Mutational analysis of the domain structure of mouse protein phosphatase 2Cβ

Kazuyuki Kusuda, Takayasu Kobayashi, Shoko Ikeda, Motoko Ohnishi, Naoki Chida, Yuchio Yanagawa, Ryuzaburo Shineha, Tetsuro Nishihira, Susumu Satomi, Akira Hiraga, Shinri Tamura

研究成果: Article査読

41 被引用数 (Scopus)

抄録

The structures of five distinct isoforms of mammalian protein phosphatase 2Cβ (PP2Cβ-1, -2, -3, -4 and -5) have previously been found to differ only at their C-terminal regions. In the present study, we performed mutational analysis of recombinant mouse PP2Cβ-1 to determine the functional domains of the molecule and elucidate the biochemical significance of the structural differences in the isoforms. Differences in affinity for [32P]phosphohistone but not for [32P]phosphocasein were observed among the five PP2Cβ isoforms. Deletion of 12 amino acids from the C-terminal end, which form a unique sequence for PP2Cβ-1, caused a 35% loss of activity against [32P]phosphohistone but no loss of activity against [32P]phosphocasein. Deletion of up to 78 amino acids from this end did not cause any further alteration in activity, whereas deletion of 100 amino acids totally eliminated the activity against both [32P]phosphohistone and [32P]phosphocasein. On the other hand, deletion of 11 amino acids from the N-terminal end caused a 97% loss of enzyme activity, and further deletions caused a total loss of activity. Substitution of any of the six specific amino acids among 16 tested in this study, which were located among the 250 N-terminal residues, caused 98-100%, loss of enzyme activity. Among these amino acids, three (Glu-38, -60 and -243) have recently been reported to be essential for the binding of metal ions in the catalytic site of the PP2C molecule. These observations indicate that PP2Cβ is composed of at least two distinct functional domains, an N-terminal catalytic domain of about 310 amino acids and the remaining C-terminal domain, which is involved in determination of substrate specificity.

本文言語English
ページ(範囲)243-250
ページ数8
ジャーナルBiochemical Journal
332
1
DOI
出版ステータスPublished - 1998 5 15

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

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