Low Molecular Weight Dual Specificity Phosphatase Gene Of Lmw-dsp2, Lmw-dsp3 The Cloning And Functional Studies

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 two cDNA clones encoding two novel dual specificity protein phosphatases. One is LMW-DSP2, which contained 1037bp. The putative protein contained 184 amino acids and a dual specificity phosphatase catalytic domain. The molecular weight was 20.9 kDa, and pI was 8.09. LMW-DSP2 showed 96.7% indentity with previously reported mouse LMW-DSP2. The gene was located in 6p22, which was related with leukemia, adenocarcinoma, multiple myeloma, Lymphoplasmacytic lymphoma and mesothelioma. LMW-DSP2 contained 8 extrons and 7 introns, spanning 58kb of human genomic DNA. The homologous ESTs were detected in the placenta, lung, brain, pancreas, testis, marrow, skin, uterus, and breast. Northern blot showed LMW-DSP2 was expressed in the heart, muscle, and pancreas. Three transcripts were detected: 1.8kb, 2.0kb and 4.0kb. The 4.0kb transcript was expressed in the heart, skeletal muscle and pancreas. 1.8kb transcript was expressed in the heart and pancreas. The 2.0kb transcript was expressed in the skeletal muscle. SAGE result showed LMW-DSP2 was expressed in many tumor cells and tissues. The GST-LMW-DSP2 fusion protein showed phosphatase activity towards p-nitrophenyl phosphate which was optimal at pH 7.0 and 37 ℃. The phosphatase activity of LMW-DSP2 was inhibited by orthovanate. Cys88 and Asp57 were conserved positions, whose mutations lead to dramatic loss of phosphatase activity. LMW-DSP2 showed phosphatase activity toward oligopeptides containing pSer/Thr and pTyr, indicating that LMW-DSP2 is a protein phosphatase with dual substrate specificity. Yeast two-hybrid screen showed LMW-DSP2 interacted with RAB11A. Using the GFP fusion expression technique, we identified the sub-cellular localization of the product of the gene,LMW-DSP2 was expressed in cytoplasm and nucleolus. It simulated the phosphorylation of JNK.The other is LMW-DSP3, which showed 84% identity with mouseLMW-DSP3 at the amino acid level. The deduced protein had a single dual-specificity phosphatase catalytic domain, and lacked a cdc25 homology domain. The molecular weight was 24.2 kDa, and pI was 6.36. LMW-DSP3 was expressed in the heart, lung, liver, and pancreas, and the expression level in the pancreas was highest. The gene was expressed in L0-2 and SMMC-7721 cell lines. The LMW-DSP3 gene was located in human chromosome 2q32, which was related with multiple myeloma,refractory anemia,diffuse large B-cell lymphoma and leukemia. It was consisted of 5 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 ℃, and the activity was enhanced by Ca2+ and Mn2+. The phosphatase activity of LMW-DSP3 was inhibited by orthovanate. Mutant LMW-DSP3 (C150S or D119A) showed lower phosphatase activity. LMW-DSP3 showed phosphatase activity toward oligopeptides containing pSer/Thr and pTyr, indicating that LMW-DSP3 is a protein phosphatase with dual substrate specificity. GFP-LMW-DSP3 fusion protein was expressed in cytoplasm. LMW-DSP3 could inactivate the activity of JNK, which suggested it was a novel MAPK phosphatase.