Regulation of protein tyrosine phosphatase 1C: opposing effects of the two src homology 2 domains.
The regulatory roles of the two src homology 2 (SH2) domains of protein tyrosine phosphatase 1C were investigated by comparing recombinant full-length PTP1C with mutants in which either the N-terminal SH2 (N-SH2) domain (PTP1C delta NSH2), the C-terminal SH2 (C-SH2) domain (PTP1C delta CSH2) or both SH2 domains were deleted (PTP1C delta NSH2 delta CSH2). This revealed that the SH2 domains have opposing and independent effects on activity: strong inhibition by N-SH2 (42-fold) and weak activation by C-SH2 (2.1-fold). C-SH2 caused activation across a wide pH range while N-SH2 inhibited most at neutral and high pH through a shift of the basic limb of the pH profile of kcat/Km, apparently via perturbation of an active-site pKa value. A phosphotyrosyl peptide derived from the erythropoietin receptor caused an approximately 30-fold activation of PTP1C and PTP1C delta CSH2 but had no effect on PTP1C delta NSH2 or PTP1C delta NSH2 delta CSH2, indicating that binding of this peptide to N-SH2 abolished its inhibition. Since C-SH2 separates N-SH2 from the catalytic domain in full-length PTP1C and activation is observed for PTP1C delta CSH2, it appears that the inhibitory effect of N-SH2 is independent of the position in the sequence and that intermolecular interactions may also be possible.
Pregel
M J
MJ
Pharmaceutical Biotechnology Sector, National Research Council of Canada, Montréal, Québec.
Shen
S H
SH
Storer
A C
AC
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010748:Phosphopeptides / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion / Q000235:genetics; D018909:src Homology Domains / Q000235:genetics*
D010748:Phosphopeptides; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases