A general assay for monitoring the activities of protein tyrosine phosphatases in living eukaryotic cells

Abstract

Protein tyrosine phosphatases (PTPs) are key signal-transduction regulators and have emerged as potential drug targets for inhibitor design. Here we report a yeast-based assay that provides a general means of assessing the activity and/or inhibition of essentially any classical PTP in living cells. The assay uses the activity of an exogenously expressed PTP to counter the activity of a co-expressed and toxic tyrosine kinase, such that only active PTPs are capable of rescuing growth. PTP activity gives rise to both increased growth and decreased phosphotyrosine levels; cellular PTP activity can therefore be monitored by either yeast-growth curves or anti-phosphotyrosine western blots. We show that four PTPs (TCPTP, Shp2, PEST, PTPα) are capable of rescuing the effects of v-Src toxicity. Since these PTPs are chosen from four distinct sub-families, it is likely that biologically and medicinally important PTPs from other subfamilies can similarly function in the cellular PTP assay. Because many small-molecule PTP inhibitors fail to penetrate cell membranes effectively, this cell-based assay has the potential to serve as a useful screening tool for determining the cellular efficacy of candidate inhibitors in a more biologically relevant context than can be provided by an in vitro PTP assay.

Defining Human Tyrosine Kinase Phosphorylation Networks Using Yeast as an In Vivo Model Substrate

“…Early indications of heterologous PTK activity in yeast were observed through growth inhibition upon overexpression of v-SRC, later explained by aberrant phosphorylation of yeast proteins (Brugge et al, 1987;Kornbluth et al, 1987;Cooper and MacAuley, 1988;Florio et al, 1994). The toxic effect of PTK activity upon overproduction in yeast was exploited for screening of kinase inhibitors or phosphatases which restore yeast growth (Montalibet and Kennedy, 2004;Koyama et al, 2006;Harris et al, 2013). Despite these overexpression toxicity issues, Nada and colleagues (Nada et al, 1991) effectively used a heterologous yeast system to discover that CSK negatively regulates SRC by C-terminal tyrosine phosphorylation.…”

Exploring absent protein function in yeast: assaying post translational modification and human genetic variation

“…The toxic effect of tyrosine kinase activity in yeast can be leveraged to study kinase activity perturbations, and as such was exploited for screening of inhibitors of kinase or phosphatases which restore yeast growth [41][42][43]. Similar to the co-expression of repressive CSK kinase in the presence of SRC [34], co-expression of the catalytical domain of PTP1B tyrosine phosphatase can rescue v-SRC tyrosine kinase growth phenotype.…”

“…The tumor suppressor p53 is one of the most frequent mutated proteins in cancer, and also one of the most intensively studied proteins. S. cerevisiae lacks a p53 homolog and expression of the human protein results in a decreased growth rate [43,[106][107][108][109]. Nigro et al showed that the expression of p53 in yeast increased the doubling time up to 250% compared to empty vector control or the expression of an inactive mutant version of p53 [108].…”

Probing the Therapeutic Potential of Marine Phyla by SPE Extraction

“…PTP-1B functions as a negative regulator of insulin and as a drug target in order to ameliorate resistance to the hormone [26]. For the screening of the MNP library, we used an enzymatic assay for the inhibition of the recombinant human PTP-1B protein together with the T Cell-PTP counter screen assay to check for enzymatic specificity [27]. We also tested 50 µg/mL extracts or fractions of the MNP library, and inhibition was calculated by comparing measurements with controls (no treatment).…”