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A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase.

ABSTRACT: CerK (ceramide kinase) produces ceramide 1-phosphate, a sphingophospholipid with recognized signalling properties. It localizes to the Golgi complex and fractionates essentially between detergent-soluble and -insoluble fractions; however, the determinants are unknown. Here, we made a detailed mutagenesis study of the N-terminal PH domain (pleckstrin homology domain) of CerK, based on modelling, and identified key positively charged amino acid residues within an unusual motif in the loop interconnecting beta-strands 6 and 7. These residues are critical for CerK membrane association and polyphosphoinositide binding and activity. Their mutagenesis results in increased thermolability, sensitivity to proteolysis, reduced apparent molecular mass as well as propensity of the recombinant mutant protein to aggregate, indicating that this loop impacts the overall conformation of the CerK protein. This is in contrast with most PH domains whose function strongly relies on charges located in the beta1-beta2 loop.

SUBMITTER: Rovina P

PROVIDER: S-EPMC1652822 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase.

Rovina Philipp P Jaritz Markus M Höfinger Siegfried S Graf Christine C Dévay Piroska P Billich Andreas A Baumruker Thomas T Bornancin Frédéric F

The Biochemical journal 20061201 2

CerK (ceramide kinase) produces ceramide 1-phosphate, a sphingophospholipid with recognized signalling properties. It localizes to the Golgi complex and fractionates essentially between detergent-soluble and -insoluble fractions; however, the determinants are unknown. Here, we made a detailed mutagenesis study of the N-terminal PH domain (pleckstrin homology domain) of CerK, based on modelling, and identified key positively charged amino acid residues within an unusual motif in the loop intercon ...[more]

PMID: 16872273

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Project description:Numerous hormones, cytokines and transforming oncogenes activate phosphoinositide 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'-phosphoinositides 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 ceramide, inhibits several PI-3K-dependent events, including insulin-stimulated glucose uptake and growth-factor-stimulated cell survival. Here we show that ceramide analogues specifically prevent the recruitment of the PtdIns(3,4,5)P(3)-binding proteins Akt/protein kinase B (PKB) or the general receptor for phosphoinositides-1 (GRP1). Specifically, the short-chain ceramide derivative C2-ceramide inhibited the platelet-derived growth factor (PDGF)-stimulated translocation of full-length Akt/PKB, as well as truncated proteins encoding only the PH domains of Akt/PKB or GRP1. C2-ceramide did not alter the membrane localization of the PH domain for phospholipase Cdelta, which preferentially binds PtdIns(4,5)P(2), nor did it affect the PDGF-stimulated production of PtdIns(3,4)P(2) or PtdIns(3,4,5)P(3). Interestingly, a glucosylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol (PDMP), shown previously to increase intracellular ceramide concentrations without affecting PI-3K [Rani, Abe, Chang, Rosenzweig, Saltiel, Radin and Shayman (1995) J. Biol. Chem. 270, 2859-2867], recapitulated the inhibitory effects of C2-ceramide on PDGF-stimulated Akt/PKB phosphorylation. These studies indicate that ceramide prevents the translocation of certain PtdIns(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 the fundamental signalling pathways initiated by PI-3K.

Dataset Information

A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase.

Publications

A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase.

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