathways are affected in our stable cell lines generated, we determined if JNK was activated in the v-crk transformed cells, and if the tyrosine phosphorylated Cas chimera was affecting this activation. JNK was immunoprecipitated and tested for activity from control NIH3T3 cells, those expressing the Cas chimera and those expressing the Cas chimera and v-crk (Fig. 6a). We found that while JNK activity was elevated in v-crk transformed NIH3T3 cells, expression of the tyrosine phosphorylated Cas chimera resulted in reduced activity of JNK (Fig. 6a, cf. lanes 1, 5 and 7). Interestingly, JNK activity was also lower in the NIH3T3 cells expressing the tyrosine phosphorylated Cas chimera than the vector transfected cells, suggesting that this chimera also affected JNK activity in those cells (Fig. 6a, cf. lanes 1 and 4). These data suggest that the tyrosine phosphorylated Cas chimera perturbs signaling via v-crk and probably via endogenous c-crk to affect the activation of the JNK pathway.

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