Proteolytic activation of protein kinase C‐ɛ

Abstract

Proteolysis of native protein kinase C-E (PKC-e) is shown to occur through tryptic attack at multiple sites within the PKC-s V2/V3 domain. Following initial cleavage of PKC-s with trypsin, the kinase activity using a synthetic peptide substrate was found to be lipid/phorbol-ester independent, as observed for other members of this kinase family. Interestingly, there is also an increase in the histone kinase activity, indicating that there is an influence of the regulatory domain of the enzyme on substrate specificity. This is discussed in the context of alternatively spliced PKC-E mRNAs that are shown to be present in brain and lung tissues.Protein kinase C has been defined as a CaZ+-and phospholipid-dependent protein kinase, which can be activated by diacylglycerol or various phorbol esters (recently reviewed [l, 21); the enzyme has been implicated in a variety of cellular processes, such as proliferation, differentiation and secretion [2]. Molecular cloning studies have shown that PKC consists of a family of closely related enzymes, PKC-a, -Dl, -pz, and -7, (see reviews [l, 31); more recently, four new members of the PKC family have been cloned, PKC-6, -6, 4, and -5 [4-61. We have recently purified and characterized one of these new members, PKC-E, by using the baculovirus insect cell expression system previously employed for the expression of PKC-1, [7]. The purified PKC-E differed in two major respects from PKC-a, -PI, -pz and -y, since it was CaZ+ independent and had a different substrate specificity; histone IIIS, a common in vitro substrate for PKC, was barely phosphorylated by . These data are in agreement with our initial studies on PKC-s expressed in COS-1 cells [5].The studies described here address the proteolytic activation of PKC-E, defining the sites of tryptic cleavage of the native enzyme and demonstrating that the catalytic fragments produced show an altered substrate specificity. The results are discussed with respect to alternatively spliced PKC-s transcripts that have been identified here and elsewhere [4].

Protein Kinase C-θ Phosphorylation of Moesin in the Actin-binding Sequence

“…A comparable pattern has been observed with PKC-␦ phosphorylation of the ␥ chain of the IgE receptor, where the rate of threonine phosphorylation was increased 2.5-fold using PI compared with PS, whereas the effect of PG was not tested (53). PKC-⑀ displays PG-stimulable activity toward a PKC-⑀ synthetic peptide resembling the PKC-⑀ pseudosubstrate site, with a serine residue at the alanine position (54). PKC-⑀ was also found to phosphorylate histones poorly, similar to the present findings with PKC-.…”

Expression and characterization of protein kinase C‐δ

“…Furthermore the catalytically active fragment of PKC-6 will phosphorylate histone 111s (see Fig. 2) implying that like PKC-E there is an influence on specificity determined by the regulatory domain [17,181. It is likely that the form of PKC-6 previously detected following expression in COS-1 cells was the proteolysed catalytic fragment [8].…”

“…With respect to the synthetic peptide based on the PKCa pseudosubstrate site (peptide-a [4]), PKC-6 shows some differences with respect to PKC-a, PI [3]. Interestingly the synthetic peptide based on the PKC-6 pseudosubstrate site (peptide-6) has a Vm,,/Km value of 153 for PKC-6 and this reflects a significantly lower apparent K, (3.5 pM); PKC-E likewise shows a low apparent K, (8 pM) for this substrate (A. R. Olivier, unpublished results).…”

Expression of protein kinase C isozymes in hippocampal neurones in culture

“…Proteolytic activation and degradation of PKC isozymes has been widely discussed as a physiological mechanism to regulate the activity and localization of PKC isozymes. Tryptic activation of PKCe increased the kinase activity towards histone about 10-fold [49], Also, Suzuki et al [50] have shown that PKCfl and PKCy isozymes are selectively associated with postsynaptic densities obtained from rat hippocampus and suggest that proteolytic activation of these isozymes could be a step in postsynaptic signal processing. It may be therefore possible that PKCfl and PKCy present in hippocampal neurones in culture are subject to specific regulation by in vivo proteolysis.…”