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.

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Type 2C protein phosphatases (PP2Cs), a class of ubiquitous and evolutionally conserved serine/threonine protein phosphatases, are encoded in at least four distinct genes and implicated in the regulation of various cellular functions. Of these four PP2C genes, the expression of the PP2Cb gene has been reported to be tissue-specific and development-dependent. To understand more precisely the regulatory mechanism of this expression, we have isolated and characterized overlapping mouse genomic l clones. A comparison of genomic sequences with PP2Cb cDNA sequences provided information on the structure and localization of intron/exon boundaries and indicated that PP2Cb isoforms with different 5 H termini were generated by alternative splicing of its pre-mRNA. The 5 H -flanking region of exon 1 had features characteristic of a housekeeping gene: it was GC-rich, lacked TATA boxes and CAAT boxes in the standard positions, and contained potential binding sites for the transcription factor SP1. In the 5 H -flanking region of exon 2, several consensus sequences were found, such as a TATA-like sequence and negative regulatory element box-1, -2 and -3. Subsequent analysis by transient transfection assay with a reporter gene showed that these regions act as distinct promoters. Analysis of PP2Cb transcripts by reverse transcriptase-PCR showed that exon-1 transcripts were expressed ubiquitously in all of the tissues examined, whereas exon-2 transcripts were predominantly expressed in the testis, intestine and liver. These results suggest that the alternative usage of two promoters within the PP2Cb gene regulates tissue-specific expression of PP2Cb mRNA.Keywords: alternative promoter; gene structure; mouse; protein phosphatase 2C; tissue-specific expression.The serine/threonine protein phosphatases can be divided into four major families (PP1, PP2A, PP2B and PP2C) based on their substrate specificity, metal ion requirements and sensitivity to inhibitors such as inhibitor-1 and -2 [1,2]. Of the four families of protein phosphatases, the PP2C family is the least well understood. It is unique because of the insensitivity of its members to okadaic acid and absolute requirement for Mg 2+ for their activity. PP2C proteins share little homology with other phosphatases in their primary structures and exist as free catalytic proteins, whereas other phosphatases are composed of catalytic and regulatory subunits. PP2C proteins are expressed by various organisms ranging from yeasts to higher eukaryotes. PP2C homologs in yeasts have been implicated in the regulation of the heat shock response, osmoregulation, tRNA splicing and cell proliferation [3±5]. The ABI1 gene product of Arabidopsis thaliana, which shares a high degree of sequence similarity with the PP2C proteins, appears to control an early step in the signaling pathway induced by the plant hormone abscisic acid [6,7]. The Caenorhabditis elegans sex-determining protein FEM-2 [8] is a protein phosphatase and is related in sequence to the PP2C proteins. Its phosphatase activity is neces...

These results suggest that elevated levels of serum soluble interleukin-2 receptor are likely to reflect activated T cells in the kidneys of patients with reflux nephropathy and may be a useful predictor of progression of renal injury in these children.