Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP<i>β</i>, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors

Cho, Hyeongjin; Krishnaraj, Ravichandran; Walsh, Christopher T.; Itoh, Michyasu; Saito, Haruo; Kitas, Eric; Bannwarth, Willi

doi:10.1002/pro.5560020611

Cited by 83 publications

(49 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These are often sluggish reactions that generally afford low stoichiometries and often show poor regioselectivity (33). Moreover, thiophosphates can indeed be hydrolyzed chemically and enzymatically, by some phosphatases almost as rapidly as phosphate substrates (34), in contrast to the nonhydrolyzable analogues used here.…”

Section: Discussionmentioning

confidence: 94%

The Role of C-terminal Tyrosine Phosphorylation in the Regulation of SHP-1 Explored via Expressed Protein Ligation

Zhang

Shen

Lü

et al. 2003

Journal of Biological Chemistry

133

113

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 94%

The Role of C-terminal Tyrosine Phosphorylation in the Regulation of SHP-1 Explored via Expressed Protein Ligation

Zhang

Shen

Lü

et al. 2003

Journal of Biological Chemistry

133

113

View full text Add to dashboard Cite

show abstract

“…Immunoprecipitated PTPs were washed with phosphatase assay buffer (100 mmol/l HEPES, pH 7.6, 1 mmol/l DTT, 2 mmol/l EDTA, 150 mmol/l NaCl, and 1 mg/ml BSA) and incubated in a final volume of 60 µl with 100 µmol/l of IR peptide TRDIpYETDpYpYRK (Biomol, Plymouth Meeting, PA). After 2 h at 22°C, phosphatase reactions were terminated by adding 40 µl aliquots to 100 µl of Biomol Green reagent, and the release of inorganic phosphate (P i ) was determined by the absorbance measured at 630 nm using the Titertek Plus 96-well plate reader (47). Measured PTP activity was linear from 0.5 to 2 times the total lysate protein concentrations used for immunoprecipitation (data not shown).…”

Section: P]atp Labelingmentioning

confidence: 94%

Decreased In Situ Insulin Receptor Dephosphorylation in Hyperglycemia-Induced Insulin Resistance in Rat Adipocytes

et al. 2001

View full text Add to dashboard Cite

The regulation of insulin receptor (IR) tyrosine (tyr) phosphorylation is a key step in the control of insulin signaling. Augmented IR tyr dephosphorylation by protein tyrosine phosphatases (PTPs) may contribute to insulin resistance. To investigate this possibility in hyperglycemia-induced insulin resistance, primary cultured rat adipocytes were rendered insulin-resistant by chronic exposure (18 h) to 15 mmol/l glucose combined with 10 -7 mol/l insulin. Insulin-resistant adipocytes showed a decrease in insulin sensitivity and a maximum response of 2-deoxyglucose uptake, which was associated with a decrease in maximum insulin-stimulated IR tyr phosphorylation in situ. To assess tyr dephosphorylation, IRs of insulin-stimulated permeabilized adipocytes were labeled with [␥-

show abstract

“…15). CD45 can dephosphorylate several protein substrates and phosphorylated peptides in vitro (6,7,16,17), yet only p56 lck and another Src family kinase, p59 fyn , have clearly been identified as potential in vivo substrates (5,8,9). Both p56 lck and, to a lesser extent, p59…”

Section: P56mentioning

confidence: 99%

Demonstration of a Direct Interaction between p56 and the Cytoplasmic Domain of CD45 in Vitro

Watts

Aebersold

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

p56lck is a potential in vivo substrate for the tyrosinespecific phosphatase, CD45. In this study, recombinant purified p56 lck was found to specifically associate with recombinant CD45 cytoplasmic domain protein, but not to the cytoplasmic domain of another related tyrosine phosphatase, receptor protein-tyrosine phosphatase ␣. Under equilibrium binding conditions, the binding was saturable and occurred at a 1:1 molar stoichiometry. A fusion protein containing only the amino-terminal region of p56 lck (residues 34 -150) also bound to recombinant CD45, and further analysis of this region indicated that glutathione S-transferase fusion proteins of the unique amino-terminal region and the SH2 domain, but not the SH3 domain of p56 lck , bound to recombinant CD45. The SH2 domain protein bound with a higher affinity than the amino-terminal region, but both were able to compete for the binding of p56 lck to CD45, and when added together worked synergistically to compete for p56 lck binding. The SH2 domain interaction with CD45 was specific as glutathione S-transferase-SH2 fusion proteins from p85␣ subunit of phosphatidylinositol 3-kinase and SHC did not bind to CD45. In addition, this interaction occurred in the absence of any detectable tyrosine phosphorylation on CD45, suggesting a nonconventional SH2 domain interaction. p56lck and CD45 (see Ref. 1 for review) are both required to generate an effective T cell antigen receptor (TCR) 1 -mediated signal, being required for the earliest detectable event to occur upon TCR stimulation, the tyrosine phosphorylation of cellular proteins (2, 3). p56lck is a member of the Src family of tyrosine kinases and is thought to be regulated by tyrosine phosphorylation at the autophosphorylation and negative regulatory sites. Tyrosine phosphorylation at the carboxyl-terminal negative regulatory site is believed to result in an intramolecular interaction with its SH2 domain which results in an inactive or inaccessible kinase (4). In the presence of CD45 this negative regulatory tyrosine residue in p56 lck is dephosphorylated (5-9), and p56lck is then thought to be able to participate in TCRmediated signal transduction events (10 -12). Exactly how dephosphorylation of p56 lck results in effective TCR-mediated signal transduction remains unclear as recent data indicate that this dephosphorylation event may not necessarily lead to increased kinase activity (13). Dephosphorylation of these kinases may also result in the release of the intramolecular binding of the carboxyl-terminal tyrosine to the SH2 domain, which would then allow the unoccupied SH2 domain to bind to other tyrosine-phosphorylated proteins. Hence CD45 may act to regulate the SH2 domain interactions of p56 lck , which itself could be crucial in mediating a TCR induced signal. Consistent with such a model is the demonstration that the SH2 domain of p56 lck also plays an active role in T cell activation (14). CD45 is a major lymphocyte glycoprotein and a transmembrane two domain tyrosine-specific phosphatase (reviewed in Ref. ...

show abstract

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTPβ, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors

Cited by 83 publications

References 38 publications

The Role of C-terminal Tyrosine Phosphorylation in the Regulation of SHP-1 Explored via Expressed Protein Ligation

The Role of C-terminal Tyrosine Phosphorylation in the Regulation of SHP-1 Explored via Expressed Protein Ligation

Decreased In Situ Insulin Receptor Dephosphorylation in Hyperglycemia-Induced Insulin Resistance in Rat Adipocytes

Demonstration of a Direct Interaction between p56 and the Cytoplasmic Domain of CD45 in Vitro

Contact Info

Product

Resources

About