title: Modulation of protein kinase C by endogenous sphingosine: inhibition of phorbol dibutyrate binding in Niemann-Pick C fibroblasts.
abstract: The abnormal and variable increase in levels of free sphingoid bases recently described in fibroblasts from Niemann-Pick C patients allowed us to investigate the modulation of protein kinase C in vivo by endogenous sphingosine. The specific binding of [20-3H]phorbol 12, 13-dibutyrate to the regulatory domain of membrane-bound protein kinase C was significantly decreased in fibroblasts from patients compared with controls. A pronounced difference between the two groups (P<0.0001) was demonstrated in low-density lipoprotein-supplemented medium, i.e. under conditions known to disclose abnormal mobilization of unesterified cholesterol in Niemann-Pick C fibroblasts. Furthermore the degree of impairment of [3H]phorbol 12,13-dibutyrate binding was highly correlated (r=0.95) with the sphingosine levels measured in fibroblasts from those patients. Scatchard analysis of the binding data indicated that Niemann-Pick C and control fibroblasts contained almost the same number of binding sites per cell. A 8-34-fold increase in Kd was measured in Niemann-Pick C fibroblasts with at least a 5-fold increase in sphingosine levels. Removal, by cell fractionation, of membrane-bound protein kinase C from the bulk of sphingosine induced a normalization of Kd values. The overall results suggest that protein kinase C inhibition is directly related to sphingosine accumulation.
authors.affiliation: Department of Biochemistry, INSERM-CNRS 189, Lyon-Sud Medical School, BP12, 69921 Oullins Cedex, France.
authors.forename: C
authors.initials: C
authors.lastname: Rodriguez-Lafrasse
authors.forename: R
authors.initials: R
authors.lastname: Rousson
authors.forename: S
authors.initials: S
authors.lastname: Valla
authors.forename: P
authors.initials: P
authors.lastname: Antignac
authors.forename: P
authors.initials: P
authors.lastname: Louisot
authors.forename: M T
authors.initials: MT
authors.lastname: Vanier
mesh_terms: D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D009542:Niemann-Pick Diseases / Q000378:metabolism* / Q000473:pathology; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D013110:Sphingosine / Q000502:physiology*; D014316:Tritium
chemical_list: D014316:Tritium; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D013110:Sphingosine