J Biochem 1996 Apr 01;1194:626-32.

Phosphorylated sites of M(r) 25,000 protein, a putative protein phosphatase 2A modulator, and phosphorylation of the synthetic peptide containing these sites by protein kinase C.

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The M(r) 25,000 protein isolated from Xenopus laevis oocytes was shown to be an effective phosphate acceptor for Ca(2+)-phospholipid-dependent protein kinase (protein kinase C) [Hashimoto, E. et al. (1995) J. Biochem. 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 muM). A stimulatory effect of M(r) 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 M(r) 25,000 protein.

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Species referenced: Xenopus laevis
Genes referenced: prss1 pth ptpa tbx2