Molecular cloning and characterization of a novel human protein phosphatase, LMW-DSP3

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Abstract

Reversible phosphorylation is recognized to be a major mechanism for the control of intracellular events in eukaryotic cells. From a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel dual specificity protein phosphatase, which showed 88% identity with previously reported mouse LMW-DSP3 at the amino acid level. The deduced protein had a single dual-specificity phosphatase catalytic domain, and lacked a cdc25 homology domain. LMW-DSP3 was expressed in the heart, lung, liver, and pancreas, and the expression level in the pancreas was highest. The LMW-DSP3 gene was located in human chromosome 2q32, and consisted of five exons spanning 21 kb of human genomic DNA. LMW-DSP3 fused to GST showed phosphatase activity towards p-nitrophenyl phosphate which was optimal at pH 7.0 and 40 °C, and the activity was enhanced by Ca2+ and Mn2+. The phosphatase activity of LMW-DSP3 was inhibited by orthovanate. LMW-DSP3 showed phosphatase activity toward oligopeptides containing pSer/Thr and pTyr, indicating that LMW-DSP3 is a protein phosphatase with dual substrate specificity.

Introduction

The mitogen activated protein kinase (MAPK) family comprises the extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SPAK), and p38/RK/CSBP(p38) as three structurally and functionally distinct enzyme classes [1], [2], [3]. ERK family members are activated by a variety of growth and differentiation factors, while the JNK/SAPK and p38 are activated preferentially by cellular stresses and inflammatory cytokines. MAPKs display a high level of evolutionary conservation and are essential for many cell functions. The activation of the MAPK cascade plays a key role in transducing various extracellular signals to the nucleus to induce responses such as gene expression, cell proliferation, differentiation, cell cycle, arrest, and apoptosis [2], [4].

Full MAPK activation needs phosphorylation on both tyrosine and threonine residues by selective upstream dual specificity kinases. Protein phosphorylation is a reversible process, and dephosphorylation by specific phosphatases may also play a critical role. Among the large number of protein tyrosine phosphatases, a new class of dual-specificity phosphatases (DSPs) has been recognized as mediating direct and specific inactivation of MAPK activity. The prototypical DSP is the VH1 gene identified as an open reading frame in vaccinia virus expressed in late stage viral infection [5]. The first mammalian DSP was identified as the mouse immediate early gene 3CH134 or its human ortholugue CL100 [6], [7], [8]. More than 10 DSP family members have been reported [9]. During our large-scale sequencing project, we identified a gene encoding a novel human DSP protein, which showed 88% identity to a mouse LMW-DSP3 at the amino acid level. It had a single DSP catalytic domain but lacked the cdc25 homology domain. It had a low molecular weight, so we termed this gene human LMW-DSP3.

Section snippets

cDNA library construction

A cDNA library was constructed in the modified pBluescript II SK(+) vector with human fetal brain mRNA purchased from Clontech. The modified pBluescipt II SK(+) vector was constructed by inserting a DNA fragment with SfiIA (5′-GGCCATTATGGCC-3′) and SfiIB (5′-GGCCGCCTCGGCC-3′) recognition sites into the EcoRI and NotI sites of pBluescript II SK(+) (Stratagene). Double-strand cDNA was synthesized using a SMART™ cDNA Library Construction Kit (Clontech) following the manufacturer’s instruction.

Identification of the Human LMW-DSP3 cDNA

The human LMW-DSP3 cDNA was cloned from human fetal brain by large-scale cDNA sequencing. The 1436 bp cDNA spanned an open reading frame from 142 to 795, encoding a putative protein with a predicted molecular mass of 24.2 kDa (Fig. 1). In a search against the GenBank™ database, the protein identified here was found to be 88% identical to the mouse LMW-DSP3 protein [11] (Fig. 2). The putative protein contained a single DSP catalytic domain but lacked the cdc25 homology domain, which is conserved

Discussion

DSPs are an emerging subclass of the protein tyrosein phosphatase family, which appears to be selective for dephosphorylating the critical phosphothreonine and phosphotyrosine within MAPKs [13]. DSPs differ in terms of substrate specificity, tissue distribution, subcelluar localization, and target gene regulation, all of which allow fine regulation of DSP by extracellular stimuli [14]. DSPs are regulated by hormone and growth factors [15], [16]. Some DSPs have been found to be restricted to a

Acknowledgements

This research was funded by a grant from the Natural Science Foundation of China (30070383).

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The nucleotide sequence reported in this paper has been submitted to the Genbank/EMBL Database with accession number AF486808.

1

Co-corresponding author.

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