The regulation of the protein tyrosine phosphatase (PTPase) SHP-2 by tyrosine phosphorylation has been difficult to elucidate because of the intrinsic instability of the phosphoprotein. In the past, expressed protein ligation has been used to site-specifically incorporate the phosphotyrosine mimic Pmp (phosphonomethylene phenylalanine) into the two tyrosine phosphorylation sites (542, 580) of SHP-2 one at a time to analyze the effects on catalytic behavior. In this study, we have incorporated two Pmps into the phosphorylation sites simultaneously and examined the effects of double SHP-2 tyrosine phosphorylation. We have found that the Pmp groups show close to additive effects on PTPase stimulation, suggesting dual SH2 domain occupancy. The relative effects of the phosphotyrosine analogue difluoromethylene phosphonophenylalanine (F2Pmp) compared to those of Pmp were also examined. It was found that the F2Pmp analogue showed slightly enhanced PTPase stimulation compared with the Pmp analogue, consistent with its higher affinity for SH2 domains. Taken together with the bis-Pmp studies, these data suggest that double phosphorylation of the SHP-2 C-terminus could give rise to a 9-fold overall PTPase activation, 30-50% of the value associated with deletion of the SH2 domains. Catalytically inactive forms of phosphorylated SHP-2 proteins were also produced by expressed protein ligation. This allowed for a systematic analysis of intermolecular autodephosphorylation of SHP-2, which revealed how conformational plasticity can modulate phosphotyrosine stability.
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