Eur. J. Biochem. 206,453-461 (1992) zyxwvutsrq 0 FEBS 1992 zyxwvutsrqpo Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain Amino acid sequence of phosphorylated forms Alex TOKER, Lynda A. SELLERS, Bob AMESS, Yasmina PATEL, Alan HARRIS and Alastair AITKEN Laboratory of Protein Structure, National Institute for Medical Research, Mill Hill, London, England (Received December 27, 1991/March 3, 1992) zyxwvutsr - EJB 91 1729 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 zyxw in zyxw vitro, but none were phosphorylated by the other protein kinases mentioned above. Protein kinase C (PKC), a calcium and phospholipid-de- pendent 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 substi- tute 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, National Institute for Medical Research, Mill Hill, London NW7 IAA, England Fax: f44819064477. Abbreviations. BNPS-skatole, 3-bromo-3-methyl-2-(2-nitro- phenylmercapto)-3H-indole; KCIP-1, protein kinase C inhibitor pro- tein-I ; PKC, protein kinase C; PKM, catalytic fragment of protein kinase C; FAB-MS, fast-atom-bombardment mass spectrometry. Enzymes. Protein kinases (EC 2.7.1.37); trypsin (EC 3.4.21.4). Note. The novel amino acid sequence data published here have been submitted to the EMBL sequence data bank(s). kinase C inhibitor protein-1 (KCIP-1) [8]. This inhibitory activity (median inhibitory concentration 1.7 pM for a mono- mer of 30 kDa) consisted of several charge forms for each of three molecular mass species (29, 30 and 33 kDa), as detected by 12.5% SDS/PAGE. The amino acid sequence of KCIP-1 has no similarity to the equally potent 17-kDa PKC inhibitor protein (termed PKC inhibitor-I) [9, 101 nor that of the 12- kDa protein referred to as PKC inhibitor-2 [lo]. This 12-Da PKC inhibitor was thought to be identical to a peptidylprolyl cis-trans isomerase [l 11, but recently it has become evident that they are in fact distinct proteins [12]. Consistent with PKC inhibitor-1, the inhibitory mechanism of KCIP-1 does not appear to involve competition with PKC cofactors (cal- cium, phosphatidylserine or diacylglycerol), its substrates or ATP. We have also shown that KCIP-1 will inhibit phos- phorylation of a range of PKC substrates, including a syn- thetic peptide corresponding to the site of phosphorylation on the 80-kDa MARCKS protein [13]. A possible mechanism for KCIP-1 has come from analysis of its primary structure, which reveals a sequence reminiscent of part of the pseudosub- strate domain of PKC [14] which is GALR in a, B and zy y iso- forms of PKC and GARR in ( PKC. The pseudosubstrate hypothesis has been proposed to account for the inhibitory sequences in the regulatory domains of a wide range of pro- tein kinases, including CAMP-dependent protein kinase, the multifunctional Ca2+/calmodulin-dependent protein kinase and myosin light-chain kinase [15]. However, in KCIP-1, the lack of basic residues at the N-terminus of the motif is no- table. Database searches of the partial amino acid sequence of KCIP-1 showed a strong similarity with that of a bovine neuronal protein termed 14-3-3 [16]. This protein has been shown to be an activator of tyrosine and tryptophan hy- droxylases, the rate-limiting enzymes involved in the synthesis of dopamine and serotonin [17, 181. Although 14-3-3 is a major protein in mammalian brain, the tissue distribution of