The purification and characterization of a human dual-specific protein tyrosine phosphatase.
An expression and purification method was developed to obtain the recombinant human dual-specific protein tyrosine phosphatase (PTPase) VHR in quantities suitable for both kinetic studies and crystallization. Physical characterization of the homogeneous recombinant protein verified the mass to be 20,500 +/- 100 by matrix-assisted laser desorption mass spectrometry, confirmed the anticipated NH2-terminal amino acid sequence and demonstrated that the protein exists as a monomer. Conditions were developed to obtain crystals which were suitable for x-ray structure determination. Using synthetic diphosphorylated peptides corresponding to MAP177-189 (mitogen-activated protein) kinase (DHTG-FLpTEpYVATR), an assay was devised which permitted the determination of the rate constants for dephosphorylation of the diphosphorylated peptide on threonine and tyrosine residues. The diphosphorylated peptides are preferred over the singly phosphorylated on tyrosine by 3-8-fold. The apparent second-order rate constant kcat/Km for dephosphorylation of phosphotyrosine on DHTGFLpTEpYVATR was 32,000 M-1 S-1 while dephosphorylation of phosphothreonine was 14 M-1 S-1 (pH 6). The reaction of DHTGFLpTEpYVATR with VHR is ordered, with rapid dephosphorylation on tyrosine occurring first followed by slow dephosphorylation on threonine. Similar results were obtained with F(NLe)(N-Le)pTPpYVVTR, a peptide corresponding to a MAP kinase-like protein (JNK1(180-189)) which is involved in the stress response signaling pathway.
Denu
J M
JM
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Zhou
G
G
Wu
L
L
Zhao
R
R
Yuvaniyama
J
J
Saper
M A
MA
Dixon
J E
JE
D000595:Amino Acid Sequence; D000838:Anions; D003460:Crystallization; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D000838:Anions; D011494:Protein Kinases; D017027:Protein Tyrosine Phosphatases