Residue 259 is a key determinant of substrate specificity of protein- tyrosine phosphatases 1B and α

Abstract

The aim of this study was to define the structural elements that determine the differences in substrate recognition capacity of two protein- tyrosine phosphatases (PTPs), PTP1B and PTPα, both suggested to be negative regulators of insulin signaling. Since the Ac-DADE(pY)L-NH2 peptide is well recognized by PTP1B, but less efficiently by PTPα, it was chosen as a tool for these analyses. Cα regiovariation analyses and primary sequence alignments indicate that residues 47, 48, 258, and 259 (PTP1B numbering) define a selectivity-determining region. By analyzing a set of DADE(pY)L analogs with a series of PTP mutants in which these four residues were exchanged between PTP1B and PTPα, either in combination or alone, we here demonstrate that the key selectivity-determining residue is 259. In PTPα, this residue is a glutamine causing steric hindrance and in PTP1B a glycine allowing broad substrate recognition. Significantly, replacing Gln259 with a glycine almost turns PTPα into a PTP1B-like enzyme. By using a novel set of PTP inhibitors and x-ray crystallography, we further provide evidence that Gln259 in PTPα plays a dual role leading to restricted substrate recognition (directly via steric hindrance) and reduced catalytic activity (indirectly via Gln262). Both effects may indicate that PTPα regulates highly selective signal transduction processes.