Abstract
Reversible phosphorylation of proteins on tyrosine residues is critical in several cellular activities. The removal of the phosphate groups from tyrosine-phosphorylated proteins is accomplished by a class of enzymes which constitute a large family of related proteins, the protein tyrosine phosphatases. We report here the isolation of a complementary DNA encoding a novel protein tyrosine phosphatase, which we termed hPTP1E. Several overlapping cDNA clones were isolated to reconstruct a sequence of 8301 nucleotides. Northern blot analysis of poly(A)+ RNA from different human tissues revealed the presence of a mRNA of 8.2-8.5 kilobases suggesting the near full-length sequence had been cloned. The composite hPTP1E cDNA contains an open reading frame encoding 2490 amino acid residues. The predicted protein (M(r) = 277,567) does not contain a signal peptide or a membrane-spanning region and possesses a single catalytic domain (amino acids 2241-2470). The recombinant phosphatase domain has been expressed in Escherichia coli, purified to near homogeneity and showed to possess specific protein tyrosine phosphatase activity. The primary structure of hPTP1E also displays homology to cytoskeleton- and membrane junction-associated proteins. Thus, the region encompassing amino acids 568-1053 is related to the cytoskeletal proteins of the band 4.1 family. In addition, hPTP1E contains five imperfect repeats possessing significant homology to the GLGF repeats of the junction-associated guanylate kinases such as the Drosophila discs-large tumor suppressor gene (dlg-1). The structural features of hPTP1E suggest that it localizes at the junction between the plasma membrane and the cytoskeleton where it may regulate signal transduction and cytoskeletal integrity.
Highlights
Reversible phosphorylation of proteins on tyrosine tyrosine phosphatases (PTPs).’ Since the isolation of PTPlB residues is critical in several cellular activities
The re- (3), the first reported enzymeof the latter group,a number of moval of the phosphate groups from tyrosine-phospho- enzymes with intrinsic tyrosine phosphatase activity have been rylated proteins is accomplished by a class of enzymes which constitute a large family of related proteins, the protein tyrosine phosphatases
We report here the isolation of a complementary DNAencodinga novel protein tyrosine phosphatase, which we termed hPTP1E
Summary
Cells and Cell Culture-The cell lines used for these studies were obtained from the American Type Culture Collection (Rockville, MD). Approximately 800,000 phages were screened following The reversible phosphorylation of proteins on tyrosine resi- standard procedures with a 1.5-kilobaseEcoRI-Sal1fragment containdues is a critical componentin the controol f many fundamental ing the two catalytic domains of the human LAR cDNA (26).The probe aspects of cellular function (1, 2). It is a major element in was labeled with 32Pusing the random priming procedure of Feinberg the conversionof a normal cell to the transformed phenotype. Isolation of hPTPlE cDNAfromZR-75-1 lbtal RNAby Anchor PCR-cDNA was prepared from ZR-75-1poly(AYRNA with random
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