Identification and Characterization of a High-Affinity Interaction between v-Crk and Tyrosine-Phosphorylated Paxillin in CT10-Transformed Fibroblasts

Raymond B. Birge, Jorge E. Fajardo, Charles Reichman, Steven E. Shoelson, Zhou Songyang, Lewis C. Cantley, Hidesaburo Hanafusa

Research output: Contribution to journalArticle

239 Citations (Scopus)

Abstract

The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. Although many additional cellular proteins became hyperphosphorylated on tyrosine in the vanadate-treated CT10-CEF, the GST-CrkSH2 fragment still bound preferentially to the paxillin and 130-kDa proteins, suggesting a high degree of specificity in the interaction of CrkSH2 with these proteins. Paxillin phosphorylation was highly sensitive to vanadate treatment in both normal CEF and CT10-CEF, and the elevation in tyrosine phosphorylation resulted in increased binding to GST-CrkSH2. Moreover, binding of full-length GST-v-Crk to tyrosine-phosphorylated paxillin in vitro protected paxillin from dephosphorylation by cellular protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.

Original languageEnglish (US)
Pages (from-to)4648-4656
Number of pages9
JournalMolecular and Cellular Biology
Volume13
Issue number8
StatePublished - Aug 1993
Externally publishedYes

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Paxillin
Tyrosine
Fibroblasts
Glutathione Transferase
Chickens
Vanadates
Embryonic Structures
Phosphorylation
Proteins
Protein Tyrosine Phosphatases
src Homology Domains
Avian Sarcoma Viruses
Phosphopeptides
Focal Adhesions
Cytoskeletal Proteins
Phosphoric Monoester Hydrolases
Phosphotransferases
Molecular Weight
Genome

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

Identification and Characterization of a High-Affinity Interaction between v-Crk and Tyrosine-Phosphorylated Paxillin in CT10-Transformed Fibroblasts. / Birge, Raymond B.; Fajardo, Jorge E.; Reichman, Charles; Shoelson, Steven E.; Songyang, Zhou; Cantley, Lewis C.; Hanafusa, Hidesaburo.

In: Molecular and Cellular Biology, Vol. 13, No. 8, 08.1993, p. 4648-4656.

Research output: Contribution to journalArticle

Birge, Raymond B. ; Fajardo, Jorge E. ; Reichman, Charles ; Shoelson, Steven E. ; Songyang, Zhou ; Cantley, Lewis C. ; Hanafusa, Hidesaburo. / Identification and Characterization of a High-Affinity Interaction between v-Crk and Tyrosine-Phosphorylated Paxillin in CT10-Transformed Fibroblasts. In: Molecular and Cellular Biology. 1993 ; Vol. 13, No. 8. pp. 4648-4656.
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abstract = "The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. Although many additional cellular proteins became hyperphosphorylated on tyrosine in the vanadate-treated CT10-CEF, the GST-CrkSH2 fragment still bound preferentially to the paxillin and 130-kDa proteins, suggesting a high degree of specificity in the interaction of CrkSH2 with these proteins. Paxillin phosphorylation was highly sensitive to vanadate treatment in both normal CEF and CT10-CEF, and the elevation in tyrosine phosphorylation resulted in increased binding to GST-CrkSH2. Moreover, binding of full-length GST-v-Crk to tyrosine-phosphorylated paxillin in vitro protected paxillin from dephosphorylation by cellular protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.",
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AU - Reichman, Charles

AU - Shoelson, Steven E.

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AU - Cantley, Lewis C.

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N2 - The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. Although many additional cellular proteins became hyperphosphorylated on tyrosine in the vanadate-treated CT10-CEF, the GST-CrkSH2 fragment still bound preferentially to the paxillin and 130-kDa proteins, suggesting a high degree of specificity in the interaction of CrkSH2 with these proteins. Paxillin phosphorylation was highly sensitive to vanadate treatment in both normal CEF and CT10-CEF, and the elevation in tyrosine phosphorylation resulted in increased binding to GST-CrkSH2. Moreover, binding of full-length GST-v-Crk to tyrosine-phosphorylated paxillin in vitro protected paxillin from dephosphorylation by cellular protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.

AB - The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. Although many additional cellular proteins became hyperphosphorylated on tyrosine in the vanadate-treated CT10-CEF, the GST-CrkSH2 fragment still bound preferentially to the paxillin and 130-kDa proteins, suggesting a high degree of specificity in the interaction of CrkSH2 with these proteins. Paxillin phosphorylation was highly sensitive to vanadate treatment in both normal CEF and CT10-CEF, and the elevation in tyrosine phosphorylation resulted in increased binding to GST-CrkSH2. Moreover, binding of full-length GST-v-Crk to tyrosine-phosphorylated paxillin in vitro protected paxillin from dephosphorylation by cellular protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.

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