MODULATION OF PROTEIN-KINASE-C BY ENDOGENOUS SPHINGOSINE - INHIBITIONOF PHORBOL DIBUTYRATE BINDING IN NIEMANN-PICK-C FIBROBLASTS

The abnormal and variable increase in levels of free sphingoid bases r ecently described in fibroblasts from Niemann-Pick C patients allowed us to investigate the modulation of protein kinase C in vivo by endoge nous sphingosine. The specific binding of [20-H-3]phorbol 12,13-dibuty rate to the regulatory domain of membrane-bound protein kinase C was s ignificantly decreased in fibroblasts from patients compared with cont rols. A pronounced difference between the two groups (P < 0.0001) was demonstrated in low-density lipoprotein-supplemented medium, i.e. unde r conditions known to disclose abnormal mobilization of unesterified c holesterol in Niemann-Pick C fibroblasts. Furthermore the degree of im pairment of [H-3]phorbol 12,13-dibutyrate binding, was highly correlat ed (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 numb er of binding sites per cell. A 8-34-fold increase in K-d was measured in Niemann-Pick C fibroblasts with at least a 5-fold increase in sphi ngosine levels. Removal, by cell fractionation, of membrane-bound prot ein kinase C from the bulk of sphingosine induced a normalization of K -d values. The overall results suggest that protein kinase C inhibitio n is directly related to sphingosine accumulation.