Protein tyrosine phosphatases (PTPs) constitute a family of receptor-like and cytoplasmic signal transducing enzymes that catalyze the dephosphorylation of phosphotyrosine residues and are characterized by homologous catalytic domains. The crystal structure of a representative member of this family, the 37-kilodalton form (residues 1 to 321) of PTP1B, has been determined at 2.8 A resolution. The enzyme consists of a single domain with the catalytic site located at the base of a shallow cleft. The phosphate recognition site is created from a loop that is located at the amino-terminus of an alpha helix. This site is formed from an 11-residue sequence motif that is diagnostic of PTPs and the dual specificity phosphatases, and that contains the catalytically essential cysteine and arginine residues. The position of the invariant cysteine residue within the phosphate binding site is consistent with its role as a nucleophile in the catalytic reaction. The structure of PTP1B should serve as a model for other members of the PTP family and as a framework for understanding the mechanism of tyrosine dephosphorylation.

https://science.sciencemag.org/content/sci/263/5152/1397.full.pdf

Crystal structure of human protein tyrosine phosphatase 1B.

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30–42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

https://www.pnas.org/content/pnas/95/13/7608.full.pdf

Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers

The coordinated action of protein tyrosine phosphatases and kinases in cell signaling

Characterization of the Mammalian YAP (Yes-associated Protein) Gene and Its Role in Defining a Novel Protein Module, the WW Domain

A number of vertebrate genes of the Dlx gene family have been cloned in mouse, frog, and zebrafish. These genes contain a homeobox related to that of Distalless, a gene expressed in the developing head and limbs of Drosophila embryos. We cloned and studied the expression of two members of this family, which we named Dlx5 and Dlx6, in human and mouse. The two human genes, DLX5 and DLX6, are closely linked in an inverted convergent configuration in a region of chromosome 7, at 7q22. Similarly, the two human genes DLX1 and DLX2 are closely linked in a convergent configuration at 2q32, near the HOXD (previously HOX4) locus. In situ hybridization experiments in mouse embryos revealed expression of Dlx5 and Dlx6 mRNA in restricted regions of ventral diencephalon and basal telencephalon, with a distribution very similar to that reported for Dlx1 and Dlx2 mRNA. A surprising feature of Dlx5 and Dlx6 is that they are also expressed in all skeletal structures of midgestation embryos after the first cartilage formation. The expression pattern of these genes, together with their chromosome localization, may provide useful cues for the study of congenital disorders in which there is a combination of craniofacial and limb defects.

https://www.pnas.org/content/pnas/91/6/2250.full.pdf

Cloning and characterization of two members of the vertebrate Dlx gene family.

A full-length cDNA for a novel isoform of the human receptor tyrosine phosphatase gamma gene (PTPRG) was overexpressed in Sf9 insect cells, and the gene product, PTP gamma, was purified and characterized. The protein was expressed as a M(r) approximately 185,000 protein accompanied by a M(r) approximately 120,000 putative cleavage product on SDS-PAGE analysis. The protein undergoes N-linked glycosylation and constitutive phosphorylation of serine residues. When assayed for tyrosine-specific phosphatase activity, PTP gamma dephosphorylated myelin basic protein at a pH optimum of 7.5 and a Km of 12.6 microM; reduced carboxyamidomethylated and maleylated lysozyme (RCM-lysozyme) at a pH optimum of 6.0 and a Km of 12 microM; and p-nitrophenylphosphate with a pH optimum of 5.5 and a Km of 3.5 mM. Phosphatase activity was inhibited by ZnCl2 and sodium orthovanadate; Mg2+, Mn2+, and Ca2+ ions were ineffective. The partially purified form of the enzyme was allosterically activated by triphosphorylated nucleosides, with a preference for purines. This activation was prevented by Mg2+ addition and did not occur when a purified form of the enzyme was utilized, suggesting that its activation depends on specific activating factors or conformational constraints. Interestingly, PTP gamma protein was specifically bound by an ATP-agarose matrix through its intracellular domain, suggesting a link between binding of nucleotides and activation of the phosphatase.

https://cancerres.aacrjournals.org/content/canres/55/21/4855.full.pdf

Characterization of the receptor protein tyrosine phosphatase gene product PTP gamma: binding and activation by triphosphorylated nucleosides.

Protein tyrosine phosphatases, on purely theoretical grounds, were suggested as possible tumor suppressor genes, and receptor protein tyrosine phosphatase γ ( PTPRG ) has been proposed, on the basis of its location at human chromosome region 3p142, specifically as a tumor suppressor gene for renal cell carcinoma

We have isolated murine genomic and complementary DNA clones for analysis and mapping of the murine Ptprg locus; interspecific backcross analysis showed that the Ptprg locus maps to the centromeric region of mouse chromosome 14 We also observed a homozygous, intragenic deletion in the Ptprg gene in all clonal derivatives of the original L-cell strain, a methylcholanthrene-treated mouse connective tissue cell line which produces sarcomas in syngeneic mice The deletion begins in the second intron of the carbonic anhydrase-like domain of the Ptprg gene and ends in the fourth intron of the carbonic anhydrase-like domain At the genomic level, perhaps several hundred kilobases of DNA are deleted; at the complementary DNA level the 400 base pairs comprising exons 2, 3, and 4 of the carbonic anhydrase-like domain are deleted By reverse transcription polymerase chain reaction, an amplified fragment is produced from L-cell mRNA which is 400 base pairs shorter than the wild type gene product, suggesting that the deleted gene is transcribed and may produce a protein product

Thus, mouse L-cells have lost one Ptprg allele and sustained an intragenic deletion in the other; such allele loss and mutation frequently occur at tumor suppressor gene loci

A Homozygous Deletion within the Carbonic Anhydrase-like Domain of the Ptprg Gene in Murine L-Cells

Because of interest in mechanisms of recombination involved in chromosomal deletions in neoplastic disease, and their relation to possible rearrangements in normal tissues, we are studying circular DNA molecules from human tissue with a long-term goal of investigating them as possible by-products of physiologically relevant intrachromosomal recombination events. Covalently closed circular (ccc) DNA from human bone marrow was cloned in bacteriophage vectors, and fourteen clones chosen randomly from the cccDNA-derived library were characterized. Five clones originated from chromosome-specific centromeric alpha-satellite DNA; two clones carried highly repetitive sequences probably derived from interspersed repetitive elements; six clones were derived from single-copy chromosome-specific sequences which detected homologous rodent sequences; and one clone (EPM10) was derived from a small chromosome 11-specific sequence family which localized to chromosome regions 11cen and 11q14. Oligonucleotide primers derived from the cccDNA clones were used in polymerase chain reaction studies to show that (1) the EPM10 clone carried the circular junction, (2) several of the single-copy products could be detected in three different bone marrow cccDNA preparations, and (3) the Alu-PCR profile for bone marrow cccDNA showed distinct bands which were similar in four bone marrow cccDNA preparations.

Zhuangwei Lou

Papers

Characterization of the receptor protein tyrosine phosphatase gene product PTP gamma: binding and activation by triphosphorylated nucleosides.

A Homozygous Deletion within the Carbonic Anhydrase-like Domain of the Ptprg Gene in Murine L-Cells

Characterization of human bone marrow-derived closed circular DNA clones.