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 experiments suggested that, only Cys1 represents the high affinity site involved in diacylglycerol-dependent activation of PKC-gamma. This raises the question 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) was expressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli and purified. In order to investigate protein-protein interaction of Cys2 domain we used affinity column and an overlay assay. Our results demonstrate that the Cys2 domain of PKC-gamma binds several proteins from rat brain extracts. In the absence of phospholipids the Cys2 domain binds some proteins in the cytosolic fraction of rat brain, but no binding was detected with the proteins extracted from particulate fraction. Ca2+ at 1 microM concentration potentiated binding of cellular proteins to Cys2 domain. In the absence of Ca2+ the Cys2 domain binds proteins in the cytosolic fraction of rat brain in the presence of phosphatidylserine and to the lesser extend in the presence of phosphatidylinositol but neither phosphatidylcholine 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 the phosphatidylserine dependent fashion, and the other proteins bind Cys-2 domain in the Ca2+ dependent and phospholipid independent manner.
Pawelczyk
T
T
Department of Molecular Medicine, Medical University of Gdansk, Poland.
Kowara
R
R
Matecki
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D003545:Cysteine; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D004075:Diglycerides; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; C087857:protein kinase C gamma; D011493:Protein Kinase C; D003545:Cysteine