Heterologous expression and catalytic properties of the C-terminal domain of starfish cdc25 dual-specificity phosphatase, a cell cycle regulator.
The 3'-terminal region of starfish Asterina pectinifera cdc25 cDNA encoding the C-terminal catalytic domain was overexpressed in Escherichia coli. The C-terminal domain consisted of 226 amino acid residues containing the signature motif HCxxxxxR, a motif highly conserved among protein tyrosine and dual-specificity phosphatases, and showed phosphatase activity toward p-nitrophenyl phosphate. The enzyme activity was strongly inhibited by SH inhibitors. Mutational studies indicated that the cysteine and arginine residues in the conserved motif are essential for activity, but the histidine residue is not. These results suggest that the enzyme catalyzes the reaction through a two-step mechanism involving a phosphocysteine intermediate like in the cases of other protein tyrosine and dual-specificity phosphatases. The C-terminal domain of Cdc25 activated the histone H1 kinase activity of the purified, inactive form of Cdc2.cyclin B complex (preMPF) from extracts of immature starfish oocytes. Synthetic diphosphorylated di- to nonadecapeptides mimicking amino acid sequences around the dephosphorylation site of Cdc2 still retained substrate activity. Phosphotyrosine and phosphothreonine underwent dephosphorylation in this order. This is the reverse order to that reported for the in vivo and in vitro dephosphorylation of preMPF. Monophosphopeptides having the same sequence served as much poorer substrates. As judged from the results with synthetic phosphopeptides, the presence of two phosphorylated residues was important for specific recognition of substrates by the Cdc25 phosphatase.
Deshimaru
Shungo
S
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan.
Miyake
Yasuo
Y
Ohmiya
Tadamasa
T
Tatsu
Yoshiro
Y
Endo
Yasuko
Y
Yumoto
Noboru
N
Toraya
Tetsuo
T
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D020134:Catalytic Domain; D002453:Cell Cycle / Q000502:physiology; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013215:Starfish / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010748:Phosphopeptides; D011994:Recombinant Proteins; C008644:nitrophenylphosphate; D001120:Arginine; D011494:Protein Kinases; C102333:histone H1 kinase; D020687:cdc25 Phosphatases; D003545:Cysteine