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.

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 ␣ and isoforms of protein kinase C, extracellular signalregulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (, ␤, ) but not others (, ). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 ␤. The and 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 threedimensional 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.Glycosphingolipids, sphingolipids, and their catabolites have been implicated as modulators of signal transduction processes (for reviews, see Refs. 1-5). Sphingosine (Sph), 1 the backbone component of all sphingolipids, was originally discovered as an inhibitor of protein kinase C (PKC), in contrast to the stimulatory effect of diacylglycerol (DAG) (6). The inhibitory effect of D-erythro-Sph and its N-methyl derivative (N,NЈ-dimethyl sphingosine; DMS) on PKC was stronger than other Sph stereoisomers and Sph derivatives (7). Subsequent studies have demonstrated effects of Sph and DMS on various protein kinases. Sph inhibits calmodulin-dependent kinase (8) and insulin receptor tyrosine kinase (9), but enhances casein kinase II (CKII) (10). DMS strongly enhances kinase activ...

The tumor suppressor protein p53 functions as a transcriptional factor that activates genes controlling cell cycle arrest and apoptosis. Here, we report that protein inhibitor of activated Stat1 (PIAS1) interacts with the tetramerization and C-terminal regulatory domains of p53 in yeast two-hybrid analyses. Endogenous PIAS1 is also associated with endogenous p53 in mammalian cells. Ectopic expression of PIAS1 activates p53-mediated expression in mouse embryonic fibroblast cells (p53 ؊/؊ ) as well as a variety of other cell lines. Furthermore, ectopic expression of PIAS1 induces p53-mediated expression of cyclin-dependent kinase inhibitor p21 and G 1 arrest of the cell cycle in H1299 cells. In addition, a PIAS1 mutant without the RING-finger domain required for sumoylation could still activate p53-mediated gene expression, indicating that activation of p53 by PIAS1 does not require the RING-finger domain. Taken together, our results suggest that PIAS1 is a novel activator of p53.The tumor suppressor protein p53 is normally very short-lived and remains at very low levels through proteasomal degradation in unstressed mammalian cells. In response to damaged DNA, nucleotide depletion, hypoxia, oncogenes, and other genotoxic stresses, p53 accumulates dramatically in nucleus and functions as a transcriptional activator (1-4). Activation of p53 leads to cell cycle arrest or apoptosis by inducing a number of genes including cyclin-dependent kinase inhibitor p21 and apoptotic genes such as Bax. Although the exact mechanisms by which some of these stress signals are transduced to p53 are not known, signaling to p53 is thought to be mediated by upstream p53 regulators in at least three distinguishable pathways (5). The first pathway transduces signals of DNA damage induced by ionizing radiation, which activates ATM, Chk2, and subsequently p53. The second pathway is mediated by p14 ARF -MDM2 interaction (6), which is triggered by oncogenic signals such as Ras and Myc. The third pathway is turned on by a wide array of chemotherapeutic drugs, UV light, and protein kinase inhibitors, which requires kinases ATR and casein kinase II. There are additional proteins that can activate p53, but the significance of some of these interactions remains to be elucidated (1).The key negative regulator of the p53 network is MDM2. Transcription of MDM2 is itself activated by p53. However, MDM2 protein can both inhibit the transcriptional activity of p53 and target p53 for ubiquitin-mediated protein degradation (7-9), forming a negative regulatory loop for p53 function. Interference of the p53 and MDM2 interaction appears to be a main convergent point for many of the stress signals, resulting in stabilization and activation of p53 (10).The discovery of p63 and p73, the p53 family members, added a new level of complexity to understanding the p53 network (11). Both p63 and p73 produce multiple transcripts, which could either activate or inhibit p53-mediated gene expression. In contrast to p53, p63 and p73 do not interact with most viral proteins...

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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.