Purification and characterization of a phosphatidylinositol 3-kinase complex from bovine brain by using phosphopeptide affinity columns

M. J. Fry, G. Panayotou, R. Dhand, F. Ruiz-Larrea, I. Gout, O. Nguyen, S. A. Courtneidge, M. D. Waterfield

Research output: Contribution to journalArticle

53 Scopus citations

Abstract

Specific phosphorylated tyrosine residues in the kinase insert region of the human platelet-derived-growth-factor β-receptor mediate the formation of multienzyme complexes with this receptor. When phosphorylated. tyrosine residue 751 within the kinase insert region mediates binding of PtdIns 3-kinase to this receptor. A 17-amino-acid peptide containing this tyrosine residue was synthesized, phosphorylated by using epidermal-growth-factor receptor and then coupled to an Actigel matrix. The tyrosine-751 phosphopeptide column is used here as a final affinity step in the purification of the PtdIns 3-kinase from bovine brain to apparent homogeneity. The active resin-bound PtdIns 3-kinase is composed of two polypeptides, p110 and p85, which are elutable with SDS-containing buffers and detectable by silver staining of polyacrylamide gels. The 85 kDa protein is shown to be identical with the recently cloned p85x. Phosphotyrosine is demonstrated to be an essential part of the structure required for binding of both of these proteins and PtdIns 3-kinase activity to this peptide. The active PtdIns 3-kinase complex from bovine brain, but not recombinant p85 subunits, shows specificity for binding to phosphopeptides containing a YXXM consensus sequence. Neither PtdIns 3-kinase activity, nor the complex of p85 and 110 kDa proteins, binds to several other phosphopeptide affinity columns lacking this sequence motif. The selectivity of binding of baculovirus-expressed free p85α subunit of bovine brain PtdIns 3-kinase, the closely related protein p85β and purified bovine brain PtdIns 3-kinase to these and other phosphopeptide columns is examined.

Original languageEnglish (US)
Pages (from-to)383-393
Number of pages11
JournalBiochemical Journal
Volume288
Issue number2
DOIs
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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