Cloning, bacterial expression, purification, and characterization of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase.
This report describes the cloning and characterization of rat leukocyte common antigen-related protein (rLAR), a receptor-like protein tyrosine phosphatase (PTPase). The recombinant cytoplasmic PTPase domain was expressed at high levels in bacteria and purified to homogeneity. Kinetic properties of the PTPase were examined along with potential modulators of PTPase activity. Several sulfhydryl-directed reagents were effective inhibitors, and a surprising distinction between iodoacetate and iodoacetamide was observed. The latter compound was an extremely poor inhibitor when compared to iodoacetate, suggesting that iodoacetate may interact selectively with a positive charge at or near the active site of the enzyme. Site-directed mutants were made at 4 highly conserved cysteine residues found at positions 1434, 1522, 1723, and 1813 within the protein. The Cys-1522/Ser mutation resulted in a 99% loss of enzymatic activity of the pure protein. This observation is consistent with greater than 99% of the PTPase activity being found in the first domain of the PTPase and demonstrates the critical importance of this cysteine residue in catalysis. The recombinant C1522S mutant phosphatase could also be phosphorylated in vitro by protein kinase C and p43v-abl tyrosine kinase. When pure recombinant PTPase was mixed with 32P-labeled tyrosine substrate and then rapidly denatured, a 32P-labeled enzyme intermediate could be trapped and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The catalytically inactive C1522S mutant did not form the phosphoenzyme intermediate.
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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Woodford
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Remboutsika
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Haun
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Dixon
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D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D006023:Glycoproteins / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D017027:Protein Tyrosine Phosphatases / Q000235:genetics*; D051381:Rats; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D011956:Receptors, Cell Surface; D016415:Sequence Alignment
D006023:Glycoproteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D011956:Receptors, Cell Surface; C008644:nitrophenylphosphate; D004247:DNA; D000803:Angiotensin I; D017027:Protein Tyrosine Phosphatases; C516845:Ptpra protein, rat; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4