Using yeast to screen for inhibitors of protein tyrosine phosphatase 1B
Section snippets
Yeast strain
YPH499: Mata ura 3-52 lys 2-801amber ade 2-101ocher trp 1-Δ63 his3-Δ200 leu2Δ1 (Stratagene).
Plasmids constructs
EcoRI and SalI were used to cut out the catalytic domain (amino acids 1-320) of human PTP1B previously cloned in a pFLAG vector [18]. The resulting fragment was gel purified and ligated to p416GAL1 (American type culture collection (ATCC) 87332) previously digested with EcoRI and SalI and de-phosphorylated. The plasmid p416GAL1-PTP1B was sequenced to confirm the correct construction. PTP1B mutants C215S
Optimization of the rescue of yeast from v-Src lethality by PTP1B
It was previously shown that expression of v-Src in yeast results in the arrest of cell growth and this cell arrest can be rescued by the co-expression of PTP1B [16]. To reproduce these results and to establish the optimal balance between this lethality and the rescue mediated by PTP1B, the expression levels of v-Src in yeast were varied by using mutated GAL promoters [19]. In fact, v-Src expression driven by any of the GAL promoters, GAL1 (wild type) or the attenuated promoters GALL or GALS
Discussion
A major issue with PTP1B drug screening programs is the lack of a robust cell-based functional assay. Typically, increased tyrosine phosphorylation of the IR or glucose uptake has been used as functional readouts for PTP1B inhibition. However, the effect on IR phosphorylation due to either PTP1B mRNA knockdown or inhibition, as measured by quantitation of Western blots, results in at most a two to three-fold increase in phosphorylation over unstimulated controls [9], [10]. This small window and
Acknowledgements
We are grateful to Ernest Asante-Appiah for helpful discussion during the development of this assay and to Kathryn Skorey for assistance with the DiFMUP assay. We would like to thank Rick Friesen, Yongxin Han, C.K. Lau and Yves Leblanc for the inhibitors used in this study. We also want to give special thanks to Ann M. English and her laboratory at Concordia University for support and critical insight. J.M. is supported by Canadian NSERC Industrial Postgraduate Scholarship, FCAR-MRST Bourse de
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A general assay for monitoring the activities of protein tyrosine phosphatases in living eukaryotic cells
2013, Analytical BiochemistryCitation Excerpt :Membranes were then exposed to film, which was developed using standard procedures. As noted above, it has been shown previously that overexpression of the tyrosine kinase v-Src in yeast leads to inhibition of cell growth and that coexpression of the phosphatase PTP1B rescues yeast growth and decreases cellular phosphotyrosine levels [8,9]. The central assumption of developing a general yeast assay for cellular PTP activity is that the previously demonstrated PTP-induced growth rescue is not specific to PTP1B and that other PTPs could function similarly in countering v-Src’s activity.
Discovery of [(3-bromo-7-cyano-2-naphthyl)(difluoro)methyl]phosphonic acid, a potent and orally active small molecule PTP1B inhibitor
2008, Bioorganic and Medicinal Chemistry LettersPTP1B-dependent insulin receptor phosphorylation/residency in the endocytic recycling compartment of CHO-IR cells
2006, Biochemical PharmacologyCitation Excerpt :In this manuscript, we describe the characterization and validation of a HCS cell-based assay that quantifies IR residency and phosphorylation in the ERC, as a measure of PTP1B inhibition. Other cell assays to screen for PTP1B inhibitors have previously been developed in Sf9 cells and yeast [66,67]. We show clearly that, compounds from multiple structural classes with nanomolar potencies on PTP1B, as well as PTP1B-specific siRNA, are capable of influencing the residency as well as the phosphorylation level of the IR found in the ERC of CHO-IR cells.
Residues distant from the active site influence protein-tyrosine phosphatase 1B inhibitor binding
2006, Journal of Biological ChemistryCitation Excerpt :p416GAL1 (ATCC 87332) and p415GALL (ATCC 87338) are low copy vectors containing the URA3 marker for growth in uracil-deficient media and the LEU2 marker for growth in leucine-deficient media, respectively. The details of the construction of p416GAL1-PTP1B-(1-320) and p415GALL-v-Src have been described previously (12). PTP1B-(1-400) was generated from the full-length construct (amino acids 1-435) by PCR using primers 5′-GAATTCATGGAGATGGAAAAGGAG-3′ and 5′-GTCGACCAGTGCCTAGTCCTCGTCCTTC-3′ to introduce EcoRI and SalI restriction sites and the following amplification program: 94 °C for 4 min and 25 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1.5 min, followed by 72 °C for 10 min.