The 14‐3‐3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14‐3‐3 proteins are cytosolic, a small but significant proportion of 14‐3‐3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform‐specific antisera we find that the ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.

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,

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The 14-3-3 protein family has received considerable attention recently in the literature, because of the finding that beta and zeta isoforms interact with and activate Raf. We had previously shown that these 14-3-3 isoforms also exist as phosphorylated forms in mammalian and avian brain. The presence of this modification enhances the activity of 14-3-3 as an inhibitor of protein kinase C nearly 2-fold. In this report we show by a combination of electrospray mass spectrometry and protein microsequencing that alpha and delta are in vivo post-translationally modified forms of beta and zeta, respectively, and the site of phosphorylation, serine 185, is in a consensus sequence motif for proline-directed kinases.

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