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

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.

Developmental regulation and extracellular release of a VSG expression-site-associated gene product from Trypanosoma brucei bloodstream forms

“…Searching available sequence databases for proteins that share this motif identified a related sequence conserved among members of the protein phosphatase PP2C family (supplementary material Fig. S3), these residues being required for Mn 2+ -binding and functional activity of these enzymes (Barford et al, 1998;Kusuda et al, 1998). Similarity to this conserved motif was also observed in members of the SRCR family of scavenger receptors.…”

Regulation of the TAK1 Signaling Pathway by Protein Phosphatase 2C

“…For bacterial expression of proteins, cDNAs encoding the proteins were subcloned into pGEX (Amersham Pharmacia Biotech) to generate glutathione S-transferase (GST) fusion proteins or into pQE31 (Qiagen, Hilden, Germany) to generate hexahistidinetagged protein and affinity-purified by standard procedures. Other expression plasmids were as described elsewhere (23,24) Cell Culture and Transfection-COS7, 293, and 293IL-1RI (25) cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum. At 50 -80% confluency the cells were transfected by the DEAE-dextran method or using LipofectAMINE (Life Technologies, Inc.).…”

PPM1H Is a p27 Phosphatase Implicated in Trastuzumab Resistance

“…After washing 3 times to remove the CDK2/cyclin A, the phosphorylated p27 was subsequently incubated with FLAG-PPM1H or FLAG-PPM1H-H153L in ATP-free buffer. The H153L mutant was predicted to disrupt enzyme activity based on a prior mutational analysis of murine PPM1B ( 32 ). Indeed, the PPM1H-H153L mutant Table 1.…”