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.

The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor c(. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 6 1 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent V,,, value of 20 nmol min-' mg-' and K , values of 4.5 pM for ATP and 1.43 mM for angiotensin 11. This corresponds to a turnover number of 1.4 mol min-' mol-' .Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin I1 as substrate. The specific activity of the intracellular domain of the EGF-R, when assayed at 20°C in the presence of 1M ammonium sulfate, was 160 nmol min-' mg-'. Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific. No activation was found when assayed using polymeric substrates. Addition of MeZ+-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDSjPAGE migration. Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2: 5 : 6 : l), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGF-R. Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme.The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to epidermal growth factor (EGF) and transforming growth factor a. The receptor consists of an extracellular hormone-binding domain, a short hydrophobic transmembrane domain and an intracellular domain which contains the protein-tyrosine kinase sequence (Carpenter, 1987; Yarden and Ullrich, 3988a). Enzymatic activity of the kinase domain is essential for signal transduction via the EGFCorrespondence to N. B. Lydon,

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