Figure 6. Raft-targeted p110α induces Akt phosphorylation upon GPCR signaling via EGFR activity.

(A) Schematic demonstration of p110α membrane microdomain targeting vectors. (B) Detergent-free fractionation of DKO+p110α-Lyn and DKO+p110α-Ras MEFs on an Opti-prep gradient followed by western blots with the indicated antibodies. TfnR; a marker for nonraft plasma membrane. Nup; a marker for nuclear membranes. Gq; a marker for membrane rafts. (C) The indicated add-back MEFs were starved and stimulated with LPA. Anti-p-Akt antibodies (for T308 and S473) mark the activation state of Akt. Anti-Akt and anti-actin immunoblots were used as loading controls. On the right, normalized density ratios of the mean fold-increase in baseline Akt phosphorylation at T308 and S473 in starved vs. LPA stimulated states. Graphs denote mean of 3 independent experiments with standard deviation. **p<0.01. (D) DKO+p110α-Lyn and DKO+p110β-wt MEFs were starved and stimulated with LPA in the presence of small molecule inhibitors targeting Gβγ, EGFR or PDGFR. Anti-p-EGFR (for Y1068), anti-p-Akt (for T308) and anti-p-S6 (for S235/236) immunoblots depict activation of EGFR, Akt and downstream signaling. Gal denotes gallein, a Gβγ inhibitor; lap depicts lapatinib, an EGFR inhibitor and cren denotes crenolanib, a PDGFR inhibitor. (E) The indicated MEFs were treated with 0, 0.1, 1, 2.5, 10, 40 or 100 μM of lapatinib or gallein in proliferation assays. Cellular growth was assessed after five days in 2% FBS-DMEM. Error bars denote standard deviation in 3 independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.17635.010

Figure 6—figure supplement 1. Membrane targeting p110α vectors selectively enrich p110α in the desired microdomain.

(A) DKO+p110α-Lyn and DKO+p110α-Ras MEFs were lysed and fractionated into triton sensitive and triton resistant membrane fractions. WCLs were analyzed to display overall levels of protein expression. Soluble, triton soluble (membrane) and resistant membrane fractions (DRM) were analyzed in immunoblots; anti-Rac1 and anti-Caveolin1 antibodies were used as markers for detergent resistant membrane (DRM) rafts, whereas anti-TfnR immunoblot depicts enrichment of nonraft membranes. Anti-tubulin immunoblot serves as a marker for soluble fractions. (B) The indicated DKO add-back MEFs were seeded on coverslips, starved and stimulated with LPA; membrane associated Akt phosphorylation at T308 and S473 was detected using anti-p-Akt T308 and p-Akt S473 antibodies (green). Anti-HA antibodies (red) depicted expression levels of the add-back vectors. DNA is shown in blue. Scale bar; 20 µm. On the right, anti-p-Akt T308 and S473 signals on the cell membrane was quantified upon LPA stimulation and the relative corrected total membrane fluorescence was depicted. Results denote mean of 3 independent experiments with standard deviation. **p<0.01.

DOI: http://dx.doi.org/10.7554/eLife.17635.011

Figure 6—figure supplement 2. Raft-targeted p110α has redundant functions with wt p110β.

(A) PC3 cells were transduced with PLKO tet.on shGFP and shp110β plasmids and samples were collected 48 hr after dox induction. p110β, p-Akt (for T308) and p-S6 (for S235/236) levels were determined in an immunoblot with the indicated antibodies. (B) PC3 cells transduced with PLKO tet.on shp110β vector were used in exogenous overexpression studies of p110α membrane targeting constructs in the presence or absence of dox. Anti-p-Akt immunoblots (for T308 and S473) depict activation of Akt, whereas anti-p-S6 antibodies (for S235/236 and S240/244) demonstrate level of downstream PI3K activation. Anti-p110β and anti-HA immunoblots determine levels of p110β and exogenously expressed p110α respectively. Akt, S6 and actin immunoblots were used as loading controls.

DOI: http://dx.doi.org/10.7554/eLife.17635.012