Ceramide dissociates 3 '-phosphoinositide production from pleckstrin homology domain translocation

Numerous hormones, cytokines and transforming oncogenes activate phosphoino sitide 3-kinase (PI-3K), a lipid kinase that initiates signal transduction cascades regulating cellular proliferation, survival, protein synthesis and glucose metabolism. PI-3K catalyses the production of the 3'-phosphoinosit ides PtdIns(3,4)P-2 and PtdIns(3,4,5)P-3, which recruit downstream effector enzymes to the membrane via their pleckstrin homology (PH) domains. Recent studies have indicated that another signalling lipid, the sphingolipid cer amide, inhibits several PI-3K-dependent events, including insulin-stimulate d glucose uptake and growth-factor-stimulated cell survival. Here we show t hat ceramide analogues specifically prevent the recruitment of the PtdIns(3 ,4,5)P-3-binding proteins Akt/protein kinase B (PKB) or the general recepto r for phosphoinositides-1 (GRP1), Specifically, the short-chain ceramide de rivative C2-ceramide inhibited the platelet-derived growth factor (PDGF)-st imulated translocation of full-length Akt/PKB, as well as truncated protein s encoding only the PH domains of Akt/PKB or GRP1. C2-ceramide did not alte r the membrane localization of the PH domain for phospholipase C delta, whi ch preferentially binds PtdIns(4,5)P-2, nor did it affect the PDGF-stimulat ed production of Ptdins(3,4)P-2 or PtdIns(3,4,5)P-3. Interestingly, a gluco sylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholinopropan -1-ol (PDMP), shown previously to increase intracellular ceramide concentra tions without affecting PI-3K [Rani, Abe, Chang, Rosenzweig, Saltiel, Radin and Shayman (1995) J. Biol. Chem. 270, 2859-2867], recapitulated the inhib itory effects of Ca-ceramide on PDGF-stimulated Akt/PKB phosphorylation. Th ese studies indicate that ceramide prevents the translocation of certain Pt dIns(3,4,5)P-3-binding proteins, despite the presence of a full complement of PtdIns(3,4)P-2 or PtdIns(3,4,5)P-3. Furthermore, these findings suggest a mechanism by which stimuli that induce ceramide synthesis could negate th e fundamental signalling pathways initiated by PI-3K.