CONSTITUTIVE ACTIVATION OF PROTEIN-KINASE-B AND PHOSPHORYLATION OF P47(PHOX) BY MEMBRANE-TARGETED PHOSPHOINOSITIDE 3-KINASE

Background: Phosphoinositide 3-kinase (PI 3-kinase) activity is requir ed for mitogenic signaling and for secretary responses. Cell activatio n is presumed to cause the translocation of PI 3-kinase from the cytos ol to the plasma membrane where the kinase interacts with its substrat e phosphatidylinositol (4,5)-bisphosphate. Thus, a membrane-targeted a nd therefore constitutively active kinase could help elucidate the rol e of PI 3-kinase in intracellular signaling. Results: The membrane-tar geting sequence of Ha-Ras, containing the consensus sequence for palmi toylation and farnesylation, was fused to the carboxyl terminus of p11 0 alpha, the catalytic subunit of PI 3-kinase. The lipid anchor direct ed PI 3-kinase to the membrane and led to constitutively elevated phos phatidylinositol (3,4,5)-trisphosphate levels in transfected cells. Ex pression of membrane-targeted PI 3-kinase resulted in the continuous a ctivation of downstream effecters, such as protein kinase B (PKB, also known as Akt/RAC), which was recently shown to regulate glycogen synt hase kinase-3. The constitutive activation of PKB was abolished by the specific PI 3-kinase inhibitor wortmannin, and PKB activation was mar ginal in transfectants expressing nonmembrane-targeted PI 3-kinase. Mu ltiple phosphorylation of the cytosolic factor p47(phox) is required f or the rapid assembly of the phagocyte NADPH oxidase upon stimulation with agonists of G-protein-coupled receptors. We show here that the ex pression of membrane-targeted PI 3-kinase in the monoblastic cell line GM-1 results in a wortmannin-sensitive continuous phosphorylation of p47(phox). Conclusions: Targeting of PI 3-kinase to the site of its pr eferred substrate leads to constitutive stimulus-independent enhanced catalysis and is sufficient to regulate different signal transduction pathways. (C) Current Biology Ltd ISSN 0960-9822