We propose a novel model for the regulation of the p85/p110␣ phosphatidylinositol 3-kinase. In insect cells, the p110␣ catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110␣ is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110␣/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110␣. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110␣ dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15-to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110␣ or by the growth of the HEK 293T cells at 30°C. These nonspecific interventions mimicked the effects of p85 on p110␣, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110␣ in mammalian cells at 30°C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110␣. Thus, we have experimentally distinguished two effects of p85 on p110␣: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110␣ is both stabilized and inhibited by dimerization with p85.Phosphatidylinositol (PI) 3Ј-kinases constitute a family of enzymes that mediate intracellular signaling initiated by receptor tyrosine kinases and heterotrimeric G-protein-coupled receptors. Activation of PI 3Ј-kinase leads to increases in the intracellular levels of PI[3,4]P 2 and PI[3,4,5]P 3 , which are presumed second messengers (4). PI 3Ј-kinases have been implicated in the control of proliferation, cytoskeletal organization, apoptosis, and vesicular trafficking (6,16,20,31,46).A classification of PI 3Ј-kinases has been described by Zvelebil and coworkers (48). The class I enzymes are heterodimeric proteins that are composed of separate regulatory and catalytic subunits and that utilize PI, PI[4]P, and PI[4,5]P 2 as substrates. Class I enzymes include the p85/p110 PI 3Ј-kinase, which is activated by binding to phosphotyrosyl proteins, and the p101/ p120 PI 3-kinase-␥ isoform, which is activated by ␤␥ subunits from trimeric G proteins (11,13,38,39). Class II PI 3Ј-kinases contain C-terminal C2 domains and preferentially utiliz...

We have examined the roles of the p85/ p110α and hVPS34 phosphatidylinositol (PI) 3′-kinases in cellular signaling using inhibitory isoform-specific antibodies. We raised anti-hVPS34 and anti-p110α antibodies that specifically inhibit recombinant hVPS34 and p110α, respectively, in vitro. We used the antibodies to study cellular processes that are sensitive to low-dose wortmannin. The antibodies had distinct effects on the actin cytoskeleton; microinjection of anti-p110α antibodies blocked insulin-stimulated ruffling, whereas anti-hVPS34 antibodies had no effect. The antibodies also had different effects on vesicular trafficking. Microinjection of inhibitory anti-hVPS34 antibodies, but not anti-p110α antibodies, blocked the transit of internalized PDGF receptors to a perinuclear compartment, and disrupted the localization of the early endosomal protein EEA1. Microinjection of anti-p110α antibodies, and to a lesser extent anti-hVPS34 antibodies, reduced the rate of transferrin recycling in CHO cells. Surprisingly, both antibodies inhibited insulin-stimulated DNA synthesis by 80%. Injection of cells with antisense oligonucleotides derived from the hVPS34 sequence also blocked insulin-stimulated DNA synthesis, whereas scrambled oligonucleotides had no effect. Interestingly, the requirement for p110α and hVPS34 occurred at different times during the G1–S transition. Our data suggest that different PI 3′-kinases play distinct regulatory roles in the cell, and document an unexpected role for hVPS34 during insulin-stimulated mitogenesis.

We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC؉LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85␣), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum-and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3-kinasemediated DNA synthesis requires both p70 S6 kinase and the p21 ras /MEK pathway.Phosphatidylinositol (PI) 3Ј-kinases are a family of enzymes with homologous catalytic subunits and varied regulatory domains or subunits (26). Isoforms of PI 3Ј-kinase include heterodimeric PI 3Ј-kinases that are stimulated by binding of regulatory SH2 domains to tyrosine phosphoproteins, an isoform form that is regulated by ␤␥ subunits from trimeric G proteins, a monomeric form that is homologous to the VPS-34 yeast PI 3Ј-kinase, and isoforms containing C2 regulatory domains (13,20,22,37,43,48,57,58,63,64). Substrate specificities of the PI 3Ј-kinases also vary: the p85-p110 and ␤␥-stimulated PI 3Ј-kinases utilize PI, PI 4-P and PI 4,5-P 2 , whereas the VPS-34-like PI kinases are specific for PI (20,58,64) and the C2-domain-containing isoforms preferentially utilize PI and PI 4-P (37, 63). The catalytic subunit of the p85-p110 PI 3Ј-kinase is also homologous to a yeast PI 4-kinase, the ataxia telangiectasia gene product, the DNA-dependent protein kinase, and the TOR2/FRAP/RAFT proteins, which are upstream regulators of p70 S6 kinase and targets of the immunosuppressant rapamycin (14,18,30,55,56).The heterodimeric PI 3Ј-kinase is composed of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110) (reviewed in reference 26). The p85 regulatory subunit...