cDNA cloning of a Novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF β-receptor

Abstract

Using immobilized PDGF receptor as an affinity reagent, we purified an 85 kd protein (p85) from cell lysates and we cloned its cDNA. The protein contains an SH3 domain and two SH2 domains that are homologous to domains found in several receptor-associated enzymes. Recombinant p85 overexpressed in mammalian cells inhibited the binding of endogenous p85 and a 110 kd protein to the receptor and also blocked the association of PI3-kinase activity with the receptor. Experiments with receptor mutants and with short peptides derived from the kinase insert region of the PDGF receptor showed that the recombinant p85 binds to a well-defined phosphotyrosine-containing sequence of the receptor. p85 appears to be the subunit of PI3-kinase that links the enzyme to the ligand-activated receptor.

Pancreastatin modulates insulin signaling in rat adipocytes: mechanisms of cross-talk.

“…The best known pathway to GLUT4 in insulin receptor signaling is PI3K (31). PI3K consists of 2 subunits: a p110 catalytic subunit (53) and a p85 regulatory subunit that contains 2 SH2 domains and 1 SH3 domain (54). In the case of the insulin receptor, the p110 catalytic subunit of PI3K is activated by interaction of SH2 domains of the p85 regulatory subunit with the tyrosine phosphorylated docking protein, IRS-1 (55), and the autophosphorylated insulin receptor ␤-subunit (56,57).…”

Purification and Characterization of Gβγ-responsive Phosphoinositide 3-Kinases from Pig Platelet Cytosol

“…Binding of the SH2 domains to phosphotyrosine residues within the sequence context, YMXM, which occurs in a wide range of activated growth factor receptors and adaptor proteins, causes the translocation and activation of the catalytic subunit (2, 15-16). More recently, a number of distinct p85 and p110 subunit isoforms have been identified (11,14,17), but the functional significance of this heterogeneity is not yet clear (18).Heterotrimeric G-protein-regulated forms of PI 3-kinase have also been identified following the observations that activation of G-protein-coupled receptors in neutrophils and platelets caused a rapid accumulation of PtdIns(3,4,[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Stephens et al (22) partially purified a G-protein ␤␥ subunit (G␤␥)-responsive PI 3-kinase from a myeloid cell line (U937).…”

“…Receptor-regulated forms of PI 3-kinase appear to prefer to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) in vivo (3-5). The product of this reaction, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), is a candidate second messenger that regulates a variety of cellular responses to growth factors perhaps through the activation of serine/threonine protein kinases such as Akt/PKB and/or certain protein kinase C isoforms and small GTP-binding proteins such as Rac 1 (6 -10).The first form of PI 3-kinase to be purified and cloned was identified as a heterodimer composed of a 110-kDa catalytic subunit with a tightly bound regulatory subunit of 85 kDa (11)(12)(13)(14). The p85 subunit possesses a number of regions with homology to recognized signaling proteins.…”

“…The first form of PI 3-kinase to be purified and cloned was identified as a heterodimer composed of a 110-kDa catalytic subunit with a tightly bound regulatory subunit of 85 kDa (11)(12)(13)(14). The p85 subunit possesses a number of regions with homology to recognized signaling proteins.…”

“…Heterotrimeric G-protein-regulated forms of PI 3-kinase have also been identified following the observations that activation of G-protein-coupled receptors in neutrophils and platelets caused a rapid accumulation of PtdIns(3,4,[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Stephens et al (22) partially purified a G-protein ␤␥ subunit (G␤␥)-responsive PI 3-kinase from a myeloid cell line (U937).…”