Cooperation of Src homology domains in the regulated binding of phosphatidylinositol 3-kinase. A role for the Src homology 2 domain

B Haefner, R Baxter, V J Fincham, C P Downes, M C Frame

Research output: Contribution to journalArticlepeer-review

Abstract

Fibroblasts transformed by the v-Src oncoprotein exhibit elevated activity of the enzyme phosphatidylinositol 3'-kinase (PI 3-kinase), which binds to, and is activated by, a wide range of receptor tyrosine kinases as well as v-Src and transforming polyoma middle T/c-Src complexes. Here we consider the role of the v-Src homology (SH) domains, SH3 and SH2, and the tyrosine kinase catalytic domain, in the stimulation of v-Src-associated PI 3-kinase activity in response to rapid activation of the oncoprotein. As shown by others, we find that the v-Src SH3 domain tightly binds the PI 3-kinase p85 regulatory subunit in normal growing chicken embryo fibroblasts. However, we also find that in transformed cells there is additional efficient binding of PI 3-kinase to the v-Src SH2 domain in a catalytically active form. Furthermore, the binding of p85 to the SH2 domain, which is almost undetectable in quiescent cells, is rapidly stimulated upon activation of temperature-sensitive v-Src and consequent cell cycle entry, demonstrating that binding is a target for regulation. We also show that v-Src-associated PI 3-kinase differs considerably from PDGF receptor-associated enzyme by a different mode of binding, a lack of substantial allosteric activation, and a dependence on the tyrosine kinase activity of v-Src. The rapidly induced binding and activation of PI 3-kinase thus provides sensitive regulation of recruitment of PI 3-kinase to its substrates and into other signaling complexes at the cell membrane, which involves all the Src homology domains.
Original languageEnglish
Pages (from-to)7937-43
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number14
Publication statusPublished - 7 Apr 1995

Keywords

  • Amino Acid Sequence
  • Animals
  • Chick Embryo
  • Enzyme Activation
  • Molecular Sequence Data
  • Oncogene Protein pp60(v-src)
  • Phosphatidylinositol 3-Kinases
  • Phosphopeptides
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor)
  • Protein Binding
  • Receptors, Platelet-Derived Growth Factor
  • Tyrosine

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