Abstract
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.
Published Version
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