title: Characterization of PEA-15, a major substrate for protein kinase C in astrocytes.
abstract: Astrocytes in the central nervous system are involved in a variety of functions including storage of glycogen, maintenance of the extracellular ionic equilibrium, and support for the migration and the differentiation of neurons. Astrocytes express membrane receptors allowing them to respond to extracellular signals. Activation of receptors induces a cascade of events, such as stimulation of protein kinases and subsequent phosphorylation of target proteins. To understand the regulatory processes underlying neuroglial interactions, attempts were made to identify major phosphorylated proteins in striatal astrocytes, grown in primary culture and labeled with [32P]phosphate. Two-dimensional gel electrophoresis revealed a major doublet, Pa and Pb, of highly labeled spots, with a low molecular weight (M(r) = 15,000) and acidic pI (pI = 5.2 and 5.3, respectively). Using an enriched, heat-stable, cytosolic fraction, Pa and Pb were eluted from semi-preparative two-dimensional gels and subjected to a limited proteolysis and partial microsequencing. The same sequences were obtained within Pa and Pb and had no homology with other known protein. Antibodies raised against corresponding synthetic peptides confirm that the doublet represents two isoelectric variants of the same protein, which also exists under a nonphosphorylated form, N. We propose to name this protein PEA-15, for Phosphoprotein Enriched in Astrocytes-15 kDa, according to its large enrichment in these cells. Treatment of intact astrocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulates protein kinase C (PKC), increased the phosphorylation of the more acidic spot (Pb) while decreasing Pa intensity. Stimulations of astrocytes known to increase PKC activity, i.e. noradrenaline, or its inhibition by decreasing extracellular calcium concentrations, staurosporine, or desensitization following long term treatment with TPA, induced a phosphorylation or a dephosphorylation of PEA-15, respectively. Using purified PKC, PEA-15 appeared to be a good substrate in vitro. Two-dimensional peptide mapping revealed that the phosphorylation site in intact cells was identical with the site phosphorylated by PKC in vitro. Mapping the phosphopeptides by HPLC following endolysine C treatment lead to the identification of a sequence, phosphorylated in intact astrocytes and in vitro by PKC, containing a consensus site for PKC: LTRIPSAKK. Antibodies raised against a synthetic peptide derived from this sequence recognized N and Pa in control conditions and Pb after its dephosphorylation. Thus, PEA-15 is an endogenous substrate for PKC, the kinase mediating the transition from Pa to Pb.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, U114 Chaire de Neuropharmacologie, Collège de France, Paris.
authors.forename: H
authors.initials: H
authors.lastname: Araujo
authors.forename: N
authors.initials: N
authors.lastname: Danziger
authors.forename: J
authors.initials: J
authors.lastname: Cordier
authors.forename: J
authors.initials: J
authors.lastname: Glowinski
authors.forename: H
authors.initials: H
authors.lastname: Chneiweiss
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011493:Protein Kinase C