A Novel Sphingosine-dependent Protein Kinase (SDK1) Specifically Phosphorylates Certain Isoforms of 14-3-3 Protein

Abstract

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...

Significance of 14-3-3 Self-Dimerization for Phosphorylation-dependent Target Binding

“…Among these, regulation of 14-3-3 function by its phosphorylation has attracted the most attention (Dubois et al, 1997a) and has been proposed to control both target binding and dimerization (Dubois et al, 1997b;Megidish et al, 1998). The enormous difference in the susceptibility of dimeric versus monomeric 14-3-3 to phosphorylation ( Figure 7) indicates that regulation of 14-3-3 by phosphorylation probably has to occur at the monomeric state rather than after dimer assembly.…”

“…These isoforms are encoded by seven separate genes, each displaying somewhat different target binding specificity and susceptibility to phosphorylation (Megidish et al, 1998). Thus, the differential tissue and development-dependent expression of the various 14-3-3 genes and the considerable tendency toward heterodimerization in addition to homodimerization suggest that small differences in binding specificity among isoforms, when combined with heterodimerization and differential regulation at the level of expression and phosphorylation of individual 14-3-3 isoforms, can combine to generate a robust regulatory apparatus.…”

“…Several studies demonstrated phosphorylation of 14-3-3 at two sites, e.g., 14-3-3 S58 and 14-3-3 T233, and proposed a regulatory role of these phosphorylations in 14-3-3 dimerization and target binding, respectively (Megidish et al, 1995;Dubois et al, 1997a,b;Megidish et al, 1998). While conducting the metabolic 32 P-labling experiments, we noticed that the dimerization-deficient 14-3-3 mutant contained significantly more 32 P than the wild-type 14-3-3; this difference was further enhanced by treatment of cells with EGF or the phosphatase inhibitor calyculin A, reaching up to 50-fold higher 32 P incorporation ( Figure 7A, compare lanes 1 and 6, basal; lanes 2 and 7, EGF; and lanes 4 and 5 with 9 and 10, calyculin A).…”

“…S58 is a 14-3-3 phosphorylation site that was identified after cell treatment with sphingosine by a putative sphingosine-dependent kinase, SDK1 (Megidish et al, 1998). This site is also a putative protein kinase C site and the surrounding sequence, i.e., RRSpSWR resembles the sequence of the peptide used to generate the pan phospho-specific 14-3-3 binding site antibody.…”

Proteomics-based Target Identification

“…In humans, seven isoforms are encoded by a multigene family. Several isoforms are post-translationally modified by phosphorylation (17,18) and N-terminal acetylation (19).…”