Chemical dissection of the effects of tyrosine phosphorylation of SHP-2.
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 (F(2)Pmp) compared to those of Pmp were also examined. It was found that the F(2)Pmp 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.
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205, USA.
Wei
W
Lu
Kui
K
Shen
Philip A
PA
Cole
D000595:Amino Acid Sequence; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
D047908:Intracellular Signaling Peptides and Proteins; D063065:Organophosphonates; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases