Endogenous substrates of sphingosine-dependent kinases (SDKs) are chaperone proteins: heat shock proteins, glucose-regulated proteins, protein disulfide isomerase, and calreticulin.

Protein kinases whose activity is detectable only in the presence of sphingosine (Sph) or N,N'-dimethyl-Sph (DMS), but not in the presence of 15 other sphingolipids, phospholipids, and glycerolipids tested (Megidish, T., et al. (1995) Biochem. Biophys. Res. Commun. 216, 739-747), have been termed "sphingosine-dependent kinases" (SDKs). We showed previously that a purified SDK (termed "SDK1") phosphorylates a specific Ser position of adapter/chaperone protein 14-3-3 isoforms beta, eta, and zeta but not tau or sigma (Megidish, T., et al. (1998) J. Biol. Chem. 273, 21834-45). In this study we found the following: (i) other SDKs with different substrate specificities are present in cytosolic and membrane extracts of mouse Balb/c 3T3 (A31) fibroblasts. (ii) The activation of these SDKs is specific to D-erythro-Sph and its N-methyl derivatives, the effect of L-threo-Sph or its N-methyl derivatives is minimal, and nonspecific cationic amphiphiles have no effect at all. An SDK separated as fractions "TN31-33" phosphorylated a 50 kDa substrate which was identified as calreticulin, as well as two endogenous substrates with molecular mass 58 and 55 kDa, both identified as protein disulfide isomerase (PDI). This SDK, which specifically phosphorylates calreticulin and PDI, both molecular chaperones found at high levels in endoplasmic reticulum, is tentatively termed "SDK2". Another SDK activity was copurified with glucose-regulated protein (GRP) and heat shock proteins (HSP). One GRP substrate had the same amino acid sequence as GRP94 (synonym: endoplasmin); another HSP substrate had the same amino acid sequence as mouse HSP86 or HSP84, the analogues of human HSP90. An SDK activity separated and present in "fraction 42" from Q-Sepharose chromatography specifically phosphorylated GRP105 (or GRP94) and HSP68 but did not phosphorylate PDI or 14-3-3. This SDK is clearly different from other SDKs in its substrate specificity and is tentatively termed "SDK3". Interestingly, substrates of all these SDKs so far identified are molecular chaperones or adapters capable of binding to enzymes and key molecules involved in signal transduction, maintaining tertiary structure of bioactive molecules, or maintaining cellular homeostasis in response to environmental stress. Thus, the essential role of Sph and DMS is to activate molecular chaperones, thereby providing a link to the mechanism by which SDK activity regulates cellular homeostasis and signal transduction.

A novel sphingosine-dependent protein kinase (SDK1) specifically phosphorylates certain isoforms of 14-3-3 protein.

Protein kinases activated by sphingosine or N,N'-dimethylsphingosine, but not by other lipids, have been detected and are termed sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun. 216, 739-747). We have now partially purified one SDK, termed SDK1, from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine kinase with molecular mass 50-60 kDa that is strongly activated by N, N'-dimethylsphingosine and sphingosine, but not by ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or glycerolipids tested. Its activity is inhibited by the protein kinase C activator phosphatidylserine. Activity of SDK1 is clearly distinct from other types of serine kinases tested, including casein kinase II, the alpha and zeta isoforms of protein kinase C, extracellular signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (eta, beta, zeta) but not others (sigma, tau). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 beta. The sigma and tau isoforms of 14-3-3 lack serine at this position, potentially explaining their lack of phosphorylation by SDK1. Interestingly, the phosphorylation site is located on the dimer interface of 14-3-3. Phosphorylation of this site by SDK1 was studied in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrate an association between an SDK and 14-3-3 in situ. Exogenous N, N'-dimethylsphingosine stimulates 14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support a biological role of SDK1 activation and consequent phosphorylation of specific 14-3-3 isoforms that regulate signal transduction. In view of the three-dimensional structure of 14-3-3, it is likely that phosphorylation by SDK1 would alter dimerization of 14-3-3, and/or induce conformational changes that alter 14-3-3 association with other kinases involved in signal transduction.

N,N-dimethylsphingosine inhibition of sphingosine kinase and sphingosine 1-phosphate activity in human platelets.

Potential sphingosine (Sph) metabolites include phosphorylated, N-acylated, and N-methylated derivatives. Phosphorylated Sph, i.e., sphingosine 1-phosphate (Sph-1-P), may act as an autocrine stimulator of blood platelets, as it is abundantly stored in platelets and released extracellularly and its exogenous addition induces platelet activation. In this study, we evaluated Sph-1-P formation and its effects in human platelets in the presence of other Sph metabolites. On addition of [3H]Sph to intact platelets, the label was rapidly converted to Sph-1-P. This conversion into [3H]Sph-1-P was inhibited by N,N-dimethylsphingosine (DMS) in a dose-dependent manner, but not by other structurally related Sph derivatives, including ceramide. The inhibition of Sph-1-P formation by DMS was reproduced using a cell-free system (Sph kinase obtained from platelet cytosolic fractions) and much stronger than that by DL-threo-dihydrosphingosine, which had been considered to be the strongest inhibitor of Sph kinase. Administration of DMS to intact platelets resulted in a decrease in Sph-1-P mass and an increase in Sph mass. Furthermore, DMS inhibited the release of Sph-1-P from platelets stimulated with 12-O-tetradecanoylphorbol 13-acetate and inhibited platelet aggregation induced by exogenous addition of Sph-1-P. Collectively, our results indicate that DMS is useful as a Sph kinase inhibitor and that Sph-1-P actions as an autocrine stimulator of platelets are inhibited by DMS.

The signal modulator protein 14-3-3 is a target of sphingosine- or N,N-dimethylsphingosine-dependent kinase in 3T3(A31) cells.

Phosphorylation of a 28 kDa protein in 3T3(A31) cells upon addition of semi-purified kinase was induced only by sphingosine (Sph) or N,N-dimethylsphingosine (DMS), but not by 17 other lipids tested. The 28 kDa protein, a major target of Sph- or DMS-dependent kinase, is now identified as 14-3-3 protein, which modulates a number of key protein kinases involved in regulation of intracellular signaling (Aitken A, Trends Biochem Sci 20:95-97, 1995; Morrison D, Science 266:56-57, 1994). Thus, Sph and DMS modulate the function of a versatile modulator controlling a number of signaling molecules.

Regulatory effect of phorbol esters on sphingosine kinase in BALB/C 3T3 fibroblasts (variant A31): demonstration of cell type-specific response--a preliminary note.

Sphingosine-1-phosphate (Sph-1-P) has been implicated as a second messenger in control of cell motility and proliferation (e.g., Sadahira Y, et al., PNAS 89:9686, 1992; Olivera A & Spiegel S, Nature 365:557, 1993). The control mechanism for its synthesis, as catalyzed by sphingosine kinase, is crucial in signal transduction. Synthesis of Sph-1-P in Balb/c 3T3 fibroblasts (A31 variant) is strongly up-regulated by brief treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA). Level of Sph-1-P in PKC-depleted cells is 10-fold higher than in undepleted cells, and a further 5-fold increase occurs after treatment with TPA. In Swiss 3T3 and B16 melanoma cells, Sph-1-P level was unaffected by TPA treatment. Thus, the effect of TPA on Sph-1-P synthesis appears to be cell type-specific.

A mutant protein kinase C that can transform fibroblasts.

Expression of normal protein kinase C (PKC) isoenzymes in fibroblasts has been shown to alter growth regulation but has failed to induce complete transformation of the recipient cells. Here we report on a murine ultraviolet-induced fibrosarcoma cell line which has an unusual PKC subcellular distribution with 87% of the PKC activity associated with the membrane. We have cloned and sequenced the alpha-PKC complementary DNA from ultraviolet-induced-fibrosarcoma cells and from mouse Balb/c brain and found four point mutations in the fibrosarcoma PKC, of which three are in the highly conserved regulatory domain and one is in the conserved region of the catalytic domain. Expression of this mutant alpha-PKC gene in normal Balb/c 3T3 fibroblasts results in a fibrosarcoma-like PKC membrane localization and in cell transformation, as judged by their formation of dense foci, anchorage-independent growth and ability to induce solid tumours when inoculated into nude mice. By contast, transfectants expressing the normal alpha-PKC cDNA do not display a morphology typical of malignant transformed cells and fail to induce tumours in vivo. These findings demonstrate that point mutations in the primary structure of PKC modulate enzyme function and are responsible for inducing oncogenicity.