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.
Pacific Northwest Research Institute, Seattle, Washington 98122 and Department of Pathobiology, University of Washington, Seattle, Washington 98195, USA.
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T
Megidish
J
J
Cooper
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L
Zhang
H
H
Fu
S
S
Hakomori
D048948:14-3-3 Proteins; D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II; D003600:Cytosol / Q000201:enzymology; D019281:Dimerization; D004789:Enzyme Activation; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051745:Protein Kinase C-delta; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D011506:Proteins; D014446:Tyrosine 3-Monooxygenase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D051745:Protein Kinase C-delta; C061800:N,N-dimethylsphingosine; D013110:Sphingosine