FBXL2- and PTPL1-mediated degradation of p110-free p85β regulatory subunit controls the PI(3)K signalling cascade

Abstract

F-box proteins are the substrate-recognition subunits of SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complexes. Purification of the F-box protein FBXL2 identified the PI(3)K regulatory subunit p85β and tyrosine phosphatase PTPL1 as interacting proteins. FBXL2 interacts with the pool of p85β that is free of p110 PI(3)K catalytic subunits and targets this pool for ubiquitylation and subsequent proteasomal degradation. FBXL2-mediated degradation of p85β is dependent on the integrity of its CaaX motif. Whereas most SCF substrates require phosphorylation to interact with their F-box proteins, phosphorylation of p85β on Tyr 655, which is adjacent to the degron, inhibits p85β binding to FBXL2. Dephosphorylation of phospho-Tyr-655 by PTPL1 stimulates p85β binding to and degradation through FBXL2. Finally, defects in the FBXL2-mediated degradation of p85β inhibit the binding of p110 subunits to IRS1, attenuate the PI(3)K signalling cascade and promote autophagy. We propose that FBXL2 and PTPL1 suppress p85β levels, preventing the inhibition of PI(3)K by an excess of free p85 that could compete with p85-p110 heterodimers for IRS1.

Figure 1

FBXL2 binds p110-free p85 regulatory subunits. (a) FBXL2 binds p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins (FBPs). 24 h post-transfection, cells were treated with MG132 for 3 h before collection for immunoprecipitation (IP) and immunoblotting as indicated. WCL, whole-cell lysate. (b) FBXL2 does not bind p110α and p110β. HEK293T and NIH3T3 cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged proteins. The experiment was performed as described in a. (c) The CaaX motif of FBXL2 is required to bind p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins. The experiment was performed as described in a. (d) Table summarizing the results presented in a–c and Supplementary Fig. S1a. NT, not tested. Uncropped images of blots are shown in Supplementary Fig. S8.

Figure 2

p85β is targeted for ubiquitylation and degradation by SCF^FBXL2. (a) HEK293T cells were transfected with an empty vector (EV) or the indicated FLAG-tagged constructs. 24 h after transfection, cells were treated with either MG132 or solvent for 3 h before collection for immunoblotting as indicated. (b) HeLa cells stably expressing FLAG–HA-tagged FBXL2 were transfected with either siRNAs targeting FBXL2 (four different siRNAs) or a non-silencing siRNA (NS). Cells were lysed, and proteins were immunoblotted as indicated. NT, non-transfected. (c) During a 72-h serum starvation, NHFs were transfected with either an siRNA targeting FBXL2 (no. 1) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition. (d) HEK293T cells were transfected with HA-tagged p85β, FLAG-tagged FBXL2, FLAG-tagged FBXL2(ΔF-box) or an empty vector (EV) as indicated. After immunopurification with anti-FLAG resin, in vitro ubiquitylation of p85β was performed in the presence of E1, E2s and ubiquitin (Ub). Where indicated, an excess of methylated ubiquitin (MeUb) was also added. Samples were analysed by immunoblotting with the indicated antibodies. The ladder of bands with a relative molecular mass of >85,000 (lane 3) corresponds to ubiquitylated p85β. Immunoblots of whole-cell lysates (WCL) are shown at the bottom. Uncropped images of blots are shown in Supplementary Fig. S8

Figure 3

Identification of p85β degron. (a) Schematic representation of p85β mutants. Binding of p85β to FBXL2 and p110α is indicated with the symbol +; NT, not tested. ‘++’ denotes enhanced binding. (b) HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated FLAG-tagged p85β mutants. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-FLAG resin, and immunocomplexes were probed with antibodies against the indicated proteins. (c) p85β(ΔSH2C) is more stable than wild-type p85β. RPE1-hTERT cells were infected with either a retrovirus expressing wild-type p85β or p85β (ΔSH2C). Cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0. (d) p85β (QR/AA) is more stable than wild-type p85β. HEK293T cells were infected with either a retrovirus expressing wild-type p85β or p85β (QR/AA). Cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0. (e) p85β (Y655A) is less stable than wild-type p85β. The experiment was performed as described in c except that p85β(Y655A) was used. In the graph, the amount of p85β(wild-type or mutant) is represented relative to the amount at time 0. Uncropped images of blots are shown in Supplementary Fig. S8.

Figure 4

p85β interaction with FBXL2 is negatively regulated by Tyr phosphorylation. (a) Lysates from cells expressing the indicated FLAG-tagged F-box proteins (FBPs) were used in binding reactions with beads coupled to either a peptide or a phospho-peptide (sequences shown on top of the panel). Beads were washed with lysis buffer, and bound proteins were eluted and subjected to SDS–PAGE and immunoblotting. (b) Denatured extracts from HEK293T cells stably expressing either FLAG-tagged wild-type p85β or FLAG-tagged p85β(Y655A) were immunoprecipitated (IP) with either an anti-FLAG resin or a 4G10 platinum resin and immunoblotted (IB) as indicated. Immunoblots of whole-cell lysates (WCL) are also shown in the last two lanes. (c) Denatured extracts from HEK293T cells expressing either FLAG-tagged wild-type p85β or the indicated FLAG-tagged p85β mutants were immunoprecipitated (IP) with an anti-FLAG resin and immunoblotted as indicated. Uncropped images of blots are shown in Supplementary Fig. S8.

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.

Figure 6

Failure to degrade p85β results in PI(3)K activation defects. (a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, p85β, both FBXL2 and p85β, or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph at the right shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition. (b) Cells were treated as in a, except that 3 h after serum re-addition, cells were lysed, immunoprecipitated (IP) with an anti-IRS1 antibody and immunoblotted as indicated. Uncropped images of blots are shown in Supplementary Fig. S8.

Figure 7

p85β degradation regulates cell autophagy. (a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2 (no. 1 or 2) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (b) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, both FBXL2 and p85β, or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (c) U2OS cells stably transfected with a doxycycline-inducible p85β construct were serum starved for 48 h. During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (d) Cells constitutively expressing GFP–LC3 were transfected with HA-tagged p85β, fixed and incubated with an anti-HA p85β antibody (red). Arrows in the top panels point to the enhanced autophagic response (LC3-II vesicles) in cells expressing high levels of p85β, as shown in the bottom panels. Uncropped images of blots are shown in Supplementary Fig. S8. Scale bars, 10 μm.

Figure 8

A model of the FBXL2- and PTPL1-dependent regulation of the PI(3)K pathway. SCF^FBXL2 interacts with and promotes the proteasome-mediated degradation of a pool of p85β that is free of p110 subunits. Phosphorylation of p85β on Tyr 655 inhibits its interaction with FBXL2 and is counteracted by the phosphatase PTPL1. Together, SCF^FBXL2 and PTPL1 ensure that free p85β does not accumulate, which would result in the inhibition of the catalytic activity of p110 and/or competition with p85–p110 heterodimers for p-Tyr docking sites (for example, in IRS1).