Purification and characterization of a phosphatidylinositol 3-kinase complex from bovine brain by using phosphopeptide affinity columns.
Specific phosphorylated tyrosine residues in the kinase insert region of the human platelet-derived-growth-factor beta-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 p85 alpha. 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 p85 alpha subunit of bovine brain PtdIns 3-kinase, the closely related protein p85 beta and purified bovine brain PtdIns 3-kinase to these and other phosphopeptide columns is examined.
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D016384:Consensus Sequence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols / Q000378:metabolism; D010770:Phosphotransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D010455:Peptides; D010716:Phosphatidylinositols; D010982:Platelet-Derived Growth Factor; D019000:Phosphotyrosine; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases