J. Biochem, 1996, Vol. 119, No. 4 626-632
© 1996 Japanese Biochemical Society
research-article |
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 C1
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*Department of Pathological Biochemistry, School of Life Sciences, Faculty of Medicine, Tottori University Nishimachi 86, Yonago 683;
Central Research Laboratories, Fukui Medical School Matsuoka, Fukui 910-11
Department of Biochemistry, Fukui Medical School Matsuoka, Fukui 910-11
Biosignal Research Center, Kobe University Kobe 657
2 To whom correspondence should be addressed.
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.
1 This work was supported in part by Grants-in-Aid for General Scientific Research, for Scientific Research on Priority Areas, and for Co-operative Research from the Ministry of Education, Science and Culture of Japan.
3Present address: Department of Biochemistry, Kobe UniversitySchool of Medicine, Kobe 650.