The p85 Regulatory Subunit Controls Sequential Activation of Phosphoinositide 3-Kinase by Tyr Kinases and Ras

Abstract

Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit that regulates a variety of cell responses, including cell division and survival. PI3K is activated following Tyr kinase stimulation and by Ras. We found that the C-terminal region of p85, including the C-Src homology 2 (C-SH2) domain and part of the inter-SH2 region, protects the p110 catalytic subunit from Rasinduced activation. Although the p110 activity associated with a C-terminal p85 deletion mutant increased significantly in the presence of an active form of Ras, purified wild type p85-p110 was only slightly stimulated by active Ras. Nonetheless, incubation of purified p85-p110 with Tyr-phosphorylated peptides, which mimic the activated platelet-derived growth factor receptor, restored Ras-induced p85-p110 activation. In conclusion, p85 inhibits p110 activation by Ras; this blockage is released by Tyr kinase stimulation, showing that the classical mechanism of class IA PI3K stimulation mediated by Tyr kinases also regulates Ras-induced PI3K activation.Phosphoinositide 3-kinases (PI3K) 1 are enzymes that transfer phosphate to position 3 of the phosphoinositide ring, regulating a variety of cell responses including survival, division, and transformation. PI3Ks are divided into three subclasses based on their primary structure and substrate specificity, but only the class I enzymes generate phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (3-poly-PtdIns) products in vivo. Basal levels of these lipids are very low in quiescent cells but increase rapidly and transiently following growth factor receptor (GFR) stimulation (for a review, see Refs. 1-4). 3-Poly-PtdIns recruits pleckstrin homology domain-containing proteins such as phosphoinositide-dependent kinase-1 and protein kinase B (PKB), which mediate PI3K signal propagation (5-8).Class IA PI3K is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit, of which there are several isoforms (1, 2, 9 -14). The prototypic p85 regulatory subunit has an SH3 domain, a BcR homology domain (BH) flanked by proline-rich sequences, and two SH2 domains separated by the so-called inter-SH2 domain, to which p110 binds (15-17). The p110 catalytic subunit contains a p85-interacting region, a Ras-binding domain, a region homologous to PI4K and the PI3K catalytic domain (4, 15). The p85 subunit protects p110 from degradation and inhibits its enzymatic activity in quiescent cells (18). When cells are stimulated by receptors with intrinsic Tyr kinase activity, such as platelet-derived growth factor receptor (PDGFR), p85 mediates p110 translocation to the cell membrane, where PI3K substrates are found (11). In addition to mediating PI3K translocation, p85 appears to transmit an activating conformational change to p110 (19 -21), because binding to p85 of Tyr-phosphorylated peptides representing the activated PDGFR increases p110 enzymatic activity (16,22). Other receptors activate PI3K by stimulating cytosolic Tyr ki...

Phosphatidylinositol 3-Kinase Regulates the CD4/CD8 T Cell Differentiation Ratio

“…This complementation may reflect that both isoforms regulate this transition physiologically or that enhanced p65 PI3K -induced PI3K activity artificially compensates for the lack of p110␥. The p65 PI3K expression nonetheless enhances basal 3-polyphosphoinositide levels only moderately and requires stimulation of a tyrosine kinase-coupled receptor to enhance PI3K activity significantly (22,23). Pre-TCR, a receptor coupled to Lck tyrosine kinase (5,35), thus may activate class IA PI3K isoforms physiologically, thereby contributing to pre-TCR-mediated differentiation, as observed in p65 PI3K Tg mice.…”

“…Lck is a Tyr kinase that is induced shortly after TCR ligation (35). Tyrosine kinases activate PI3K and MAPK (14,23,25,45); thus, these two pathways may cooperate to control the CD4 ϩ /CD8 ϩ cell differentiation ratio. In support of this view, MAPK and Lck control pre-TCR-mediated differentiation and CD4/CD8 lineage commitment.…”

“…In addition, to examine the role of the p85/p110 PI3K isoforms (class IA) in T cell development, we analyzed the phenotype of mice expressing an activating mutation of the p85 regulatory subunit, p65 PI3K , in T cells. This mutant regulatory subunit associates with the p110 catalytic subunit, moderately increasing basal PI-3,4,5-P 3 levels and significantly enhancing the transient PI3K activation that follows receptor activation (22,23). p65 PI3K potentially acts on all three class IA catalytic subunits, constituting a useful tool for the study of the role of class IA p85/p110 PI3K isoforms in T cell development.…”

Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD

“…It has been reported that PI3K activity is regulated by PTK through tyrosine phosphorylation (4,15). The catalytic activity of p110␣ is suppressed by association with an 85-kDa regulatory subunit (p85␣).…”

“…However, detailed mechanisms by which PTK-dependent signal transduction pathways regulate ROMK channel activity are still not completely understood. PTK has been shown to regulate PI3K activity by tyrosine phosphorylation (3,4,15). Moreover, PI3K or PI3K-dependent lipid products such as phosphatidylinositol phosphates are involved in remodeling of the cytoskeleton (6), which has been shown to regulate ROMK channel activity (35).…”