title: The presence of phorbol ester responsive and non-responsive forms of the zeta isozyme of protein kinase C in mouse epidermal cells.
abstract: The possible involvement of zeta isozyme of protein kinase C (PKC zeta) in phorbol ester-induced signal transduction was investigated in mouse epidermal cells. Western blot analysis of RESOURCE Q column chromatography eluates obtained from 105,000 g supernatants and particulate fractions of epidermal cells was performed using anti-PKC zeta specific antibody. Anti-PKC zeta antibody recognised proteins in low salt range corresponding to 25-125 mM NaCl (low salt-eluted PKC zeta; 1-PKC zeta) as well as high salt range corresponding to 175-300 mM NaCl (high salt-eluted PKC zeta; h-PKC zeta) in both subcellular fractions. 1-PKC zeta and h-PKC zeta were detected as a doublet protein of 79,000 and 85,000 M(r) in 105,000 g supernatants, but as a 79,000 M(r) protein in particulate fractions. Immunoprecipitated 1-PKC zeta and h-PKC zeta with anti-PKC zeta specific antibody possessed phosphatidylserine (PS)-dependent protein kinase activity, but neither 1-PKC zeta nor h-PKC zeta were further activated by 40 nM phorbol 12-myristate 13-acetate (PMA) in the presence of PS. Furthermore, 1-PKC zeta and h-PKC zeta can be autophosphorylated, indicating that both 1-PKC zeta and h-PKC zeta are PKC zeta. Treatment of intact epidermal cells with PMA or other PKC activators caused the apparent shift of 79,000 M(r) 1-PKC zeta to the 85,000 M(r) from in particulate fractions. Prolonged treatment of the cells with PMA induced the downregulation of both forms of 1-PKC zeta in particulate fractions. Under the same condition, 1-PKC zeta in 105,000 g supernatants and h-PKC zeta in both fractions did not respond to PMA. This apparent shift was reversible and the content ratio of 85,000 to 75,000 M(r) 1-PKC zeta was decreased by acid phosphatase treatment, indicating that the apparent shift results at least in part from phosphorylation of 79,000 M(r) 1-PKC zeta. Total activity of 1-PKC zeta was increased in association with the apparent shift from the 79,000 to 85,000 M(r) form in response to PMA treatment of intact epidermal cells. All of these results indicate that PKC zeta is present as multiple forms in mouse epidermal cells, and that especially 1-PKC zeta in particulate fractions play a significant role(s) in PMA-induced signal transduction in mouse epidermal cells.
authors.affiliation: Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Nishikawa
authors.forename: S
authors.initials: S
authors.lastname: Yamamoto
authors.forename: H
authors.initials: H
authors.lastname: Nagumo
authors.forename: K
authors.initials: K
authors.lastname: Maruyama
authors.forename: R
authors.initials: R
authors.lastname: Kato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects / Q000201:enzymology*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D007527:Isoenzymes; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate