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 [Das, Helps, Cohen and Barford (1996) EMBO J. 15, 6798–6809]. 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.
Mutational analysis of the domain structure of mouse protein phosphatase 2Cβ
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Kazuyuki KUSUDA, Takayasu KOBAYASHI, Shoko IKEDA, Motoko OHNISHI, Naoki CHIDA, Yuchio YANAGAWA, Ryuzaburo SHINEHA, Tetsuro NISHIHIRA, Susumu SATOMI, Akira HIRAGA, Shinri TAMURA; Mutational analysis of the domain structure of mouse protein phosphatase 2Cβ. Biochem J 15 May 1998; 332 (1): 243–250. doi: https://doi.org/10.1042/bj3320243
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