There is accumulating evidence that glial cells actively modulate neuronal synaptic transmission. We identified a glia-derived soluble acetylcholine-binding protein (AChBP), which is a naturally occurring analogue of the ligand-binding domains of the nicotinic acetylcholine receptors (nAChRs). Like the nAChRs, it assembles into a homopentamer with ligand-binding characteristics that are typical for a nicotinic receptor; unlike the nAChRs, however, it lacks the domains to form a transmembrane ion channel. Presynaptic release of acetylcholine induces the secretion of AChBP through the glial secretory pathway. We describe a molecular and cellular mechanism by which glial cells release AChBP in the synaptic cleft, and propose a model for how they actively regulate cholinergic transmission between neurons in the central nervous system.

Phosphatidic acid (PA) is produced rapidly in agonist-stimulated cells, but the physiological function of this PA is unknown. We have examined the effects of PA on distinct isoforms of protein kinase C (PKC) using a new cell-free assay system. Addition of PA to cytosol from COS cells overexpressing PKC-alpha, -epsilon or -zeta differentially-activated all three isotypes, as shown by PKC autophosphorylation, and prominent phosphorylation of multiple endogenous substrates. In the absence of Ca2+, the diacylglycerol-insensitive zeta-isotype of PKC was most strongly activated by both PA and bisPA, a newly identified product of activated phospholipase D, with each lipid inducing its own profile of protein phosphorylation. BisPA was also a strong activator of PKC-epsilon, but a weak activator of PKC-alpha. Ca2+, at > or = 0.1 microM, inhibited PA and bisPA activation of PKC-zeta, but did not affect PKC-epsilon activation. In contrast, PKC-alpha was strongly activated by PA only in the presence of Ca2+. BisPA-induced phosphorylations mediated by PKC-zeta could be mimicked in part by other acidic phospholipids and unsaturated fatty acids. PA activation of PKC-zeta was unique in that PA not only stimulated PKC-zeta-mediated phosphorylation of distinctive substrates, but also caused an upward shift in electrophoretic mobility of PKC-zeta, which was not observed with other acidic lipids or with PKC-alpha or -epsilon. We have presented evidence that this mobility shift is not caused by PKC-zeta autophosphorylation, but it coincides with physical binding of PA to PKC-zeta. These results suggest that in cells stimulated under conditions where intracellular Ca2+ is at (or has returned to) basal level, PA may be a physiological activator of PKC-zeta.

PKC-e was isolated from a murine brain eDNA library. The clone, 261PKC-e, encoded a polypeptide of 737 amino acids that is homologous to other PKCs. Northern analysis showed that the 7 kb mRNA for this eDNA is widely expressed. The protein when expressed in COS-1 cells displayed phorbol ester-binding activity. However in order to detect the kinase activity of PKC-e, it was necessary to employ a synthetic peptide substrate based upon the pseudosubstrate site. Subsequent analysis demonstrated that PKC-e, while showing certain properties characteristic of the PKC family, has a quite distinct substrate specificity and is independent of Ca 2 +.

Diacylglycerol kinases are involved in cell signaling, either as regulators of diacylglycerol levels or as intracellular signal‐generating enzymes. However, neither their role in signal transduction nor their biochemical regulation has been elucidated. Hepatocyte growth factor (HGF), upon binding to its tyrosine kinase receptor, activates multiple signaling pathways stimulating cell motility, scattering, proliferation and branching morphogenesis. Herein we demonstrate that: (i) the enzymatic activity of α‐diacylglycerol kinase (αDgk) is stimulated by HGF in epithelial, endothelial and αDgk‐transfected COS cells; (ii) cellular expression of an αDgk kinase‐defective mutant inhibits activation of endogenous αDgk acting as dominant negative; (iii) specific inhibition of αDgk prevents HGF‐induced cell movement of endothelial cells; (iv) HGF induces the association of αDgk in a complex with Src, whose tyrosine kinase activity is required for αDgk activation by HGF; (v) Src wild type stimulates αDgk activity in vitro; and (vi) αDgk can be tyrosine phosphorylated in intact cells.

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Diacylglycerol kinase (DGK) attenuates levels of second messenger diacylglycerol in cells and produces another (putative) messenger, phosphatidic acid. We have previously purified a 110-kDa DGK from rat brain (Kato, M., and Takenawa, T. (1990) J. Biol. Chem. 265, 794 -800). Here we report the cDNA cloning from human brain and retina cDNA libraries. The cDNA encodes a novel DGK isotype, termed DGK, of 941 amino acids with an apparent molecular mass of 110 kDa. DGK contains a C-terminal putative catalytic domain, which is present in all eukaryotic DGKs. In contrast to other DGK isotypes, DGK contains three cysteine-rich domains instead of two. The third cysteine-rich domain is most homologous to the second one in other DGK isotypes. This particular sequence homology extends C-terminally beyond the typical cysteine/histidine core structure and is DGKspecific. DGK furthermore contains various domains for protein-protein interaction, such as a proline-and glycine-rich domain with a putative SH3 domain-binding site and a pleckstrin homology domain with an overlapping Ras-associating domain. DGK is expressed in the brain and, to a lesser extent, in the small intestine, duodenum, and liver. In situ hybridization of DGK mRNA in adult rat brain reveals high expression in the cerebellar cortex and hippocampus. DGK activity in COS cell lysates is optimal toward diacylglycerols containing an unsaturated fatty acid at the sn-2 position.

Diacylglycerol (DG) kinase attenuates the level of the second messenger DG in signal transduction, and therefore possibly modulates protein kinase C (PKC). DG kinase was purified to homogeneity from human white blood cells, showing an M 1 of 86 kDa as determined by SDS‐PAGE and gel filtration. Two amino acid sequences of tryptic peptides from DG kinase were determined and degenerate oligonucleotides were prepared and used in the polymerase chain reaction. An amplified DNA fragment was subsequently used to clone the full‐length human DG kinase cDNA. This sequence is the human homolog of porcine DG kinase cDNA sequence reported recently [1]. The sequence contains a double EF‐ hand structure typical for Ca2+ binding proteins. DG kinase further contains a double cysteine repeat that is present in all PKC isoforms, where it constitutes the phorbol ester (and most likely diacylglycerol) binding site. Therefore we speculate that the double cysteine repeat in DG kinase is involved in DG binding. DG kinase is transcribed as a single mRNA of 3.2 kb, that is highly expressed in T‐lymphocytes. The human DG kinase cDNA when transfected in mammalian cells (COS‐7) results in a 6–7‐fold increase of DG kinase activity.

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].