Characterization of protein tyrosine phosphatase SH-PTP2. Study of phosphopeptide substrates and possible regulatory role of SH2 domains.
The src homology 2 (SH2) domain containing protein-tyrosine-phosphatase SH-PTP2, was over-expressed in Escherichia coli for a kinetic study employing a set of synthetic 13- to 14-mer phosphopeptide substrates. The full-length SH-PTP2 protein, as well as a truncated form, lacking the two amino terminus SH2 domains (SH-PTP2(delta SH2)), exhibited Michaelis-Menten kinetics, and demonstrated striking substrate preferences on phosphopeptides having sequences based on sites of intracellular protein tyrosine phosphorylation. For example, while a KM of 59 microM and kcat/KM of 1.1 x 10(5) were obtained using SH-PTP2(delta SH2) and PDGFRY1021, a phosphorylation site within the platelet-derived growth factor receptor, other peptides revealed no detectable phosphate release. PDGFRY1009, modeled after a sequence identified as an in vivo binding site for SH-PTP2, was also a good substrate for this enzyme. The truncated form, lacking the SH2 domains demonstrated higher catalytic efficiency than the full-length enzyme. Interestingly, soluble SH2 domains were found to inhibit the catalytic activity of SH-PTP2 in a concentration-dependent manner. There was also evidence of a non-phosphotyrosine-mediated association between the two domains. These observations suggested that the SH2 domains have a direct role in regulating the catalytic activity of SH-PTP2.
Dechert
U
U
Biomedical Research Centre, University of British Columbia, Canada.
Adam
M
M
Harder
K W
KW
Clark-Lewis
I
I
Jirik
F
F
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D010748:Phosphopeptides; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases