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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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Related in: MedlinePlus

v-crk specifically interacts with and enhances the tyrosine phosphorylation status of the 3' portion of the Cas SD chimera in cells (SrcKM/Cas(SD)).a) Cos-7 cells were transfected with vector coding for the SrcKM/Cas(SD) chimera alone (lane 1), or along with mutants of v-crk, WT (lane 2), SH2 mutant (lane 3) or SH3 mutant (lane 4). The SrcKM/Cas(SD) chimera was then immunoprecipitated with antibodies to HA and probed with anti-phosphotyrosine (top panel). The blot was then stripped and then probed with antibodies to HA to detect the SrcKM/Cas(SD) chimera (middle panel). The bottom panel shows the expression of the v-crk mutants in the transfection as determined by anti-gag blotting. Arrows point to the SrcKM/Cas(SD) chimera and v-crk in the top, middle and bottom panels respectively. b) Cos-7 cells were transfected with the following plasmids, and immunoprecipitated with anti-HA antibodies. SrcKM/Cas(5'SD) chimera (lane 1), SrcKM/Cas(3'SD) chimera (lane 2), and SrcKM/Cas(SD) chimera (lane 3) alone, or along with WT v-crk (lanes 4–6). The top panel was probed with antibodies against phosphotyrosine, and the bottom panel with antibodies against HA to detect the chimeras. Probing whole cell lysates with antibodies to v-crk (anti-gag) demonstrated that cells leading to lanes 4–6 expressed equivalent levels of v-crk (data not shown).
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Figure 4: v-crk specifically interacts with and enhances the tyrosine phosphorylation status of the 3' portion of the Cas SD chimera in cells (SrcKM/Cas(SD)).a) Cos-7 cells were transfected with vector coding for the SrcKM/Cas(SD) chimera alone (lane 1), or along with mutants of v-crk, WT (lane 2), SH2 mutant (lane 3) or SH3 mutant (lane 4). The SrcKM/Cas(SD) chimera was then immunoprecipitated with antibodies to HA and probed with anti-phosphotyrosine (top panel). The blot was then stripped and then probed with antibodies to HA to detect the SrcKM/Cas(SD) chimera (middle panel). The bottom panel shows the expression of the v-crk mutants in the transfection as determined by anti-gag blotting. Arrows point to the SrcKM/Cas(SD) chimera and v-crk in the top, middle and bottom panels respectively. b) Cos-7 cells were transfected with the following plasmids, and immunoprecipitated with anti-HA antibodies. SrcKM/Cas(5'SD) chimera (lane 1), SrcKM/Cas(3'SD) chimera (lane 2), and SrcKM/Cas(SD) chimera (lane 3) alone, or along with WT v-crk (lanes 4–6). The top panel was probed with antibodies against phosphotyrosine, and the bottom panel with antibodies against HA to detect the chimeras. Probing whole cell lysates with antibodies to v-crk (anti-gag) demonstrated that cells leading to lanes 4–6 expressed equivalent levels of v-crk (data not shown).

Mentions: The adaptor protein Cas has a N-terminal SH3 domain, a large number of tyrosines that serve as potential kinase substrates within its substrate domain and a C-terminal Src binding domain (Fig. 1). Understanding the function of this substrate domain and the tyrosines within it could lead to a better understanding of the signaling pathways regulated by this protein. Previously, investigators have created dominant negative versions of this protein lacking its substrate domain and shown that it is required for cell adhesion and migration. However, these studies depend on upstream signals and so it is not clear which signals are directly emanating from the substrate domain itself, as this domain requires the activation of upstream kinases for its phosphorylation and function. We have tried to address this problem by attempting to create a dominant active substrate domain of Cas, i.e. a Cas substrate domain that is constitutively tyrosine phosphorylated and able to send signals independently of upstream signals. We did this by fusing the SD of Cas to a Src kinase domain that had been attenuated by mutating the activation tyrosine (Y416) to phenylalanine (Src*/Cas(SD), Fig. 2). This Src mutant is inactive against exogenous substrates [34-36]. We also generated fusions of the Cas SD with a Src kinase inactive mutant (K295M) that has no enzymatic activity (SrcKM/Cas(SD)), fusions of either the attenuated kinase domain or kinase inactive domain to the 5' or 3' regions of the Cas SD (Src*/Cas(5'SD), Src*/Cas(3'SD), SrcKM/Cas(5'SD), SrcKM/Cas(3'SD)), as well as the attenuated Src kinase (Src*) alone without the attached Cas SD (Fig. 2). Transient expression of the Src*/Cas(SD) in Cos-7 cells followed by analysis of total cell lysates using anti-phosphotyrosine antibodies demonstrated that while the attenuated Src kinase domain alone did not increase total cellular tyrosine phosphorylated proteins as expected, expression of the Src*/Cas(SD) fusion resulted in tyrosine phosphorylation of a protein of the expected size of the chimera, as well as other proteins (Fig. 3a, 3b). Expression of the Cas(SD) or the SrcKM/Cas(SD), fusion did not result in any increase in total phosphotyrosine containing proteins nor in the tyrosine phosphorylation of the Cas SD (data not shown and see Fig. 4).


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

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

v-crk specifically interacts with and enhances the tyrosine phosphorylation status of the 3' portion of the Cas SD chimera in cells (SrcKM/Cas(SD)).a) Cos-7 cells were transfected with vector coding for the SrcKM/Cas(SD) chimera alone (lane 1), or along with mutants of v-crk, WT (lane 2), SH2 mutant (lane 3) or SH3 mutant (lane 4). The SrcKM/Cas(SD) chimera was then immunoprecipitated with antibodies to HA and probed with anti-phosphotyrosine (top panel). The blot was then stripped and then probed with antibodies to HA to detect the SrcKM/Cas(SD) chimera (middle panel). The bottom panel shows the expression of the v-crk mutants in the transfection as determined by anti-gag blotting. Arrows point to the SrcKM/Cas(SD) chimera and v-crk in the top, middle and bottom panels respectively. b) Cos-7 cells were transfected with the following plasmids, and immunoprecipitated with anti-HA antibodies. SrcKM/Cas(5'SD) chimera (lane 1), SrcKM/Cas(3'SD) chimera (lane 2), and SrcKM/Cas(SD) chimera (lane 3) alone, or along with WT v-crk (lanes 4–6). The top panel was probed with antibodies against phosphotyrosine, and the bottom panel with antibodies against HA to detect the chimeras. Probing whole cell lysates with antibodies to v-crk (anti-gag) demonstrated that cells leading to lanes 4–6 expressed equivalent levels of v-crk (data not shown).
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Figure 4: v-crk specifically interacts with and enhances the tyrosine phosphorylation status of the 3' portion of the Cas SD chimera in cells (SrcKM/Cas(SD)).a) Cos-7 cells were transfected with vector coding for the SrcKM/Cas(SD) chimera alone (lane 1), or along with mutants of v-crk, WT (lane 2), SH2 mutant (lane 3) or SH3 mutant (lane 4). The SrcKM/Cas(SD) chimera was then immunoprecipitated with antibodies to HA and probed with anti-phosphotyrosine (top panel). The blot was then stripped and then probed with antibodies to HA to detect the SrcKM/Cas(SD) chimera (middle panel). The bottom panel shows the expression of the v-crk mutants in the transfection as determined by anti-gag blotting. Arrows point to the SrcKM/Cas(SD) chimera and v-crk in the top, middle and bottom panels respectively. b) Cos-7 cells were transfected with the following plasmids, and immunoprecipitated with anti-HA antibodies. SrcKM/Cas(5'SD) chimera (lane 1), SrcKM/Cas(3'SD) chimera (lane 2), and SrcKM/Cas(SD) chimera (lane 3) alone, or along with WT v-crk (lanes 4–6). The top panel was probed with antibodies against phosphotyrosine, and the bottom panel with antibodies against HA to detect the chimeras. Probing whole cell lysates with antibodies to v-crk (anti-gag) demonstrated that cells leading to lanes 4–6 expressed equivalent levels of v-crk (data not shown).
Mentions: The adaptor protein Cas has a N-terminal SH3 domain, a large number of tyrosines that serve as potential kinase substrates within its substrate domain and a C-terminal Src binding domain (Fig. 1). Understanding the function of this substrate domain and the tyrosines within it could lead to a better understanding of the signaling pathways regulated by this protein. Previously, investigators have created dominant negative versions of this protein lacking its substrate domain and shown that it is required for cell adhesion and migration. However, these studies depend on upstream signals and so it is not clear which signals are directly emanating from the substrate domain itself, as this domain requires the activation of upstream kinases for its phosphorylation and function. We have tried to address this problem by attempting to create a dominant active substrate domain of Cas, i.e. a Cas substrate domain that is constitutively tyrosine phosphorylated and able to send signals independently of upstream signals. We did this by fusing the SD of Cas to a Src kinase domain that had been attenuated by mutating the activation tyrosine (Y416) to phenylalanine (Src*/Cas(SD), Fig. 2). This Src mutant is inactive against exogenous substrates [34-36]. We also generated fusions of the Cas SD with a Src kinase inactive mutant (K295M) that has no enzymatic activity (SrcKM/Cas(SD)), fusions of either the attenuated kinase domain or kinase inactive domain to the 5' or 3' regions of the Cas SD (Src*/Cas(5'SD), Src*/Cas(3'SD), SrcKM/Cas(5'SD), SrcKM/Cas(3'SD)), as well as the attenuated Src kinase (Src*) alone without the attached Cas SD (Fig. 2). Transient expression of the Src*/Cas(SD) in Cos-7 cells followed by analysis of total cell lysates using anti-phosphotyrosine antibodies demonstrated that while the attenuated Src kinase domain alone did not increase total cellular tyrosine phosphorylated proteins as expected, expression of the Src*/Cas(SD) fusion resulted in tyrosine phosphorylation of a protein of the expected size of the chimera, as well as other proteins (Fig. 3a, 3b). Expression of the Cas(SD) or the SrcKM/Cas(SD), fusion did not result in any increase in total phosphotyrosine containing proteins nor in the tyrosine phosphorylation of the Cas SD (data not shown and see Fig. 4).

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