Akt-PDK1 complex mediates epidermal growth factor-induced membrane protrusion through Ral activation

Abstract

We studied the spatiotemporal regulation of Akt (also called protein kinase B), phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] by using probes based on the principle of fluorescence resonance energy transfer. On epidermal growth factor (EGF) stimulation, the amount of PtdIns(3,4,5)P3 was increased diffusely in the plasma membrane, whereas that of PtdIns(3,4)P2 was increased more in the nascent lamellipodia than in the plasma membrane of the central region. The distribution and time course of Akt activation were similar to that of increased PtdIns(3,4)P2 levels, which were most prominent in the nascent lamellipodia. Moreover, we found that upon EGF stimulation 3-phosphoinositide-dependent protein kinase-1 (PDK1) was also recruited to nascent lamellipodia in an Akt-dependent manner. Because PDK1 is known to activate Ral GTPase and because Ral is required for EGF-induced lamellipodial protrusion, we speculated that the PDK1-Akt complex may be indispensable for the induction of lamellipodia. In agreement with this idea, EGF-induced lamellipodia formation was promoted by the overexpression of Akt and inhibited by an Akt inhibitor or a Ral-binding domain of Sec5. These results identified the Akt-PDK1 complex as an upstream positive regulator of Ral GTPase in the induction of lamellipodial protrusion.

Figure 1.

Development of a FRET probe for Akt. (A) Schematic representation of Akt Indicator (Akind). YFP and CFP denote a yellow-emitting mutant of GFP and cyan-emitting mutant of GFP, respectively. PH and CD indicate the pleckstrin homology domain and the catalytic domain of Akt, respectively. In this probe design, the FRET level increases when the probe is activated on the plasma membrane. (B) Akind-transfected NIH3T3 cells were analyzed by Western blotting with anti-Akt or anti-phospho-Thr³⁰⁸ of Akt before and after PDGF stimulation. (C) Akind-HA, Flag-Akt-WT, and Flag-Akt-3A expressed in PDGF-stimulated or nonstimulated NIH3T3 cells were immunoprecipitated with an anti-HA antibody or anti-FLAG antibody. Kinase activity was measured by using recombinant GSK3 as a substrate, of which phosphorylation was detected with anti-phospho-GSK3 antibody. A small aliquot of the immunoprecipitates were also analyzed with anti-FLAG antibody.

Figure 2.

Akt activation in the nascent lamellipodia as monitored by Akind. (A) Akind-expressing NIH3T3 cells were serum starved for 24 h and stimulated with 50 ng/ml PDGF. CFP (excitation [ex] 440 nm/emission [em] 480 nm) and sensitized FRET (ex 440 nm/em 530 nm) images were obtained every 1 min with a time-lapse epifluorescent microscope. Ratio image of FRET/CFP was prepared to demonstrate the level of FRET (also available as Supplemental Video 1). At least 20 similar images were obtained. In the right panel, the net intensities of CFP and FRET in each cell were measured to calculate the averaged emission ratio (FRET/CFP). Then, the emission ratio values were normalized to those of the record-starting time. Data from more than 10 independent cells are shown with SE. Phosphorylation of endogenous Akt was also examined by immunoblotting and quantitated values are overlaid to the graph. Similar experiments were performed by using EGF-stimulated Cos7 cells (B and Supplemental video 2) and insulin-stimulated Cos7 cells (C). (D) Cell images used in B were used to negate the correlation between the FRET level at the zenith and the concentration of the probe. (E) Cos7 cells expressing Akt wild type or mutant (3A, 2A, KA, and TA) were serum starved for 24 h and stimulated with 50 ng/ml EGF as described in D. Bars, 10 μm.

Figure 3.

Semiquantitative analysis of plasma membrane translocation of Akind. Signal-dependent translocation of CFP-tagged cytosolic proteins to the plasma membrane was measured semiquantitatively by using RFP as a reference (for details, see Supplemental Material). (A) The translocation index and the FRET level of Cos7 cells expressing Akind were plotted against time. (B) Shown are time courses of the translocation index of cells expressing wild type, KA mutant, and 3A mutant of Akind.

Figure 4.

High Akt activity and accumulation of PtdIns(3,4)P₂ in the nascent lamellipodia. (A) Schematic representation of Pippi-PI(3,4)P₂. TAPP1-PH indicates the pleckstrin homology domain of TAPP1. The PH domain is sandwiched is by CFP and Venus. Venus and the PH domain are connected by means of rigid α-helical linkers consisting of repeated EAAAR sequences, between which is a single Gly-Gly motif introduced as a hinge. At the C terminus of Venus is the hypervariable region of K-Ras used as a plasma membrane-anchoring motif. Thus, the probes are designed to propagate high FRET signal when the PH domain binds to PtdIns(3,4)P₂. (B) NIH3T3 cells expressing Pippi-PI(3,4)P₂ were serum starved for 24 h and stimulated with 50 ng/ml PDGF. FRET imaging was performed as described in Figure 2. (C) Pippi-PI(3,4)P₂-expressing NIH3T3 cells untreated or treated with 40 μM LY294002 were stimulated with 50 ng/ml PDGF. The time course of the FRET level was obtained as in Figure 2. Cells were transfected with pSuper-PTEN, SHIP2, or luciferase, an shRNA vector, and selected by puromycin. Sixty hours posttransfection, FRET imaging was performed as described above. Averaged data from five cells are shown with SD. (D) Cos7 cells expressing Akind, Pippi-PI(3,4)P₂, or Pippi-PI(3,4,5)P₃ were serum starved for 24 h and stimulated with 50 ng/ml EGF. FRET images were obtained by a conventional epifluorescent microscope (D and Supplemental Videos 3 and 4). The FRET/CFP ratio image was used to show the FRET level in the intensity-modulated display mode. (E) Along the white lines in D, FRET/CFP values are plotted. (F) Cos7 cells prepared as described in D were imaged with a spinning disk confocal microscope. X-Y and Y-Z sections are shown. Bar, 10 μm.

Figure 5.

Accumulation of PDK1–Akt complex in nascent lamellipodia. (A and B) Cos7 cells expressing Akt-YFP and CFP-PDK1 were stimulated with 50 ng/ml EGF. Three images, CFP (ex 440 nm/em 480 nm), sensitized FRET (ex 440 nm/em 530 nm), and YFP (ex 510 nm/em 565 nm) were obtained every 30 s for 1 h. Corrected FRET (cFRET) images prepared as described in the text are shown in a pseudocolor mode (A). Net intensities of CFP and YFP in each cell were measured to calculate the averaged cFRET/CFP. (C) The Venus/CFP ratio values obtained from each pixel are plotted against cFRET. Pixels with YFP/CFP value over 1.0 corresponds those in the nucleus. (D) Cos7 cells expressing CFP-PDK1 in the presence or absence of Akt were stimulated with 50 ng/ml EGF and time-lapse imaged. (E) From each cell image, the translocation index of CFP-PDK1 were determined and plotted against time (n ≥ 18).

Figure 6.

Requirement of Akt for EGF-induced RalA activation. (A) Cos7 cells transfected with or without an expression vector for a constitutively active mutant of PI3K, p110-x. Cells were serum starved for 8 h and left untreated or treated with LY294002 or Akt inhibitor IV for 30 min. The levels of endogenous GTP-Ral were assayed by Bos' pull-down method and quantitated with an LAS-1000 image analyzer. Averaged data from two independent experiments are shown with SE. (B) Cos7 cells transfected with or without an expression vector for GFP-RalA and a constitutively active mutant of Akt mDPH-Akt were serum starved for 8 h and left untreated or treated with LY294002. Cells with or without 50 ng/ml EGF stimulation were analyzed as described in A. (C) Cos7 cells expressing GFP-RalA with or without Myc-RalGDS were serum starved for 8 h and left untreated or treated with Aki inhibitor IV for 30 min before stimulation with 50 ng/ml EGF for 10 min. The level of GTP-bound GFP-RalA was analyzed as described in text.

Figure 7.

Requirement of Akt for EGF-induced membrane protrusion. (A) Parent and Akt-expressing Cos7 cells were stimulated with 50 ng/ml EGF. Differential interference contrast images of these cells were obtained every 1 min (top). White and black lines indicated the outlines of the cells before and 10 min after EGF stimulation, respectively. Bar, 10 μm. (B) Averaged extended cell areas, which was obtained by the reduction of black-lined area from white-lined area, are shown with SE (bottom). n ≥ 12.

Figure 8.

Association of Akt and RalGDS in a PDK1-dependent manner. Cos7 cells expressing Akt and Myc-RalGDS with or without HA-PDK1 were serum starved for 8 h and stimulated with 50 ng/ml EGF for 10 min. Cells were lysed and immunoprecipitated with anti-Myc monoclonal antibody (mAb), anti-HA rat mAb, or anti-Akt rabbit antibody. The immunoprecipitates (IP) and total cell lysates (TLC) were analyzed by immunoblotting.