title: Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets.
abstract: Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.
authors.forename: Y A
authors.initials: YA
authors.lastname: Hannun
authors.forename: C R
authors.initials: CR
authors.lastname: Loomis
authors.forename: A H
authors.initials: AH
authors.lastname: Merrill
authors.forename: R M
authors.initials: RM
authors.lastname: Bell
mesh_terms: D000588:Amines / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D004075:Diglycerides / Q000494:pharmacology; D006801:Humans; D008823:Micelles; D008954:Models, Biological; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000097:blood*; D010718:Phosphatidylserines / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D013110:Sphingosine / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors
chemical_list: D000588:Amines; D004075:Diglycerides; D008823:Micelles; D010703:Phorbol Esters; D010718:Phosphatidylserines; C044295:1,2-dioctanoylglycerol; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D013917:Thrombin; C009317:stearylamine; D013110:Sphingosine; C008699:octylamine; C013965:diolein