Sphingosine kinase type 2 activation by ERK-mediated phosphorylation.
Sphingosine 1-phosphate (S1P), a potent lipid mediator, is a ligand for a family of five G protein-coupled receptors (S1P(1-5)) that have been shown to regulate a variety of biological responses important for cancer progression. The cellular level of S1P is low and tightly regulated in a spatio-temporal manner through its synthesis catalyzed by two sphingosine kinases, denoted SphK1 and SphK2. Many stimuli activate and translocate SphK1 to the plasma membrane by mechanisms that are dependent on its phosphorylation. Much less is known about activation of SphK2. Here we demonstrate that epidermal growth factor (EGF) as well as the protein kinase C activator, phorbol ester, induce rapid phosphorylation of hSphK2 which was markedly reduced by inhibition of MEK1/ERK pathway. Down-regulation of ERK1 blocked EGF-induced phosphorylation of SphK2. Recombinant ERK1 phosphorylated hSphK2 in vitro and increased its enzymatic activity. ERK1 also was found to be in a complex with hSphK2 in vivo. Site-directed mutagenesis indicated that hSphK2 is phosphorylated on Ser-351 and Thr-578 by ERK1 and that phosphorylation of these residues is important for EGF-stimulated migration of MDA-MB-453 cells. These studies provide the first clues to the mechanism of agonist-mediated SphK2 activation and enhance understanding of the regulation of SphK2 activity by phosphorylation and its role in movement of human breast cancer cells toward EGF.
Hait
Nitai C
NC
Department of Biochemistry and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298, USA.
Bellamy
Andrea
A
Milstien
Sheldon
S
Kordula
Tomasz
T
Spiegel
Sarah
S
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry; D000099157:Sphingosine Kinase
D004815:Epidermal Growth Factor; D048049:Extracellular Signal-Regulated MAP Kinases; D048052:Mitogen-Activated Protein Kinase 3; D017853:Phosphotransferases (Alcohol Group Acceptor); D011493:Protein Kinase C; D013912:Threonine; D000099157:Sphingosine Kinase