Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.
Sphingosine-1-phosphate, a sphingolipid metabolite, is involved in the mitogenic response of platelet-derived growth factor (PDGF) and is formed by activation of sphingosine kinase. We examined the effect of PDGF on sphingosine kinase activation in TRMP cells expressing wild-type or various mutant betaPDGF receptors. Sphingosine kinase was stimulated by PDGF in cells expressing wild-type receptors but not in cells expressing kinase-inactive receptors (R634). Cells expressing mutated PDGF receptors with phenylalanine substitutions at five major tyrosine phosphorylation sites 740/751/771/1009/1021 (F5 mutants), which are unable to associate with PLCgamma, phosphatidylinositol 3-kinase, Ras GTPase-activating protein, or protein tyrosine phosphatase SHP-2, not only failed to increase DNA synthesis in response to PDGF but also did not activate sphingosine kinase. Moreover, mutation of tyrosine-1021 of the PDGF receptor to phenylalanine, which impairs its association with PLCgamma, abrogated PDGF-induced activation of sphingosine kinase. In contrast, PDGF was still able to stimulate sphingosine kinase in cells expressing the PDGF receptor mutated at tyrosines 740/751 and 1009, responsible for binding of phosphatidylinositol 3-kinase and SHP-2, respectively. In agreement, PDGF did not stimulate sphingosine kinase activity in F5 receptor 'add-back' mutants in which association with the Ras GTPase-activating protein, phosphatidylinositol 3-kinase, or SHP-2 was individually restored. However, a mutant PDGF receptor that was able to bind PLCgamma (tyrosine-1021), but not other signaling proteins, restored sphingosine kinase sensitivity to PDGF. These data indicate that the tyrosine residue responsible for binding of PLCgamma is required for PDGF-induced activation of sphingosine kinase. Moreover, calcium mobilization downstream of PLCgamma, but not protein kinase C activation, appears to be required for stimulation of sphingosine kinase by PDGF.-Olivera, A., Edsall, J., Poulton, S., Kazlauskas, A., Spiegel, S. Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.
Olivera
A
A
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007, USA. spiegel@bc.georgetown.edu
Edsall
L
L
Poulton
S
S
Kazlauskas
A
A
Spiegel
S
S
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004285:Dogs; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D051966:Phospholipase C gamma; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine; D000099157:Sphingosine Kinase
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002118:Calcium; D002614:Chelating Agents; D004247:DNA; D004533:Egtazic Acid; D007527:Isoenzymes; D010649:Phenylalanine; D051966:Phospholipase C gamma; D017853:Phosphotransferases (Alcohol Group Acceptor); D010982:Platelet-Derived Growth Factor; D020794:Receptor Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D011994:Recombinant Proteins; D019284:Thapsigargin; D010738:Type C Phospholipases; D014443:Tyrosine; D000099157:Sphingosine Kinase; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C099852:PDGF receptor tyrosine kinase