The ultraviolet studies on protein-lipid interaction of a protein kinase C-gamma phorbol-binding domain

Family of protein kinase C (PKC) isozymes play a key role in transducing a vast number of signals into the cells. The members of classical PKC family are activated by binding of various lipid ligands to one of the several cys teine-rich domains of the enzyme. Second cysteine-rich (Cys2) domain of PKC -gamma was expressed in Escherichia coli as a fusion protein with glutathio ne-S-transferase (GST) using the cDNA sequence from rat brain. The Cys2 pro tein after cleavage from GST was purified to homogeneity using glutathione- agarose and Mono-S cation exchanger column. In order to investigate the int eraction of lipids and calcium with Cys2 protein we used UV spectroscopy. T he UV spectrum of Cys2 protein exhibited a maximum at 205 nn. Exposition of Cys2 protein to phosphatidylserine (PS) vesicles resulted in significant d ecrease in the absorbance in the 210 nm region. Changes in UV spectrum of C ys2 protein induced by phorbol 12,13-dibutyrate (PDB) were smaller than tho se induced by PS, and addition of PDB with PS had no effect on the PS induc ed changes in UV spectrum of Cys2. Neither phosphatidylcholine (PC) nor pho sphatidylethanolamine (PE) affected UV spectrum of Cys2 but in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol 4- phosphate (PIP) vesicles some changes were observed. Calcium ions alone or in the presence of PS had no effect on the UV spectrum of Cys2 protein. The se data indicate that PS comparing to PDB, interacts with a larger area of Cys2 protein, and that the binding sites for these two molecules are at lea st overlapping. The site of PIP and PIP2 interaction with PKC-gamma is dist inct from that of phorbol ester binding site.