SPECIFIC ACTIVATION OF P85-P110 PHOSPHATIDYLINOSITOL 3'-KINASE STIMULATES DNA-SYNTHESIS BY RAS-DEPENDENT AND P70 S6 KINASE-DEPENDENT PATHWAYS

We have developed a polyclonal antibody that activates the heterodimer ic p85-p110 phosphatidylinositol (PI) 3'-kinase in vitro and in microi njected cells, Affinity purification revealed that the activating anti body recognized the N-terminal SH2 (NSH2) domain of p85, and the antib ody increased the catalytic activity of recombinant p85-p110 dimers th reefold in vitro. To study the role of endogenous PI 3'-kinase in inta ct cells, the activating anti-NSH2. antibody was microinjected into GR C+LR73 cells, a CHO cell derivative selected for tight quiescence duri ng 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 activat ion of Pf 3'-kinase, as the effect was blocked by coinjection of the a ppropriate antigen (glutathione S-transferase-NSH2 domains from p85 al pha), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study sign als downstream from PI 3'-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorp oration, coinjection with an anti-p70 S6 kinase antibody effectively b locked anti-NSH2-stimulated DNA synthesis. We also found that coinject ion of inhibitory anti-ras antibodies blocked both serum- and anti-NSH 2-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 physiolog ical levels of PI 3'-kinase is sufficient to stimulate DNA synthesis i n quiescent cells, PI 3'-kinase-mediated DNA synthesis requires both p 70 S6 kinase and the p21(ras)/MEK pathway.