Galphaq binds to p110alpha/p85alpha phosphoinositide 3-kinase and displaces Ras

Abstract

Several studies have reported that activation of G(q)-coupled receptors inhibits PI3K (phosphoinositide 3-kinase) signalling. In the present study, we used purified proteins to demonstrate that Galpha(q) directly inhibits p110alpha/p85alpha PI3K in a GTP-dependent manner. Activated Galpha(q) binds to the p110alpha/p85alpha PI3K with an apparent affinity that is seven times stronger than that for Galpha(q).GDP as measured by fluorescence spectroscopy. In contrast, Galpha(q) did not bind to the p110gamma PI3K. Fluorescence spectroscopy experiments also showed that Galpha(q) competes with Ras, a PI3K activator, for binding to p110alpha/p85alpha. Interestingly, co-precipitation studies using deletion mutants showed that Galpha(q) binds to the p85-binding domain of p110alpha and not to the Ras-binding domain. Expression of constitutively active Galpha(q)Q209L in cells inhibited Ras activation of the PI3K/Akt pathway but had no effect on Ras/Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] signalling. These results suggest that activation of G(q)-coupled receptors leads to increased binding of Galpha(q).GTP to some isoforms of PI3K, which might explain why these receptors inhibit this signalling pathway in certain cell types.

Figure 1. Inhibition of PI3K by Gα_q

Gα_q·GDP and Gα_q·GTP[S], along with an equal volume of GDP buffer or GTP[S] buffer as controls, were diluted in PI3K assay buffer and concentrated using a Microcon 30 microconcentrator (Amicon, Beverly, MA, U.S.A.). This was repeated three more times to remove nucleotides and CHAPS, which interferes with the PI3K assay ([26] and results not shown). PI3K assays contained 25 nM purified p110α/p85α complex and either 15 nM Gα_q or an equal volume of control buffer. L-α-phosphatidylinositol (Sigma) and [γ-³²P]ATP (PerkinElmer LifeSciences) were used as substrates. The autoradiogram shows a typical result from four experiments. Abbreviations: GTPγS, GTP[S]; PI-3P, phosphatidylinositol 3-phosphate.

Figure 2. Gα_q binds to PI3K

Purified recombinant p110α/p85α complex was titrated into a solution containing 5 nM coumarin-labelled Gα_q·GTP[S] (●) or Gα_q·GDP (○) reconstituted into 80 μM POPC/PS (2:1) LUVs. Recombinant p110γ was added to a solution containing 5 nM coumarin-labelled Gα_q·GTP[S] (■) in LUVs. Fluorescence measurements and data analysis were performed as described in the Materials and methods section. The results shown are from three independent experiments performed in triplicate.

Figure 3. Gα_q competes with Ras for binding to PI3K

LUVs were reconstituted with 10 nM coumarin-labelled Ras·GDP (○) or coumarin–Ras·GTP[S] without (●) or with (▲) 10 nM Gα_q·GTP[S]. Purified recombinant p110α/p85α was titrated into the solutions and fluorescence measurements were made. The results shown are from three independent experiments performed in triplicate. GTPγS, GTP[S].

Figure 4. Gα_q binds to the p85-binding domain (p85 BD) of p110α

(A) Purified GST–p110α fusion proteins encompassing individual domains of p110α (50 ng) were immobilized on glutathione–Sepharose beads and mixed at 4 °C for 1 h with 250 μg of lysate proteins from FreeStyle 293 cells expressing HA–Gα_qQ209L. Cells were lysed in a buffer containing 1% Nonidet P40 plus 0.25% sodium deoxycholate (see the Materials and methods section). The beads were washed with lysis buffer and bound proteins were analysed on a Western blot probed with HA antibody. Native GST protein immobilized on beads was used as a negative control (first lane). The last lane is a sample of cell lysate expressing HA–Gα_qQ209L. The experiment was repeated with similar results. (B) COS7 cells were co-transfected with HA–Gα_qQ209L and either empty vector, FLAG–p110α amino acids 1–116, FLAG–p110α amino acids 118–1068 or FLAG–p110γ. Cell lysates were incubated with FLAG antibody and the immunoprecipitates were analysed on a Western blot probed with HA antibody (top panel). Western-blot analysis of total cell lysate proteins showed appropriate expression of HA–Gα_qQ209L and the various FLAG-tagged p110 proteins (bottom two panels). IP, immunoprecipitate; WB, Western blot. The experiment was repeated with similar results.

Figure 5. Specific inhibition of the Ras/PI3K/Akt pathway by Gα_q

HEK-293 cells were co-transfected with (A) Akt–HA or (B) HA–MEK1 with or without V12Ras in the presence or absence of Gα_qQ209L. Cells were incubated in serum-free medium overnight prior to lysate preparation. (A) Akt activity was assayed in HA immunoprecipitates of cell lysates (mean±S.E.M.; n=3). The inset shows a Western blot of cell lysates probed with HA antibody. (B) Activation of MEK1 was assessed on a Western blot probed with phospho-MEK1/2 antibody (upper panel). The blot was reprobed with HA antibody (lower panel).