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 cysteine-rich domains of the enzyme. Second cysteine-rich (Cys2) domain of PKC-gamma was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) using the cDNA sequence from rat brain. The Cys2 protein after cleavage from GST was purified to homogeneity using glutathione-agarose and Mono-S cation exchanger column. In order to investigate the interaction of lipids and calcium with Cys2 protein we used UW spectroscopy. The UV spectrum of Cys2 protein exhibited a maximum at 205 nm. Exposition of Cys2 protein to phosphatidylserine (PS) vesicles resulted in significant decrease in the absorbance in the 210 nm region. Changes in UW spectrum of Cys2 protein induced by phorbol 12,13-dibutyrate (PDB) were smaller than those induced by PS, and addition of PDB with PS had no effect on the PS induced changes in UV spectrum of Cys2. Neither phosphatidylcholine (PC) nor phosphatidylethanolamine (PE) affected UV spectrum of Cys2 but in the presence of phosphatidylinositol 4,5 bisphosphate (PIP2) or phosphatidyliinositol 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. These 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 least overlapping. The site of PIP and PIP2 interaction with PKC-gamma is distinct from that of phorbol ester binding site.
Kowara
R
R
Department of Molecular Medicine, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Poland.
Gryckiewicz
E
E
Matecki
A
A
Pawelczyk
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014466:Ultraviolet Rays
D018076:DNA, Complementary; D007527:Isoenzymes; D010743:Phospholipids; D011506:Proteins; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; C087857:protein kinase C gamma; D011493:Protein Kinase C; D003545:Cysteine