Fig. 4: Localized deactivation of PTP1BPS.
From: Minimally disruptive optical control of protein tyrosine phosphatase 1B
a A biosensor for PTP1B activity. Src-mediated phosphorylation of the substrate domain causes it to bind to the SH2 domain, triggering a conformational change that decreases FRET; dephosphorylation by PTP1B increases FRET. b Src increases the donor/acceptor emission ratio in vitro (normalized by the buffer-only condition); EDTA or PTP1B prevent this increase. Error bars denote propagated SE for measurements of n = 3 independent experiments (measurements are normalized to a to a buffer-only condition). c The percent change in donor/acceptor emission ratio over 1 min within 5-μm circular regions located in the cytosol and nucleus of COS-7 cells activated with 457 nm light. Each condition includes the interquartile average, associated data points, and SE for n = 11 biological replicates. The p-values correspond to a two-tailed Student’s t test. d An image of localized illumination (405 nm) of a COS-7 cell expressing both PTP1BPS and biosensor. Circles delineate irradiated (red) and secondary (blue) regions, and colors show the donor/acceptor emission ratio (scale bar, 10 μm). e Time courses of FRET in irradiated and secondary regions. Shading highlights 5-s periods before (gray), during (blue), and after (gray) illumination. f A depiction of a HEK293T/17 cell expressing PTP1BPS**. Insulin stimulates phosphorylation of the membrane-bound insulin receptor (IR); PTP1B dephosphorylates it. g ELISA-based measurements of IR phosphorylation in (i) wild-type HEK293T/17 cells and (ii) HEK293T/17 cells stably expressing PTP1BPS** or PTP1BPS**(C450M). Insulin-mediated simulation of IR, BBR-mediated inhibition of PTP1B, and photoinactivation of PTP1B all increase IR phosphorylation. The dark state of PTP1BPS** and the dark and light states of PTP1BPS**(C450M), by contrast, leave IR phosphorylation unaltered from its levels in the wild-type strain (DMSO). The plotted data depict the mean, propagated SE, and associated data points for measurements of n = 3 biological replicates (relative to a buffer-only condition). Source data are provided as a Source Data file.
