title: Mutational analysis of the domain structure of mouse protein phosphatase 2Cbeta.
abstract: The structures of five distinct isoforms of mammalian protein phosphatase 2Cbeta (PP2Cbeta-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 PP2Cbeta-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 PP2Cbeta isoforms. Deletion of 12 amino acids from the C-terminal end, which form a unique sequence for PP2Cbeta-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 PP2Cbeta 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.
authors.affiliation: Department of Biochemistry, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seryomachi, Aoba-ku, Sendai 980, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kusuda
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: S
authors.initials: S
authors.lastname: Ikeda
authors.forename: M
authors.initials: M
authors.lastname: Ohnishi
authors.forename: N
authors.initials: N
authors.lastname: Chida
authors.forename: Y
authors.initials: Y
authors.lastname: Yanagawa
authors.forename: R
authors.initials: R
authors.lastname: Shineha
authors.forename: T
authors.initials: T
authors.lastname: Nishihira
authors.forename: S
authors.initials: S
authors.lastname: Satomi
authors.forename: A
authors.initials: A
authors.lastname: Hiraga
authors.forename: S
authors.initials: S
authors.lastname: Tamura
mesh_terms: D000818:Animals; D019556:COS Cells / Q000166:cytology / Q000201:enzymology; D004252:DNA Mutational Analysis; D000939:Epitopes / Q000276:immunology; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics; D010750:Phosphoproteins / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000939:Epitopes; D007527:Isoenzymes; D010750:Phosphoproteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C087358:PTC1 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C