Ceramide induces translocation of protein kinase C-alpha to the Golgi compartment of human embryonic kidney cells by interacting with the C2 domain.
Ceramide is a lipid second messenger produced by sphingolipid metabolism in cells exposed to a limited number of agonists and in turn triggers important cell responses including protein kinase C (PKC)-alpha activation. Using a fusion protein comprising bovine PKCalpha and the green fluorescent protein (GFP), we transfected human embryonic kidney (HEK) cells and investigated to which subcellular compartment ceramide triggers PKCalpha redistribution. Stimulation of HEK cells with exogenous C16-ceramide or bacterial sphingomyelinase (bSMase), which leads to increased endogenous ceramide formation, evokes a translocation of PKCalpha to the Golgi compartment. By using deletion mutants of PKCalpha lacking distinct domains in the regulatory region, it is shown that the Ca(2+)-dependent lipid binding C2 domain, but not one of the C1 domains is essentially required for the ceramide-triggered translocation of PKCalpha to the Golgi complex. In contrast, the C2 domain is not required for phorbol ester (TPA) binding and translocation of PKCalpha to the plasma membrane. In addition, evidence is provided that TPA requires only one of the two C1 subdomains to trigger translocation to the plasma membrane.In summary, our data provide evidence that ceramide either directly or indirectly interacts with the Ca(2+)-dependent lipid binding C2 domain of PKCalpha and thereby induces translocation of the enzyme to the Golgi compartment.
Aschrafi
Armaz
A
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfort on the Main, Germany.
Franzen
Rochus
R
Shabahang
Soheyla
S
Fabbro
Doriano
D
Pfeilschifter
Josef
J
Huwiler
Andrea
A
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D002518:Ceramides; D011993:Recombinant Fusion Proteins; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha