Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) using synthetic phosphopeptides.
The intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) (44 kDa) was expressed in bacteria, purified using epitope 'tagging' immunoaffinity chromatography, and characterized with respect to kinetic profile, substrate specificity and potential modulators of enzyme activity. A chromogenic assay based on the Malachite Green method was employed for the detection of inorganic phosphate (Pi) released from phosphopeptides by HPTP beta. This assay, modified so as to improve its sensitivity, was adapted to a 96-well microtitre plate format, and provided linear detection between 50 and 1000 pmol of Pi. The cytoplasmic domain of HPTP beta was strongly inhibited by vanadate, molybdate, heparin, poly(Glu, Tyr) (4:1) and zinc ions. In order to explore the substrate preferences of this PTPase, we generated 13-residue synthetic phosphotyrosine-containing peptides that corresponded to sites of physiological tyrosine phosphorylation. HPTP beta demonstrated kcat. values between 76 and 258 s-1 using four different phosphopeptides. The substrate preference of HPTP beta was in the order srcTyr-527 > PDGF-RTyr-740 > ERK1Tyr-204 >> CSF-1RTyr-708 with Km values ranging from 140 microM to greater than 10 mM. The variations in affinity were probably due to differences among the four phosphopeptides compared, particularly with respect to the character of the charged amino acids flanking the phosphotyrosine residue.
Harder
K W
KW
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Owen
P
P
Wong
L K
LK
Aebersold
R
R
Clark-Lewis
I
I
Jirik
F R
FR
D000595:Amino Acid Sequence; D001483:Base Sequence; D004396:Coloring Agents; D017931:DNA Primers; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012394:Rosaniline Dyes; D013379:Substrate Specificity
D004396:Coloring Agents; D017931:DNA Primers; D010748:Phosphopeptides; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; C005095:malachite green; D017027:Protein Tyrosine Phosphatases