Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85.

Abstract

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85.Phosphatidylinositol 3-kinase (PI 3-kinase), an activity that phosphorylates PI at the 3'-OH of the inositol ring (63), was first identified as an activity that associates with a variety of viral oncoproteins (36,54,65). Studies with various mutants of polyomavirus middle T antigen demonstrated a correlation between association with PI 3-kinase and the ability to transform cells (15,26,58,65). The finding that PI 3-kinase also associates with activated growth factor receptor tyrosine kinases (14, 25, 61) implicated this activity as a potential participant in mitogenic signaling.PI 3-kinase phosphorylates PI, PI-4-P, and PI-4,5-P2 in vitro (8,38). Measurements of polyphosphoinositides in virally transformed cells and in cells treated with growth factors demonstrate elevations in levels of 4,9,13,20,24,34,43,44,46,52,58,60,61). Cells expressing nontransforming mutants of viral oncoproteins do not exhibit elevated levels of PI-3,4-P2 or 4,46,58,60), corroborating previous correlations between PI 3-kinase activation and the transformed phenotype. However, the observation that PI 3-kinase products are synthesized in response to nonmitogenic stimuli (31,55,56) suggests that PI 3-kinase is involved in physiological processes unrelated to mitogenesis. Since the products of PI 3-kinase are poor substrates for phospholipase C (35, 47), it has been suggested that they may constitute a second messenger pathway distinct from that involving the hydrolysis of PI-4,5-P2 to generate diacylglycerol and inositol trisphosphate (4).The association of PI 3-kinase activity with polyomavirus middle T antigen and antiphosphotyrosine immunoprecipitates from platelet-derived growth factor (PDGF)-treated fibroblasts correlated with the presence of an 85-kDa phosphoprotein (15, 25), suggesting th...

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

“…Like the related Class IB catalytic subunit p110␥ (17), p110␣ contains Ras-binding, C2, helical, and kinase domains. In the p110␣ structure, p110␣ is anchored by the binding of the N-terminal ABD to the far end of the rod-like iSH2 domain, consistent with previous biochemical studies ( [18][19][20]. The kinase and C2 domains drape over the iSH2 domain like a saddle, with the Ras-binding domain facing upward above the ABD.…”

Lower Phosphoinositide 3-Kinase (PI 3-kinase) Activity and Differential Expression Levels of Selective Catalytic and Regulatory PI 3-Kinase Subunit Isoforms in Prefrontal Cortex and Hippocampus of Suicide Subjects

“…Subclass IA PI 3-kinase enzymes are heterodimers composed of a catalytic subunit of 110 kDa and an adapter regulatory p85 subunit (Hiles et al, 1992;Hu et al, 1993).…”

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).…”

“…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).…”