Constitutive activation of protein kinase B and phosphorylation of p47phox by a membrane-targeted phosphoinositide 3-kinase.
BACKGROUND
Phosphoinositide 3-kinase (PI 3-kinase) activity is required for mitogenic signaling and for secretory responses. Cell activation is presumed to cause the translocation of PI 3-kinase from the cytosol to the plasma membrane where the kinase interacts with its substrate phosphatidylinositol (4,5)-bisphosphate. Thus, a membrane-targeted and therefore constitutively active kinase could help elucidate the role of PI 3-kinase in intracellular signaling.


RESULTS
The membrane-targeting sequence of Ha-Ras, containing the consensus sequence for palmitoylation and farnesylation, was fused to the carboxyl terminus of p110 alpha, the catalytic subunit of PI 3-kinase. The lipid anchor directed PI 3-kinase to the membrane and led to constitutively elevated phosphatidylinositol (3,4,5)-trisphosphate levels in transfected cells. Expression of membrane-targeted PI 3-kinase resulted in the continuous activation of downstream effectors, such as protein kinase B (PKB, also known as Akt/RAC), which was recently shown to regulate glycogen synthase kinase-3. The constitutive activation of PKB was abolished by the specific PI 3-kinase inhibitor wortmannin, and PKB activation was marginal in transfectants expressing non-membrane-targeted PI 3-kinase. Multiple phosphorylation of the cytosolic factor p47phox is required for the rapid assembly of the phagocyte NADPH oxidase upon stimulation with agonists of G-protein-coupled receptors. We show here that the expression of membrane-targeted PI 3-kinase in the monoblastic cell line GM-1 results in a wortmannin-sensitive continuous phosphorylation of p47phox.


CONCLUSIONS
Targeting of PI 3-kinase to the site of its preferred substrate leads to constitutive stimulus-independent enhanced catalysis and is sufficient to regulate different signal transduction pathways.
Didichenko
S A
SA
Theodor Kocher-Institut University of Bern, Switzerland.
Tilton
B
B
Hemmings
B A
BA
Ballmer-Hofer
K
K
Thelen
M
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038341:Glycogen Synthase Kinases; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D019255:NADPH Oxidases / Q000096:biosynthesis; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D038341:Glycogen Synthase Kinases; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D016283:Proto-Oncogene Proteins p21(ras); D000077191:Wortmannin; C060974:phosphatidylinositol 3,4,5-triphosphate; C060505:neutrophil cytosolic factor 1; C494918:AKT1 protein, human; C501009:HRAS protein, human