Protein kinase C-gamma phorbol-binding domain involved in protein-protein interaction

Protein kinase C-gamma (PKC-gamma) contains two cysteine-rich regions (Cys1 , Cys2) responsible for interaction with phospholipids. However, previous e xperiments suggested that, only Cys1 represents the high affinity site invo lved in diacylglycerol-dependent activation of PKC-gamma. This raises the q uestion whether Cys2 might participate in other functions of the PKC-gamma regulatory domain. The purpose of our studies was to examine the ability of Cys2 domain to bind cellular proteins. The Cys2 domain (residues 92-173) w as expressed as a fusion protein with glutathione-S-transferase (GST) in Es cherichia coli and purified. In order to investigate protein-protein intera ction of Cys2 domain we used affinity column and an overlay assay. Our resu lts demonstrate that the Cys2 domain of PKC-gamma binds several proteins fr om rat brain extracts. In the absence of phospholipids the Cys2 domain bind s some proteins in the cytosolic fraction of rat brain, but no binding was detected with the proteins extracted from particulate fraction. Ca2+ at 1 m u M concentration potentiated binding of cellular proteins to Cys2 domain. In the absence of Ca2+ the Cys2 domain binds proteins in the cytosolic frac tion of rat brain in the presence of phosphatidylserine and to the lesser e xtend in the presence of phosphatidylinositol but neither phosphatidylcholi ne nor phosphatidylethanolamine. These results suggest that the Cys2 domain of PKC-gamma has the ability to interact with two classes of proteins. One class binds the Cys2 domain in t he phosphatidylserine dependent fashion, and the other proteins bind Cys-2 domain in the Ca2+ dependent and phospholipid independent manner.