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. Author manuscript; available in PMC: 2013 Dec 17.
Published in final edited form as: Nat Cell Biol. 2013 Apr 21;15(5):10.1038/ncb2731. doi: 10.1038/ncb2731

Figure 5.

Figure 5

PTPL1 dephosphorylates p85β, promoting its binding to FBXL2 and degradation. (a) The CaaX motif of FBXL2 is not required to bind PTPL1. HEK293T cells were transfected with either FLAG-tagged wild-type FBXL2 or FLAG-tagged FBXL2(CaaX/SaaX). 24 h post-transfection, cells were collected and whole-cell lysates (WCL) were immunoprecipitated (IP) and immunoblotted as indicated. (b) Schematic representation of p85β mutants. Binding of p85β to PTPL1 and p110α is indicated with the symbol +. (c) PTPL1 stimulates the binding of FBXL2 to wild-type p85β, but not p85β(Y655A). HEK293T cells were transfected with GFP-tagged FBXL2 and either FLAG-tagged p85β or FLAG-tagged p85β(Y655A). Where indicated, HA-tagged PTPL1 or HA-tagged PTPL1(C/S) were also transfected. The experiment was performed as described in a. (d) PTPL1 silencing inhibits the FBXL2–p85β interaction. HeLa cells stably expressing FLAG-tagged FBXL2 were transfected with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). Forty-eight hours post-transfection, cells were collected and whole-cell lysates (WCL) were immunoprecipitated (IP) and immunoblotted as indicated. (e) During a 72-h serum starvation, NHFs were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were subsequently re-stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (f) HEK293T cells were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were transfected with p85β(Y655A) and 16 h after cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. (g) During a 48-h serum starvation, U2OS cells stably transfected with a doxycycline-inducible p85β construct were transfected twice with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently re-stimulated with media containing serum and collected 30 min later. Denatured cell lysates were immunoprecipitated with an anti-FLAG resin and immunoblotted as indicated. The asterisk indicates a non-specific band. Uncropped images of blots are shown in Supplementary Fig. S8.