SELECTIVE CERAMIDE BINDING TO PROTEIN-KINASE-C-ALPHA AND PROTEIN-KINASE-C-DELTA ISOENZYMES IN RENAL MESANGIAL CELLS

Ceramide is an important lipid second messenger produced by sphingolip id metabolism in cells exposed to a limited number of agonists and in turn triggers several cell responses in a protein kinase C (PKC)-depen dent manner, Stimulation of mesangial cells with a radioiodinated phot oaffinity labeling analogue of ceramide, (N-[3-[[[2-(I-125)iodo-4-[3-( trifluoromethyl) carbonyl]propanoyl]-D-erythro-sphingosine) ([I-125]-T ID-ceramide), defines PKC-alpha and PKC-delta as direct targets of cer amide. No binding of ceramide to PKC-epsilon and PKC-zeta could be det ected. Moreover, TID-ceramide selectively binds to recombinant PKC-alp ha and -delta but not to PKC-epsilon and -zeta isoenzymes. In vitro ki nase activity assays reveal that only the binding of ceramide to PKC-a lpha is accompanied by an increase in kinase activity. In contrast, th ere is no change in in vitro kinase activity of the other isoforms tes ted, i.e., PKC-delta, -epsilon, and -zeta, toward any of the conventio nal substrates tested. However, it is noteworthy that PKC-delta shows a decreased autophosphorylation upon ceramide binding. In vivo, activa tion of PKC-alpha by ceramide is monitored by a delayed translocation of the isoform from the cytosol to the membrane fraction, detectable a fter 1 h of stimulation. In contrast, neither PKC-delta, nor -epsilon nor -zeta is redistributed by ceramide, One functional cell response m ediated by PKC-alpha in mesangial cells is a negative feedback regulat ion of ligand-stimulated phosphoinositide hydrolysis. When cells are p retreated with ceramide. ATP-induced inositol trisphosphate formation is time-dependently reduced, A maximal inhibition is observed after 2 h of ceramide exposure. In summary, these results suggest that ceramid e selectively interacts with the alpha-and delta-isoforms of PKC in me sangial cells. Whereas PKC-alpha is activated with pronounced inhibiti on of hormone-stimulated phosphoinositide signaling, PKC-delta display s a decrease in its autophosphorylation, suggesting a negative role of ceramide binding on PKC-delta activity.