Inhibitory Properties of the Regulatory Domains of Human Protein Kinase Cα and Mouse Protein Kinase Cε

Abstract

Two fusion proteins in which the regulatory domains of human protein kinase C␣ (R␣; amino acids 1-270) or mouse protein kinase C⑀ (R⑀; amino acids 1-385) were linked in frame with glutathione S-transferase (GST) were examined for their abilities to inhibit the catalytic activities of protein kinase C␣ (PKC␣) and other protein kinases in vitro. Both GST-R␣ and GST-R⑀ but not GST itself potently inhibited the activities of lipid-activated rat brain PKC␣. In contrast, the fusion proteins had little or no inhibitory effect on the activities of the Ser/ Thr protein kinases cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, myosin light chain kinase, and mitogen activated protein kinase or on the src Tyr kinase. GST-R␣ and GST-R⑀, on a molar basis, were 100 -200-fold more potent inhibitors of PKC␣ activity than was the pseudosubstrate peptide PKC 19 -36 . In addition, a GST-R␣ fusion protein in which the first 32 amino acids of R␣ were deleted (including the pseudosubstrate sequence from amino acids 19 -31) was an effective competitive inhibitor of PKC␣ activity. The three GST-R fusion proteins also inhibited protamine-activated PKC␣ and proteolytically activated PKC␣ (PKM), two lipid-independent forms of PKC␣; however, the IC 50 values for inhibition were 1 order of magnitude greater than the IC 50 values obtained in the presence of lipid. These results suggest that part of the inhibitory effect of the GST-R fusion proteins on lipidactivated PKC␣ may have resulted from sequestration of lipid activators. Nonetheless, as evidenced by their abilities to inhibit the lipid-independent forms of the enzyme, the GST-R fusion proteins also inhibited PKC␣ catalytic activity through direct interactions. These data indicate that the R domains of PKC␣ and PKC⑀ are specific inhibitors of protein kinase C␣ activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.

Regulation of PKCα Activity by C1-C2 Domain Interactions

“…However, similar to the effects on intact PKC␣ activity, the presence of the ␣C1A-C1B domain concentrations greater than 10 nM also resulted in an inhibition of catalytic subunit activity. This observation indicates that the inhibitory effects of high levels of the ␣C1A-C1B domain may involve interactions with the catalytic domain, which is consistent with the findings of a recent study that showed that GST fusion proteins containing the regulatory domains of PKC␣ and PKC⑀ each inhibited the activity of the catalytic subunit of PKC␣ (46). The possibility that the activation of PKC␣ by the ␣C1A-C1B domain may have involved GST or (His) 6 was ruled out by the finding that PKC␣ activity was unaffected by a fusion peptide containing these moieties alone (Fig.…”

Increased protein kinase Cδ in mammary tumor cells: relationship to transformation and metastatic progression

“…In vitro studies indicate that RD inhibition is at least partially due to the N-terminal autoinhibitory pseudosubstrate sequence (Parissenti et al, 1998). However, since RDs are approximately 1000-fold more potent than the 11 amino acid pseudosubstrate peptide at inhibiting PKC activity in vitro, pseudosubstrate inhibition is only part of the mechanism (Chapline et al, manuscript in preparation).…”

Regulation of Protein Kinase C by the Cytoskeletal Protein Calponin

“…This antibody recognized all calponin fragments. Expression vectors encoding the regulatory domain of human PKC␣ (16) and murine PKC⑀ (17) fused to glutathione S-transferase (GST) were supplied by Drs. Bernard P. Schimmer and Amadeo Parissenti.…”

“…RegPKC⑀ and regPKC␣ were expressed in the DH5␣ or BL21 strains of Escherichia coli following the procedure of Parissenti (17). This expression system consistently yielded fulllength GSTregPKC␣.…”