caa a22 4500 397557515 CHVBK 20180308164807.0 cr unu---uuuuu 161202e199604 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021288 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021288 Phosphorylated Sites of Mr 25,000 Protein, a Putative Protein Phosphatase 2A Modulator, and Phosphorylation of the Synthetic Peptide Containing These Sites by Protein Kinase C [Elektronische Daten] [Eikichi Hashimoto, Naoki Kobayashi, Norika Kubota, Yukie Tanaka, Hirohei Yamamur] The Mr 25,000 protein isolated from Xenopus laevis oocytes was shown to be an effective phosphate acceptor for Ca2+-phospholipid-dependent protein kinase (protein kinase C) [Hashimoto, E. et al. (1995) J. Biochenu 118,453-460]. In this study, the sites of this protein phosphorylated by protein kinase C were determined and the mechanism of substrate recognition was studied using a synthetic peptide containing the phosphorylation sites. After incorporation of about 2 mol of phosphate per mol of this protein, the radioactive protein was digested with trypsin and the phosphopeptides were purified by a series of column chromatographies. The amino acid sequence of the major radioactive peptide was shown to be Ser-Arg-Val-Ser-Lys-Arg. This and previous results suggest that the two serine residues at the amino-terminal region were phosphorylated by protein kinase C. To confirm this, the phosphorylated protein was directly analyzed for the amino acid sequence. The percent distribution of dithiothreitol adduct of the phenylthiohydantoin derivative of serine (PTH-serine) compared with that of PTH-serine increased at the first and fourth cycles of the sequence analysis. When the synthetic peptide composed of the amino-terminal eleven amino acids was employed as phosphate acceptor, the Km value was unexpectedly high (1.1 mM) compared with that of the native protein (0.5 μM). A stimulatory effect of Mr 25,000 protein on the activity of protein phosphatase 2A was further enhanced after phosphorylation by protein kinase C. These results suggest that the two serine residues recognized by protein kinase C may have some role in the regulation of this Mr 25,000 protein. © by the Journal of Biochemistry Regular Papers nationallicence phosphorylated site nationallicence protein kinase C nationallicence protein phosphatase 2A nationallicence synthetic peptide nationallicence Xenopus laevis oocytes nationallicence Hashimoto Eikichi Department of Pathological Biochemistry, School of Life Sciences, Faculty of Medicine, Tottori University Nishimachi 86, Yonago 683 aut Kobayashi Naoki Department of Pathological Biochemistry, School of Life Sciences, Faculty of Medicine, Tottori University Nishimachi 86, Yonago 683 aut Kubota Norika Department of Pathological Biochemistry, School of Life Sciences, Faculty of Medicine, Tottori University Nishimachi 86, Yonago 683 aut Tanaka Yukie Central Research Laboratories, Fukui Medical School Matsuoka, Fukui 910-11 aut Yamamur Hirohei Department of Biochemistry, Fukui Medical School Matsuoka, Fukui 910-11 aut The Journal of Biochemistry Oxford University Press 119/4(1996-04), 626-632 0021-924X 119:4<626 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021288 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021288 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hashimoto Eikichi Department of Pathological Biochemistry, School of Life Sciences, Faculty of Medicine, Tottori University Nishimachi 86, Yonago 683 aut NATIONALLICENCE 700 1- Kobayashi Naoki Department of Pathological Biochemistry, School of Life Sciences, Faculty of Medicine, Tottori University Nishimachi 86, Yonago 683 aut NATIONALLICENCE 700 1- Kubota Norika Department of Pathological Biochemistry, School of Life Sciences, Faculty of Medicine, Tottori University Nishimachi 86, Yonago 683 aut NATIONALLICENCE 700 1- Tanaka Yukie Central Research Laboratories, Fukui Medical School Matsuoka, Fukui 910-11 aut NATIONALLICENCE 700 1- Yamamur Hirohei Department of Biochemistry, Fukui Medical School Matsuoka, Fukui 910-11 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/4(1996-04), 626-632 0021-924X 119:4<626 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford