THE PURIFICATION AND CHARACTERIZATION OF A HUMAN DUAL-SPECIFIC PROTEIN-TYROSINE-PHOSPHATASE

An expression and purification method was developed to obtain the reco mbinant human dual specific protein tyrosine phosphatase (PTPase) VHR in quantities suitable for both kinetic studies and crystallization. P hysical characterization of the homogeneous recombinant protein verifi ed the mass to be 20,500 +/- 100 by matrix-assisted laser desorption m ass spectrometry, confirmed the anticipated NH2-terminal amino acid se quence and demonstrated that the protein exists as a monomer. Conditio ns were developed to obtain crystals which were suitable for x-ray str ucture determination. Using synthetic diphosphorylated peptides corres ponding to MAP(177-189) (mitogen-activated protein) kinase (DHTGFLpTEp YVATR), an assay was devised which permitted the determination of the rate constants for dephosphorylation of the diphosphorylated peptide o n threonine and tyrosine residues. The diphosphorylated peptides are p referred over the singly phosphorylated on tyrosine by 3-8-fold. The a pparent second-order rate constant k(cat)/K-m for dephosphorlyation of phosphotyrosine on DHTGFLpTEpYVATR was 32,000 M(-1) s(-1) while depho sphorylation of phosphothreonine was 14 M(-1) s(-1) (pH 6). The reacti on of DHTGFLpTEpYVATR with VHR is ordered, with rapid dephosphorylatio n on tyrosine occurring first followed by slow dephosphorylation on th reonine. Similar results were obtained with F(NLe) (NLe)pTPpYVVTR, a p eptide corresponding to a MAP kinase-like protein (JNK1(180-189)) whic h is involved in the stress response signaling pathway.