Expression of a functional protein kinase C-gamma using a baculovirus vector: purification and characterisation of a single protein kinase C iso-enzyme.
The cloning of complementary DNAs for protein kinase C (PKC) has revealed a multi-gene family of closely related protein kinases [Parker et al. (1986) Science 233, 853-859; Coussens et al. (1986) Science 233, 859-866]. In vivo, the distribution of the PKC isoenzymes follows a fairly tissue-specific pattern suggesting that functional differences exist between the members of this kinase family. To initiate a detailed characterisation of the individual isoenzymes, and as an alternative approach to purifying and separating the individual PKC types and their splice variants from mammalian tissues, we have expressed the bovine PKC type gamma in insect cells using a baculovirus expression vector. The bovine protein constitutes one of the major proteins in infected cells and can be purified to near homogeneity by a 2-step procedure. Analysis of the purified protein confirms that it has authentic mammalian PKC characteristics with respect to phospholipid dependence and phorbol ester binding. The bovine PKC gamma purified from infected cells is post-translationally modified and resolves into a doublet of molecular weights 82,000 and 84,000 upon SDS-polyacrylamide gel electrophoresis. These two size classes of polypeptides appear to result from differential phosphorylation as demonstrated by sensitivity to protein phosphatase treatment. The applicability and the potential of this system for the analysis of the various mammalian PKC isoenzymes is discussed.
Patel
G
G
Imperial Cancer Research Fund, Gene Regulation Laboratory, Lincoln's Inn Fields, London.
Stabel
S
S
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009036:Moths; D005810:Multigene Family; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D007527:Isoenzymes; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases