Characterization of the receptor protein tyrosine phosphatase gene product PTP gamma: binding and activation by triphosphorylated nucleosides.
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.
Sorio
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Jefferson Cancer Institute, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Mendrola
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J
Lou
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LaForgia
S
S
Croce
C M
CM
Huebner
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism* / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D012685:Sepharose; D018411:Spodoptera / Q000378:metabolism / Q000821:virology
D000906:Antibodies; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D012685:Sepharose; C516850:PTPRG protein, human; D017027:Protein Tyrosine Phosphatases; C516851:Ptprg protein, mouse; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5