Multiple isoforms of a protein kinase C inhibitor (KCIP‐1/14‐3‐3) from sheep brain

Abstract

A potent inhibitor of protein kinase C (PKC), inhibitor protein-1 (KCIP-l), isolated from sheep brain has been shown to consist of eight isoforms by reverse-phase HPLC. Direct protein sequence analysis has revealed these to be the same as those of 14-3-3 protein, described as an activator of tyrosine and tryptophan hydroxylases involved in neurotransmitter biosynthesis. The N-termini of KCIP-1 isoforms were shown to be acetylated, and secondary structure predictions revealed a high degree of a-helix with an amphipathic nature. KCIP-1 showed no inhibitory activity towards protein kinase M (the catalytic fragment of PKC) and had no effect on the activities of three other protein kinases, CAMP-dependent protein kinase, Ca2 c calmoddin-dependent protein kinase I1 and casein kinase 2. Four forms of KCIP-1 were shown to be substrates for PKC in vitro, but none were phosphorylated by the other protein kinases mentioned above.Protein kinase C (PKC), a calcium and phospholipid-dependent enzyme, is activated by diacylglycerol, hydrolyzed from inositol phospholipids by phospholipase C in response to a variety of extracellular signals [l]. This results in the phosphorylation of a wide range of proteins leading to the regulation of many physiological processes. A large family of PKC isoforms with multiple subspecies have now been identified which show subtle individual characteristics and specificity for substrates [2] which may suggest different roles for some of the isoforms.The role of PKC in regulating cellular function has been studied using specific activators (phorbol esters) which substitute for the physiological second messenger diacylglycerol [3], as well as naturally occurring and synthetic inhibitors. H-7 and K-252 inhibit the enzyme by competing with ATP [4,5] but the use of these compounds is limited in studies of regulatory mechanisms by their lack of specificity towards PKC. One of the most potent PKC inhibitors recently described is the microbial alkaloid staurosporine, with a Ki of 2.7 nM [6]. Endogenous sphingosine and lysosphingolipids may play a role in cellular regulation and have been proposed to act as negative effectors of PKC [7].There have been few reports of mammalian proteins that have been shown to have potent inhibitory activity towards PKC. We recently described the isolation and characterization of a PKC inhibitor protein from sheep brain named protein Correspondence to A. Aitken, Laboratory of Protein Structure,

The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family.

“…At the nucleotide level, there are only 14 differences between this human PLA2 and the bovine FAS genes within the coding region. Similarly, the published peptide sequences of ; isoform from sheep and rat are identical (26), and these isoforms differ from the deduced bovine FAS sequence only at two positions (Ala-109---Arg and Ala-112--Pro, double-underlined in Fig. 1).…”

Identification of a novel interaction between integrin β1 and 14-3-3β

“…For example, binding of 14-3-3b with signaling molecules such as PI-3K (Bonnefoy-Berard et al, 1995), PKC (Morgan and Burgoyne, 1992;Toker et al, 1992), and Raf (Fantl et al, 1994;Freed et al, 1994;Fu et al, 1994;Irie et al, 1994) might alter their activities and/or subcellular localizations. Signaling pathways from these molecules could then modulate integrin a nity resulting in increased cell spreading and migration.…”

Antibodies against the major brain isofbrms of 14‐3‐3 protein

“…Lower case letters in the a and S sequences indicate tentative assignments. The sequences are from references [2,3,4,8,10,18,19,20,29,30]. AC denotes N-a&y1 group and the threonine suggested as the first residue in the B sequence is shown in italics.…”

“…14-3-3 proteins all have a p1 of around 4.5 and M, of 30,000 Da on SDS-PAGE but 60,000 on size exclusion chromatography [2]. There are seven to eight mammalian brain isoforms of 14-3-3, named cl_r after their respective elution positions on HPLC [3].…”

“…Biochem., in press). The studies of our own group have focussed on 14-3-3 as a anase C Inhibitor Protein [2,6] that we named KCIP-1. A --14-3-3 protein member(s) called EXOl stimulates calcium dependent exocytosis in permeabilized adrenal medulla cells [7].…”