MUTATIONAL ANALYSIS OF THE DOMAIN-STRUCTURE OF MOUSE PROTEIN PHOSPHATASE 2C-BETA

The structures of five distinct isoforms of mammalian protein phosphat ase 2C beta (PP2C beta-1, -2, -3, -4 and -5) have previously been foun d to differ only at their C-terminal regions. In the present study, we performed mutational analysis of recombinant mouse PP2C beta-1 to det ermine the functional domains of the molecule and elucidate the bioche mical significance of the structural differences in the isoforms. Diff erences in affinity for [P-32]phosphohistone but not for [P-32]phospho casein were observed among the five PP2C beta isoforms. Deletion of 12 amino acids from the C-terminal end, which form a unique sequence for PP2C beta-1, caused a 35% loss of activity against [P-32]phosphohisto ne but no loss of activity against [P-32]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 th e activity against both [P-32]phosphohistone and [P-32]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 t otal loss of activity. Substitution of any of the six specific amino a cids among 16 tested in this study, which were located among the 250 N -terminal residues, caused 98-100% loss of enzyme activity. Among thes e amino acids, three (Glu-38, -60 and -243) have recently been reporte d 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 PP2C beta is composed of at least two distinct functional domains, an N-terminal catalytic dom ain of about 310 amino acids and the remaining C-terminal domain, whic h is involved in determination of substrate specificity.