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

作者：Harris Leigh K; Frumm Stacey M; Bishop Anthony C^*

来源：Analytical Biochemistry, 2013, 435(2): 99-105.

DOI：10.1016/j.ab.2012.12.025

• Protein tyrosine phosphatases
• Yeast
• Cell-based assay
• TCPTP
• Shp2
• PEST
• PTP alpha

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 coexpressed 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, PTP alpha) are capable of rescuing the effects of v-Src toxicity. Since these PTPs are chosen from four distinct subfamilies, 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.

• 出版日期2013-4-15