Expression and partial characterization of rat protein kinase C-delta and protein kinase C-zeta in insect cells using recombinant baculovirus

J Cell Biochem. 1992 Jul;49(3):239-50. doi: 10.1002/jcb.240490306.

Abstract

Expression of rat protein kinase C-delta (PKC-delta) and PKC-zeta in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype-specific antisera. Although the PKC-zeta cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC-delta were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC-alpha. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC-zeta. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC-delta or PKC-zeta when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC-delta and PKC-zeta. In contrast to PKC-zeta, the PKC-delta enzyme activity phosphorylated MBP or histone in a phosphatidylserine-(PS)/diacylglycerol(DG)-dependent manner, albeit not to the same extent as PKC-alpha. Lack of stimulation of the enzyme activity of PKC-zeta by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC-zeta, whereas several insect cell proteins were phosphorylated by PKC-delta in a PS/DG-dependent manner, including a protein of 78 kD. Our data demonstrate that the 76 kD PKC-zeta, in contrast to PKC-delta, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS or PS/DG. In addition, staurosporine was about 2-4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC-delta and PKC-zeta when compared to PKC-alpha.

MeSH terms

  • Alkaloids / pharmacology
  • Amino Acid Sequence
  • Animals
  • Baculoviridae / genetics*
  • Base Sequence
  • Blotting, Western
  • Cell Line
  • Cloning, Molecular
  • DNA
  • Molecular Sequence Data
  • Moths
  • Phorbol Esters / metabolism
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / chemistry
  • Protein Kinase C / genetics*
  • Protein Kinase C / metabolism
  • Rats
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Staurosporine

Substances

  • Alkaloids
  • Phorbol Esters
  • Recombinant Proteins
  • DNA
  • Protein Kinase C
  • Staurosporine