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.2012 Apr;23(8):1486-99.
doi: 10.1091/mbc.E11-02-0124. Epub 2012 Feb 29.

The endosomal adaptor protein APPL1 impairs the turnover of leading edge adhesions to regulate cell migration

Affiliations

The endosomal adaptor protein APPL1 impairs the turnover of leading edge adhesions to regulate cell migration

Joshua A Broussard et al. Mol Biol Cell.2012 Apr.

Abstract

Cell migration is a complex process that requires the integration of signaling events that occur in distinct locations within the cell. Adaptor proteins, which can localize to different subcellular compartments, where they bring together key signaling proteins, are emerging as attractive candidates for controlling spatially coordinated processes. However, their function in regulating cell migration is not well understood. In this study, we demonstrate a novel role for the adaptor protein containing a pleckstrin-homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1) in regulating cell migration. APPL1 impairs migration by hindering the turnover of adhesions at the leading edge of cells. The mechanism by which APPL1 regulates migration and adhesion dynamics is by inhibiting the activity of the serine/threonine kinase Akt at the cell edge and within adhesions. In addition, APPL1 significantly decreases the tyrosine phosphorylation of Akt by the nonreceptor tyrosine kinase Src, which is critical for Akt-mediated cell migration. Thus, our results demonstrate an important new function for APPL1 in regulating cell migration and adhesion turnover through a mechanism that depends on Src and Akt. Moreover, our data further underscore the importance of adaptor proteins in modulating the flow of information through signaling pathways.

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Figures

FIGURE 1:
FIGURE 1:
APPL1 regulates cell migration. (A) A schematic with the domain structure of APPL1. (B) HT1080 cells were transfected with either GFP or GFP-APPL1, plated on fibronectin, and subjected to time-lapse microscopy. Migration was quantified for individual cells, and Rose plots of migration tracks are shown (left). Quantification of the migration speed for GFP- and GFP-APPL1–expressing cells is shown (right). Error bars represent the standard error of the mean (SEM) for 49–64 cells from at least three separate experiments (*p< 0.0001). (C) HT1080 cells were transfected with empty pSUPER vector, a scrambled siRNA (Src siRNA), or APPL1 siRNA. Cell lysates were subjected to immunoblot analysis to determine the levels of endogenous APPL1 and β-actin (as a loading control; left). Quantification of the endogenous levels of APPL1 in cells transfected with the indicated constructs is shown (right). Error bars represent the SEM from four separate experiments (*p< 0.0001, **p = 0.0002). (D) Cells were transfected with empty pSUPER vector, a scrambled siRNA (Src siRNA), or APPL1 siRNA and used in migration assays 3 d later. Left, Rose plots with migration tracks for cells transfected with the indicated constructs. Right, quantification of the migration speed of cells transfected with the indicated constructs. Error bars represent the SEM for 46–64 cells from three individual experiments (*p< 0.0009). For C and D, asterisks indicate a statistically significant difference compared with pSUPER-transfected cells.
FIGURE 2:
FIGURE 2:
APPL1 localizes to vesicular structures, which is critical in its regulation of cell migration. (A) Wild-type HT1080 cells were immunostained for endogenous APPL1 (left) or transfected with either GFP-APPL1 (middle) or GFP-APPL1 in which three basic residues (147, 153, and 155) within the BAR domain were mutated to alanines (GFP-APPL1-AAA; right). Unlike GFP-APPL1 or the endogenous protein, GFP-APPL1-AAA did not localize to endosomal structures. Scale bar, 15 μm. (B) High-magnification images of the boxed regions from A. Scale bar, 15 μm. (C) GFP- and GFP-APPL1-AAA–transfected cells were imaged using time-lapse microscopy, and migration was analyzed. Rose plots with migration tracks for these cells are shown. (D) Quantification of the migration speed of GFP- and GFP-APPL1-AAA–expressing cells. Error bars represent the SEM for 52–66 cells from at least three separate experiments.
FIGURE 3:
FIGURE 3:
APPL1 impairs adhesion turnover at the leading edge of cells. (A) Cells were transfected with either GFP or GFP-APPL1 and immunostained for the adhesion marker paxillin. Scale bar, 10 μm. (B) HT1080 cells were cotransfected with mCherry-paxillin and either GFP or GFP-APPL1 and imaged using time-lapse microscopy. In GFP-APPL1–expressing cells, the leading edge adhesions assemble and disassemble more slowly than those in control cells expressing GFP (arrows). Scale bar, 5 μm. (C) Quantification of the apparentt1/2 for adhesion assembly and thet1/2 for adhesion disassembly for cells expressing the indicated constructs. Error bars represent the SEM for 26–30 adhesions from four to six cells from at least three independent experiments (*p < 0.0001, **p ≤ 0.007, ***p < 0.013). Asterisks indicate a statistically significant difference compared with GFP cells.
FIGURE 4:
FIGURE 4:
Akt plays an important role in the APPL1-mediated regulation of cell migration. (A) HT1080 cells were cotransfected with GFP or GFP-APPL1 and empty vector, constitutively active Akt (CA-Akt), or dominant-negative Akt (DN-Akt) and used in migration assays. Rose plots with individual migration tracks for cells transfected with the indicated constructs are shown. (B) Quantification of the migration speed of cells transfected with the indicated constructs. Error bars represent the SEM of 35–65 cells from at least three individual experiments (*p< 0.0001). (C) Lysates from HT1080 cells transiently transfected with GFP-APPL1 (Transients) and HT1080 cells stably expressing GFP-APPL1 (Stables) were subjected to immunoblot analysis to determine the levels of total APPL1 (endogenous and exogenously expressed GFP-APPL1; top). Quantification of the relative amounts of GFP-APPL1 compared with endogenous APPL1 is shown (bottom). Error bars represent the SEM from at least three separate experiments (*p< 0.05). Asterisks indicate a statistically significant difference compared with endogenous APPL1. (D) Stable HT1080 cells expressing GFP were transfected with empty vector (GFP). Stable HT1080 cells expressing GFP-APPL1 were transfected with empty vector (GFP-APPL1), 1.5 μg of CA-Akt cDNA (GFP-APPL1 + CA-Akt), or 3 μg of CA-Akt cDNA (GFP-APPL1 + 2X CA-Akt). Left, cell lysates were subjected to immunoblot analysis to determine the levels of total Akt (endogenous and exogenously expressed CA-Akt) and β-actin (as a loading control). Right, quantification of the relative amount of Akt expression compared with that observed in control GFP cells. Error bars represent the SEM from three separate experiments (*p ≤ 0.03). Asterisks indicate a statistically significant difference compared with control GFP cells. (E) Stable HT1080 GFP or GFP-APPL1 cells were transfected as described in D and used in migration assays. Quantification of the migration speed of transfected cells is shown. Error bars represent the SEM of 80–91 cells from three individual experiments (*p< 0.0001). Asterisks indicate a statistically significant difference compared with GFP cells.
FIGURE 5:
FIGURE 5:
Akt knockdown inhibits cell migration. (A) HT1080 cells were cotransfected with empty pSUPER vector, a scrambled siRNA (Scr siRNA), or Akt siRNA and either GFP or GFP-APPL1 and imaged using time-lapse microscopy 3 d later. Rose plots are shown for the migration tracks of cells expressing the indicated constructs. (B) Quantification of the migration speed of cells transfected with the indicated constructs is shown. Error bars represent the SEM of 56–76 cells from three separate experiments (*p< 0.0001).
FIGURE 6:
FIGURE 6:
APPL1 decreases Akt activity in cells. (A) HT1080 cells were cotransfected with either mCherry or mCherry-APPL1 and the Akt activity FRET probe Akind. Ratio images of FRET/CFP are shown in pseudocolor coding (left). Scale bar, 10 μm. Quantification of the average FRET/CFP ratio for cells cotransfected with either mCherry or mCherry-APPL1 and Akind is shown (right). Error bars represent the SEM from >34 cells from at least three individual experiments (*p< 0.0001). (B) HT1080 cells were cotransfected with either mCherry or mCherry-APPL1 and Akind. Ratio images of FRET/CFP at the cell edge are shown in pseudocolor coding (left). Scale bar, 5 μm. A line-scan analysis was performed on the boxed region, which represents an area 5 μm long and 1.3 μm wide. Quantification of the line-scan analysis is shown (right). Error bars represent the SEM of 52–59 total line scans from 20 cells from three separate experiments.
FIGURE 7:
FIGURE 7:
APPL1 reduces the amount of active Akt in adhesions. (A) HT1080 cells were transfected with CFP and either empty vector (Control) or FLAG-APPL1 (APPL1), fixed, and immunostained for active Akt, using a phospho-Thr-308–specific antibody (P-Akt), and paxillin. TIRF images of paxillin (left) and P-Akt (middle) are shown. P-Akt images are shown in pseudocolor coding that indicates the range of fluorescence intensities to the assigned color (P-Akt; right). Overlays of paxillin and P-Akt are shown (far right). Scale bar, 2 μm. (B) Quantification of phospho–Thr-308 Akt levels in adhesions in cells transfected with the constructs from A . Error bars represent the SEM of >4782 adhesions from 58–63 cells from three separate experiments (*p = 0.0251).
FIGURE 8:
FIGURE 8:
Src mediates tyrosine phosphorylation of Akt. (A) FLAG-Akt transfected HT1080 cells were incubated with the indicated concentrations of PP2 for 1.5 h. Left, FLAG-Akt protein was immunoprecipitated from cell lysates, and FLAG-Akt samples were subjected to immunoblot analysis to determine the levels of total FLAG-Akt, using FLAG M2 antibody (FLAG), and tyrosine phosphorylated Akt with 4G10 monoclonal antibody (4G10). Right, quantification of the amount of Akt tyrosine phosphorylation relative to the control (0 μM PP2). Error bars represent the SEM from three separate experiments (*p ≤ 0.0031). (B) HT1080 cells were cotransfected with FLAG-Akt and either GFP (Control) or GFP-CA-Src (CA-Src). Left, immunoprecipitated FLAG-Akt protein samples were immunoblotted for total FLAG-Akt (FLAG) and tyrosine phosphorylated Akt (4G10). Right, quantification of the relative amount of Akt tyrosine phosphorylation compared with control. Error bars represent the SEM from three separate experiments (*p = 0.025). (C) FLAG-Akt was immunoprecipitated from lysates of cells expressing FLAG-Akt and either GFP (Control) or GFP-APPL1 (APPL1). Left, samples were subjected to immunoblot analysis to determine the levels of total FLAG-Akt (FLAG) and tyrosine phosphorylated Akt (4G10). Right, quantification of the relative amount of Akt tyrosine phosphorylation compared with control. Error bars represent the SEM from three separate experiments (*p ≤ 0.0001). (D) HT1080 cells were cotransfected with FLAG-Akt and either mCherry + GFP-CA-Src (CA-Src) or mCherry-APPL1 + GFP-CA-Src (CA-Src + APPL1). Left, immunoprecipitated FLAG-Akt protein samples were subjected to immunoblot analysis to determine the levels of total FLAG-Akt (FLAG) and tyrosine phosphorylated Akt (4G10). Right, quantification of the relative amount of Akt tyrosine phosphorylation compared to that observed in control cells from B. Error bars represent the SEM from three separate experiments (*p = 0.048). Asterisk indicates a statistically significant difference compared with CA-Src–transfected cells.
FIGURE 9:
FIGURE 9:
Tyrosine phosphorylation of Akt regulates its activation and function. (A) HT1080 cells were cotransfected with FLAG-Akt and mCherry + GFP (Control), mCherry-APPL1 + GFP (APPL1), mCherry + GFP-CA-Src (CA-Src), or mCherry-APPL1 + GFP-CA-Src (CA-Src + APPL1). Left, after 24 h, FLAG-Akt was immunoprecipitated from cell lysates and subjected to immunoblot analysis to determine the levels of total FLAG-Akt (FLAG) and T308 phosphorylated Akt (T308). Right, quantification of the relative amount of T308 phosphorylated Akt compared with control. Error bars represent the SEM from at least 10 separate experiments (*p< 0.0001, **p ≤ 0.002, ***p = 0.031). (B) HT1080 cells were transfected with FLAG-Akt (Wt-Akt) or FLAG-Akt-Y315F/Y326F (Akt-Y315F/Y326F). Top, immunoprecipitated FLAG-Akt protein was subjected to immunoblot analysis to determine the levels of total FLAG-Akt (FLAG) and tyrosine phosphorylated Akt (4G10). Bottom, quantification of the relative amount of Akt tyrosine phosphorylation compared with Wt-Akt. Error bars represent the SEM from four separate experiments (*p< 0.0001). (C) HT1080 cells were transfected with GFP-CA-Src (CA-Src) and either FLAG-Akt (Wt-Akt) or FLAG-Akt-Y315F/Y326F (Akt-Y315F/Y326F). Top, after 24 h, FLAG-Akt protein was immunoprecipitated from cell lysates, and samples were subjected to immunoblot analysis to determine the levels of total FLAG-Akt (FLAG) and tyrosine phosphorylated Akt (4G10) . Bottom, quantification of the relative amount of Akt tyrosine phosphorylation compared with that observed in cells transfected with Wt-Akt + CA-Src. Error bars represent the SEM from three separate experiments (*p< 0.0001). (D) HT1080 cells were cotransfected with GFP and empty vector (Control), constitutively active Akt (CA-Akt), or CA-Akt-Y315F/Y326F and used in migration assays. Left, Rose plots with migration tracks for these cells. Right, quantification of the migration speed for cells transfected with the indicated constructs. Error bars represent the SEM for at least 56 cells from at least three separate experiments (*p< 0.0001, **p = 0.0019).
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