The Role of C-terminal Tyrosine Phosphorylation in the Regulation of SHP-1 Explored via Expressed Protein Ligation
The protein-tyrosine phosphatase SHP-1 plays a variety of roles in the ânegativeâ 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...
Saved in:
Published in | The Journal of biological chemistry Vol. 278; no. 7; pp. 4668 - 4674 |
---|---|
Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
14.02.2003
|
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | The protein-tyrosine phosphatase SHP-1 plays a variety of roles in the ânegativeâ 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. |
---|---|
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M210028200 |