The Ras GTPase-activating protein (GAP) is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase, Hck

S. D. Briggs, S. S. Bryant, R. Jove, Sam Sanderson, T. E. Smithgall

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Abstract

The Ras GTPase-activating protein (GAP) is a target for protein tyrosine kinases of both the receptor and cytoplasmic classes and may serve to integrate tyrosine kinase and Ras signaling pathways. In this report, we provide evidence that GAP is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase Hck, which has been implicated in the regulation of myeloid cell growth, differentiation, and function. Wild-type (WT) or kinase-inactive (K269E) mutant Hck proteins were co-expressed with bovine GAP using the baculovirus/Sf-9 cell system. GAP was readily phosphorylated on tyrosine by WT but not K269E Hck. GAP was present in WT Hck immunoprecipitates from the coinfected cells, indicative of Hck·GAP complex formation. Unexpectedly, GAP also associated with the kinase-inactive mutant of Hck, suggesting that tyrosine autophosphorylation of Hck is not required for complex formation. The WT and K269E forms of Hck also associated with GAP mutants lacking either the C-terminal catalytic domain (ΔCAT) or the Src homology region (ΔSH), indicating that these GAP domains are dispensable for complex formation. Recombinant GST fusion proteins containing the Hck, Src, Fyn, or Lck SH3 domains associated with full-length GAP, ΔCAT, and ΔSH, all of which share an N-terminal proline-rich region resembling an SH3-binding motif (PPLPPPPPQLP). Deletion of the highly conserved YXY sequence from the Hck SH3 domain abolished binding. GAP-SH3 interaction was also inhibited by the proline-rich peptide GFPPLPPPPPQLPTLG, which corresponds to N-terminal amino acids 129-144 of bovine GAP. An N-terminal deletion mutant of GAP lacking this proline-rich region did not bind to the Hck SH3 domain. These data implicate the Hck SH3 domain in GAP interaction, and suggest a general function for the SH3 domains of Src family kinases in recognition of GAP via its proline-rich N-terminal domain.

Original languageEnglish (US)
Pages (from-to)14718-14724
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number24
DOIs
StatePublished - Jan 1 1995

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ras GTPase-Activating Proteins
GTPase-Activating Proteins
src Homology Domains
src-Family Kinases
Protein-Tyrosine Kinases
Carrier Proteins
Substrates
Proline
Tyrosine
Catalytic Domain
Phosphotransferases
Recombinant Fusion Proteins
Conserved Sequence
Baculoviridae
Receptor Protein-Tyrosine Kinases
Cell growth
Myeloid Cells
Mutant Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The Ras GTPase-activating protein (GAP) is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase, Hck. / Briggs, S. D.; Bryant, S. S.; Jove, R.; Sanderson, Sam; Smithgall, T. E.

In: Journal of Biological Chemistry, Vol. 270, No. 24, 01.01.1995, p. 14718-14724.

Research output: Contribution to journalArticle

Briggs, S. D. ; Bryant, S. S. ; Jove, R. ; Sanderson, Sam ; Smithgall, T. E. / The Ras GTPase-activating protein (GAP) is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase, Hck. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 24. pp. 14718-14724.
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