Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways.
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 alpha), 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'-kinase-mediated DNA synthesis requires both p70 S6 kinase and the P21ras/MEK pathway.
McIlroy
J
J
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Chen
D
D
Wjasow
C
C
Michaeli
T
T
Backer
J M
JM
D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D016466:CHO Cells; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D002417:Cattle; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D048728:MAP Kinase Kinase Kinase 1; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011090:Polyenes / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D019893:Ribosomal Protein S6 Kinases; D015398:Signal Transduction / Q000502:physiology*; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases
D000906:Antibodies; D002352:Carrier Proteins; D004247:DNA; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007328:Insulin; D048728:MAP Kinase Kinase Kinase 1; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011090:Polyenes; D017346:Protein Serine-Threonine Kinases; D016283:Proto-Oncogene Proteins p21(ras); D011993:Recombinant Fusion Proteins; D019893:Ribosomal Protein S6 Kinases; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; C546842:MTOR protein, human; C482576:MAP3K1 protein, human; C501009:HRAS protein, human; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one