Isolation and characterization of a human dual specificity protein-tyrosine phosphatase gene.

S.P Kwak,D.J Hakes,K.J Martell,J.E Dixon

doi:10.1016/s0021-9258(17)41905-3

Abstract

Vaccinia phosphatase VH-1 and its mammalian counterparts, including protein-tyrosine phosphatases (PTPase) CL100 and VHR, constitute a novel subfamily of protein-tyrosine phosphatases that exhibits dual substrate specificity for phosphotyrosine- and phosphoserine/threonine-containing substrates. The expression of human VH-1-like PTPase CL100 is rapidly inducible by mitogen stimulation and oxidative stress, suggesting that this gene is transcriptionally regulated. In order to study the mechanism underlying this transcriptional regulation, we isolated the first human gene of this subfamily, the CL100 gene, and characterized its promoter. The gene consists of four exons intervened by three short introns 400-500 base pairs in length. Analysis of the protein sequence encoded by each exon revealed that there is a second region of similarity between CL100 protein and cdc25 in addition to the PTPase catalytic domain. Promoter analysis of the CL100 gene indicates that an 800-base pair region flanking the transcriptional initiation site is sufficient to confer a transcriptional response to serum and 12-O-tetradecanoylphorbol-13-acetate stimulation. The CL100 gene is expressed in numerous tissues, including nonmitotic cells in the brain. Within the brain, CL100 mRNA is localized in discrete neuronal populations, suggesting that this PTPase is likely to play a key role in neurotransmission as well as in mitotic signaling. Finally, although extracellular signal-regulated kinase has recently been shown to act as substrate for CL100 in vitro, we find no clear correspondence between the distribution of extracellular signal-regulated kinase and CL100 mRNA in the brain. The potential significance of a second cdc25 homology domain of CL100 is discussed.

Highlights

Vaccinia phosphatase VH-1and its mammalian coun- ily of enzymes that exhibit a n absolute substrate requirement terparts, including protein-tyrosine phosphatases for phosphotyrosines
Extracellular signal-regulated kinase has recently been showtno act as substratefor CLlOO in vitro, we find no clear correspondence betweenthe distribution of extracellular signal-regulatedkinase and CLlOO mRNA in the brain
The transcriptional start site was identified by primer extension analysis using primers complementary to nucleotide position 210 and 234 of the cDNA (Fig. 3A).A control reaction consisting of RNAharvested from unstimulated Humancervical carcinoma (HeLa) cells low in CLlOO mRNA was run on adjacent lanes

Summary

Chromosomal assignment"

Genomic Library Screening-Specific primers 24 nt inlength coding for the N terminus peptide sequence PVEILPFL (S'GGTGGAAATCCTGCCCTITCT3') and complementary to the C-terminal peptide sequence PVSIPVHS (5'AGTGGACAGGcATGGAGACGGB')of pCLl00 were used to amplify a 560-bp CLlOO cDNA fragment from a human placental cDNA library (Stratagene, La Jolla, CA). The amplified fragment containing the catalytic region of pCLl00 was subsequently la-

Uncharacterized clones

RESULTS

NTDRRMNSHRYPFLYYP REVDRKLNSNIYPRCDYE

CRE CRE

DISCUSSION