Expression and partial characterization of rat protein kinase C‐δ and protein kinase C‐ξ in insect cells using recombinant baculovirus

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.

UCN‐01, an anti‐tumor drug, is a selective inhibitor of the conventional PKC subfamily

“…There is also the possibility that another kinase is the target for the anti-tumor action of UCN-01. The inhibition of aPKC( by staurosporine is less efficient than that of cPKC~, consistent with the previous results using recombinant aPKC( [15,16]. The ICs0 value of staurosporine determined using aPKC~" partially purified from murine epidermis was lower (16 nM) [17], although this aPKC~" preparation was crude and its kinase activity significantly weak.…”

“…1). In addition, staurosporine shows almost the same sensitivity for cPKC and nPKC isozymes when the synthetic peptides MBP 4 14 [9] and peptide-y [19] are used as substrates, although there is a 100-fold selectivity for cPKCc~ over nPKC~ when protamine sulfate is used [15]. These results suggest that staurosporine interacts with the substrate binding domain of PKC.…”

“…However, calphostin C inhibits not only cPKC and nPKC but also aPKC( with only a 5-to 30-fold higher ICs0, suggesting that calphostin C recognizes the regulatory domain of aPKC( as well as those of cPKCs and nPKCs, cPKC and nPKC isozymes have the C1 domains with repeated cysteine-rich sequences and are dependent on phorbol esters, while aPKC( contains only one cysteine-rich sequence and its kinase activity is independent of phorbol esters or diacylglycerols [7,8]. Recombinant aPKC( produced by baculovirus-insect cell systems has been reported to exhibit constitutive kinase activity which is independent of phosphatidylserine and diacylglycerol [ 15,16], although aPKC( purified from bovine kidney showed kinase activity dependent on phosphatidylserine [23]. Our recombinant aPKC( also showed phosphatidylserine dependency and this dependency disappeared by limited fragmentation of aPKC( with calpain treatment, which produced a free catalytic fragment of aPKC( (Ueda, Y. et al, manuscript in preparation).…”

Characterisation of protein kinase C isoforms and enzymic activity from the αT3‐1 gonadotroph‐derived cell line

“…The IC,, values obtained in our inhibitor studies for either basal, PDBu-evoked or PDBu + Ca"-evoked activity are in agreement with several other reports of the potency of some of these inhibitors on the c, E and a isoforms, respectively [22,27,35]. Our I&,, values for H7 also reflect the spectrum of IC,, values that we have found in several physiological models that involve gonadotrophs [7,3638], with the exception of PDBu (100 nM)-induced LH secretion, which is particularly sensitive to H7 (ICY,,, value 1.7 + 1.5 PM) [7].…”

Extracellular Simian Virus 40 Induces an ERK/MAP Kinase-independent Signalling Pathway that Activates Primary Response Genes and Promotes Virus Entry

“…The mechanism for the activation of the atypical isoforms of PKC is not known. Unlike the classical isoforms, the atypical ones require neither Ca 2+ nor association with membrane diacylglycerol for their activation (McGlynn et al, 1992;Nakanishi & Exton, 1992), perhaps accounting for our results.…”