The protein-tyrosine phosphatase SHP-1 plays a variety of roles in the "negative" regulation of cell signaling. The molecular basis for the regulation of SHP-1 is incompletely understood. Whereas SHP-1 has previously been shown to be phosphorylated on two tail tyrosine residues (Tyr(536) and Tyr(564)) by several protein-tyrosine kinases, the effects of these phosphorylation events have been difficult to address because of the intrinsic instability of the linkages within a protein-tyrosine phosphatase. Using expressed protein ligation, we have generated semisynthetic SHP-1 proteins containing phosphotyrosine mimetics at the Tyr(536) and Tyr(564) sites. Two phosphonate analogues were installed, phosphonomethylenephenylalanine (Pmp) and difluorophosphonomethylenephenylalanine (F(2)Pmp). Incorporation of Pmp at the 536 site led to 4-fold stimulation of the SHP-1 tyrosine phosphatase activity whereas incorporation at the 564 site led to no effect. Incorporation of F(2)Pmp at the 536 site led to 8-fold stimulation of the SHP-1 tyrosine phosphatase activity and 1.6-fold at the 564 site. A combination of size exclusion chromatography, phosphotyrosine peptide stimulation studies, and site-directed mutagenesis led to the structural model in which tyrosine phosphorylation at the 536 site engages the N-Src homology 2 domain in an intramolecular fashion relieving basal inhibition. In contrast, tyrosine phosphorylation at the 564 site has the potential to engage the C-Src homology 2 domain intramolecularly, which can modestly and indirectly influence catalytic activity. The finding that phosphonate modification at each of the 536 and 564 sites can promote interaction with the Grb2 adaptor protein indicates that the intramolecular interactions fostered by post-translational modifications of tyrosine are not energetically strong and susceptible to intermolecular competition.