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

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 21kb 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 degrees C, and the activity was enhanced by Ca(2+) and Mn(2+). 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.

[1]  A. Ashworth,et al.  The Dual Specificity Phosphatases M3/6 and MKP-3 Are Highly Selective for Inactivation of Distinct Mitogen-activated Protein Kinases* , 1996, The Journal of Biological Chemistry.

[2]  J. Avruch,et al.  Sounding the Alarm: Protein Kinase Cascades Activated by Stress and Inflammation* , 1996, The Journal of Biological Chemistry.

[3]  T. Sugimoto,et al.  Mitogen-activated protein kinase phosphatase: a negative regulator of the mitogen-activated protein kinase cascade. , 1999, European journal of pharmacology.

[4]  C. Moskaluk,et al.  PAC-1: a mitogen-induced nuclear protein tyrosine phosphatase. , 1993, Science.

[5]  M. Cobb,et al.  Mitogen-activated protein kinase pathways. , 1997, Current opinion in cell biology.

[6]  S. Keyse,et al.  Oxidative stress and heat shock induce a human gene encoding a protein-tyrosine phosphatase , 1992, Nature.

[7]  A. Ashworth,et al.  Molecular Cloning and Functional Characterization of a Novel Mitogen-activated Protein Kinase Phosphatase, MKP-4* , 1997, The Journal of Biological Chemistry.

[8]  N. Tonks,et al.  Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1 , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Muda,et al.  Induction of the mitogen‐activated protein kinase phosphatase MKP3 by nerve growth factor in differentiating PC12 , 1998, FEBS letters.

[10]  Piero Carninci,et al.  Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. , 2000, Genome research.

[11]  Lin Chen,et al.  Structure, mapping, and expression of erp, a growth factor-inducible gene encoding a nontransmembrane protein tyrosine phosphatase, and effect of ERP on cell growth , 1993, Molecular and cellular biology.

[12]  K. Takagaki,et al.  Characterization of a novel low-molecular-mass dual-specificity phosphatase-3 (LDP-3) that enhances activation of JNK and p38. , 2004, The Biochemical journal.

[13]  J. Dixon,et al.  hVH‐5: A Protein Tyrosine Phosphatase Abundant in Brain that Inactivates Mitogen‐Activated Protein Kinase , 1995, Journal of neurochemistry.

[14]  R. Davis,et al.  MAPKs: new JNK expands the group. , 1994, Trends in biochemical sciences.

[15]  J. Dixon,et al.  A Tyr/Ser protein phosphatase encoded by vaccinia virus , 1991, Nature.

[16]  M. Camps,et al.  Dual specificity phosphatases: a gene family for control of MAP kinase function , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[17]  L. Mahadevan,et al.  Parallel signal processing among mammalian MAPKs. , 1995, Trends in biochemical sciences.

[18]  L. Lau,et al.  cDNA sequence of a growth factor-inducible immediate early gene and characterization of its encoded protein. , 1992, Oncogene.

[19]  N. Aoki,et al.  Molecular Cloning and Characterization of a Novel Dual Specificity Phosphatase, LMW-DSP2, That Lacks the Cdc25 Homology Domain* , 2001, The Journal of Biological Chemistry.

[20]  J. M. Mulvaney,et al.  Activation of mitogen-activated protein kinase phosphatase 2 by gonadotropin-releasing hormone , 2001, Molecular and Cellular Endocrinology.

[21]  J. C. Clemens,et al.  Mutational analysis of a Ser/Thr phosphatase. Identification of residues important in phosphoesterase substrate binding and catalysis. , 1994, The Journal of biological chemistry.

[22]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[23]  Masahiko Watanabe,et al.  MKP-7, a Novel Mitogen-activated Protein Kinase Phosphatase, Functions as a Shuttle Protein* , 2001, The Journal of Biological Chemistry.