Alternative promoters direct tissue‐specific expression of the mouse protein phosphatase 2Cβ gene

Abstract

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...

Protein Phosphatases

“…Analysis of PP2CP transcripts showed that exon-1 transcripts were expressed ubiquitously in several tissues, whereas exon-2 transcripts were predominantly expressed in the testis, intestine and liver. These results suggested that the alternative usage of two promoters within the PP2CP gene regulates tissue-specific expression of PP2CP mRNA (Ohnishi et al, 1999).…”

Serine/Threonine Phosphatases: Classification, Roles and Pharmacological Regulation

“…As PP2C exists as multiple isoforms this allows for tissue specific expression (53). PP2C isoforms are broadly expressed but most abundant in heart and skeletal muscle (54). PP2C is insensitive to okadaic acid and other naturally-occurring protein phosphatase inhibitors and the lack of pharmacological agents able to modulate PP2C activity has hindered the understanding of its character in regulating cell function.…”

Wnt-16a, a Novel Wnt-16 Isoform, Which Shows Differential Expression in Adult Human Tissues

“…There are several precedents in the literature to support this, Van den Wijngaard et al (14) described two promoters involved in the transcriptional regulation of the human BMP-4 gene, one upstream of exon 1, the second located in intron 1, upstream of exon 2, giving rise to different transcripts in a cell type and differentiation-dependent manner. In another example, Ohnishi et al (15) isolated a type 2C protein phosphatase (PP2Cbeta) with different 5Ј termini. Subsequent analysis of the 5Ј flanking regions of exon 1 and 2 by reporter gene showed that these regions acted as distinct promoters.…”

“…Subsequent analysis of the 5Ј flanking regions of exon 1 and 2 by reporter gene showed that these regions acted as distinct promoters. Analysis of PP2Cbeta transcripts by RT-PCR showed that exon-1 and exon-2 derived transcripts were differentially expressed, suggesting that alternative promoter usage regulated tissue-specific expression of PP2Cbeta (15).…”