Sphingosine activation of protein kinases in Jurkat T cells. In vitro phosphorylation of endogenous protein substrates and specificity of action.
Sphingosine displays multiple biochemical and biological effects, in particular inhibition and activation of protein kinases. To determine the predominant interaction of sphingosine with cellular kinases, the effects of sphingosine on endogenous protein phosphorylation in Jurkat T lymphoblastic cells were investigated in vitro. Sphingosine was found to cause prominent phosphorylation of a number of cytosolic proteins ranging in molecular mass from 18 to 165 kDa. Phosphorylation was calcium-independent. Phosphorylation of substrates was increased in response to concentrations of sphingosine as low as 10 microM and peaked at concentrations of 20-200 microM. Multiple lines of evidence suggested that sphingosine activated more than one protein kinase: 1) the concentration dependence on sphingosine differed from substrate to substrate, 2) phosphorylation of one group of substrates required ATP as the phosphate donor, whereas a second group showed no preference between ATP and GTP, and 3) phosphorylation of some substrates was inhibited by heparin, whereas other substrates were resistant. Activation of these kinases demonstrated a very specific requirement for D-erythro-sphingoid bases. DL-erythro-dihydrosphingosine was partially active, whereas DL-threo-dihydrosphingosine was not. Other related molecules such as stearylamine, sphingomyelin, and C2-ceramide were not active. Sphingosine-activated kinase(s) were distinct from protein kinase C, cyclic nucleotide-activated kinases, and calcium-dependent kinases. These observations demonstrate the existence of multiple sphingosine-activated protein kinases with high specificity for D-erythro-sphingosine, suggesting physiologic regulation of protein phosphorylation by sphingosine.
Pushkareva MYu
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Khan
W A
WA
Alessenko
A V
AV
Sahyoun
N
N
Hannun
Y A
YA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D002478:Cells, Cultured; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013110:Sphingosine / Q000494:pharmacology*; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology*
D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006493:Heparin; D011494:Protein Kinases; D013110:Sphingosine