Cloning and characterization of a protein kinase A anchoring protein (AKAP)-related protein that interacts with and regulates sphingosine kinase 1 activity.
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has novel dual actions. S1P is the ligand for a family of G protein-coupled receptors known as S1PRs that mediate various physiological functions. Growth factors rapidly activate sphingosine kinase type 1 (SPHK1) resulting in phosphorylation of sphingosine to form S1P, which plays important roles in cell growth regulation and protection from apoptosis. However, little is known of the mechanism(s) by which SPHK activity is regulated. Using a yeast two-hybrid screening approach, we cloned a 3-kb cDNA encoding a SPHK1-interacting protein (SKIP). BLAST analysis revealed that SKIP corresponded to the C-terminal region of a larger ( approximately 7 kb) cDNA that encoded a protein with a high degree of similarity to a family of protein kinase A anchor proteins (AKAP). In confirmation of the yeast two-hybrid assay, glutathione S-transferase (GST)-SPHK1 specifically pulled down SKIP, whereas GST did not. Moreover, immunoprecipitation of in vitro translated SPHK1 and SKIP revealed that SKIP and SPHK1 are tightly associated. Furthermore, SKIP overexpression in NIH 3T3 fibroblasts reduced SPHK1 activity and interfered with its biological functions. The apoptotic-sparing effect of SPHK1 against serum deprivation was reduced when co-transfected with SKIP. In addition, SPHK1-enhanced cell proliferation was also abolished by SKIP, with a corresponding decrease in activation of ERK. Taken together, these results indicate that SKIP is a novel protein likely to play a regulatory role in the modulation of SPHK1 activity.
Lacaná
Emanuela
E
Department of Biochemistry and Molecular Biology, Georgetown University Medical School, Washington, D. C. 20007, USA.
Maceyka
Michael
M
Milstien
Sheldon
S
Spiegel
Sarah
S
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000099157:Sphingosine Kinase
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D018076:DNA, Complementary; D005982:Glutathione Transferase; D020928:Mitogen-Activated Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D000099157:Sphingosine Kinase; C466999:SPHKAP protein, human