LAPTM4B is a PtdIns(4,5)P 2 effector that regulates EGFR signaling, lysosomal sorting, and degradation
2015; Springer Nature; Volume: 34; Issue: 4 Linguagem: Inglês
10.15252/embj.201489425
ISSN1460-2075
AutoresXiaojun Tan, Yue Sun, Narendra Thapa, Yihan Liao, Andrew C. Hedman, Richard A. Anderson,
Tópico(s)RNA Interference and Gene Delivery
ResumoArticle14 January 2015free access Source Data LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling, lysosomal sorting, and degradation Xiaojun Tan Xiaojun Tan Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Yue Sun Yue Sun Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Narendra Thapa Narendra Thapa Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Yihan Liao Yihan Liao Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Andrew C Hedman Andrew C Hedman Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Richard A Anderson Corresponding Author Richard A Anderson Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Xiaojun Tan Xiaojun Tan Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Yue Sun Yue Sun Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Narendra Thapa Narendra Thapa Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Yihan Liao Yihan Liao Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Andrew C Hedman Andrew C Hedman Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Richard A Anderson Corresponding Author Richard A Anderson Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA Search for more papers by this author Author Information Xiaojun Tan1, Yue Sun1, Narendra Thapa1, Yihan Liao1, Andrew C Hedman1 and Richard A Anderson 1 1Program in Molecular and Cellular Pharmacology, University of Wisconsin–Madison, School of Medicine and Public Health, Madison, WI, USA *Corresponding author. Tel: +1 608 262 7436; E-mail: [email protected] The EMBO Journal (2015)34:475-490https://doi.org/10.15252/embj.201489425 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Lysosomal degradation is essential for the termination of EGF-stimulated EGF receptor (EGFR) signaling. This requires EGFR sorting to the intraluminal vesicles (ILVs) of multi-vesicular endosomes (MVEs). Cytosolic proteins including the ESCRT machineries are key regulators of EGFR intraluminal sorting, but roles for endosomal transmembrane proteins in receptor sorting are poorly defined. Here, we show that LAPTM4B, an endosomal transmembrane oncoprotein, inhibits EGF-induced EGFR intraluminal sorting and lysosomal degradation, leading to enhanced and prolonged EGFR signaling. LAPTM4B blocks EGFR sorting by promoting ubiquitination of Hrs (an ESCRT-0 subunit), which inhibits the Hrs association with ubiquitinated EGFR. This is counteracted by the endosomal PIP kinase, PIPKIγi5, which directly binds LAPTM4B and neutralizes the inhibitory function of LAPTM4B in EGFR sorting by generating PtdIns(4,5)P2 and recruiting SNX5. PtdIns(4,5)P2 and SNX5 function together to protect Hrs from ubiquitination, thereby promoting EGFR intraluminal sorting. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B, PtdIns(4,5)P2 signaling, and the ESCRT complex and define a mechanism by which the oncoprotein LAPTM4B can transform cells and promote tumor progression. Synopsis The oncogene LAPTM4B prolongs EGFR signalling. LAPTM4B promotes ubiquitination of ESCRT component Hrs, preventing endosomal EGFR sorting, which is counteracted by phosphoinositide PtdIns(4,5)P2. LAPTM4B inhibits EGF-stimulated EGFR-intraluminal sorting and degradation and enhances EGFR signaling LAPTM4B promotes Hrs ubiquitination by the E3 ubiquitin ligase Nedd4 LAPTM4B interacts with PIPKIγi5, an enzyme generating PtdIns(4,5)P2 at endosomes LAPTM4B is a PtdIns(4,5)P2 effector, and PtdIns(4,5)P2 binding alleviates the inhibitory effect of LAPTM4B on EGFR degradation Introduction Epidermal growth factor receptor (EGFR) plays fundamental roles not only in physiological cellular processes, but also in diseases such as cardiovascular hypertrophy and cancers (Kagiyama et al, 2002; Eguchi et al, 2003; Mendelsohn & Baselga, 2006). Therefore, EGFR expression levels and signaling strength must be tightly controlled. One key mechanism to downregulate EGFR signaling is the lysosomal trafficking and degradation of the activated receptor. Upon ligand binding, activated EGFR is rapidly internalized to endosomes, where ligand-bound EGFR continues to signal until it is sorted to intraluminal vesicles (ILVs) in the multi-vesicular endosomes (MVEs) or late endosomes (Wiley, 2003; Sorkin & Goh, 2008). Finally, the MVE fuses with the lysosome, resulting in EGFR degradation (Eden et al, 2009). Intraluminal sorting of EGFR is an essential step that terminates EGFR signaling, which is mediated by the endosomal sorting complex required for transport (ESCRT) machineries (Williams & Urbé, 2007; Raiborg & Stenmark, 2009; Henne et al, 2011). The ESCRT-mediated EGFR ILV sorting pathway requires ubiquitination of EGFR (Williams & Urbé, 2007). Upstream ESCRT subunits, including Hrs and TSG101, contain ubiquitin-interacting motifs (UIM) that recognize ubiquitinated EGFR, and cooperate with downstream ESCRT complexes for EGFR ILV sorting (Raiborg & Stenmark, 2009). Hrs, like other ESCRT subunits, is a cytosolic protein that is recruited to the endosome by phosphoinositides and protein–protein interactions (Di Paolo & De Camilli, 2006; Lindmo & Stenmark, 2006; Henne et al, 2013). The function of Hrs is also regulated by the E3 ubiquitin ligases Nedd4-1 and Nedd4-2 that ubiquitinate Hrs and trigger an intramolecular interaction between the Hrs-UIM and ubiquitin (Katz et al, 2002; Hoeller et al, 2006; Persaud et al, 2009). This interaction inhibits Hrs function by preventing it from binding to ubiquitinated cargos, like EGFR. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is a lipid messenger that regulates many cellular processes, including actin and focal adhesion dynamics, endocytosis, exocytosis, and gene expression (Anderson & Marchesi, 1985; Ling et al, 2002; Mellman et al, 2008; Thapa et al, 2012; Balla, 2013; Sun et al, 2013c). PtdIns(4,5)P2 has been traditionally thought to be largely at the plasma membrane (Di Paolo & De Camilli, 2006), but a broader intracellular distribution and synthesis have been recently revealed (Sun et al, 2013c). Type I phosphatidylinositol-4-phosphate (PIP) 5-kinases (PIPKIα, β, and γ) are the major enzymes for PtdIns(4,5)P2 generation in cells (Heck et al, 2007). PIPKI has critical functions in various protein trafficking processes, including endocytosis, exocytosis, and endosomal trafficking (Bairstow et al, 2006; Schramp et al, 2012; Thapa et al, 2012; Sun et al, 2013a,b). There are six splice variants of human PIPKIγ (i1-i6), each with distinct C-terminal extensions that mediate specific protein–protein interactions, leading to distinct intracellular targeting of each isoform (Schill & Anderson, 2009; Xia et al, 2011). PIPKIγi5 is targeted to endosomes and generates phosphoinositide signals that control EGFR intraluminal sorting (Sun et al, 2013b). This pathway requires an interaction between PIPKIγi5 and sorting nexin 5 (SNX5). PIPKIγi5 and its kinase activity regulate the interaction of SNX5 with Hrs to protect Hrs from ubiquitination and promote the Hrs association with EGFR. Thus, PIPKIγi5, its kinase activity, and SNX5 control Hrs function in EGFR intraluminal sorting and degradation (Sun et al, 2013b). All the ESCRT subunits including Hrs are cytosolic proteins recruited to endosomal surface during ILV sorting. The roles for resident endosomal transmembrane proteins in the regulation of ESCRT complexes and ILV sorting are poorly defined. A family of the resident proteins is the mammalian lysosomal-associated protein transmembrane (LAPTM) that has three members, LAPTM4A, LAPTM4B, and LAPTM5, with ~36% sequence similarities. All LAPTMs are multi-transmembrane proteins primarily localized to the late endosome/lysosome (Adra et al, 1996; Hogue et al, 2002; Shao et al, 2003; Pak et al, 2006; Milkereit & Rotin, 2011). LAPTM4B has four transmembrane domains, with two cytoplasmic termini (Shao et al, 2003). LAPTM4B is upregulated in a wide variety of human cancers, including breast, liver, lung, colon, uterine, and ovarian cancers (Shao et al, 2003; Kasper et al, 2005; Li et al, 2010b). LAPTM4B overexpression in cancers correlates with poor prognosis (Yang et al, 2010b; Kang et al, 2012). Further, ectopic expression of LAPTM4B induces transformation and tumorigenesis of normal human cells (Li et al, 2011) and promotes proliferation and migration of cancer cells in vitro and in vivo (Yang et al, 2010a). The underlying mechanisms for the LAPTM4B oncogenesis are not defined, but LAPTM4B overexpression enhances AKT activation (Li et al, 2010a). Here, we report that LAPTM4B blocks EGF-stimulated EGFR intraluminal sorting and degradation. In this pathway, LAPTM4B binds to PIPKIγi5 and its product PtdIns(4,5)P2, and this neutralizes LAPTM4B inhibition of EGFR trafficking. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B and phosphoinositide signaling and may represent the underlying mechanism for LAPTM4B oncogenesis. Results PIPKIγi5 interacts with endosomal transmembrane protein LAPTM4B The endosomal PIP kinase, PIPKIγi5, generates the lipid messenger PtdIns(4,5)P2 and is required for EGFR intraluminal sorting and degradation (Sun et al, 2013b). Based on a yeast two-hybrid screen using the C-terminal 223 amino acids of PIPKIγi5 as bait (Sun et al, 2013b), the lysosomal-associated protein Transmembrane 4B (LAPTM4B) was identified as a PIPKIγi5 interactor. The interaction between endogenous LAPTM4B and PIPKIγi5 was confirmed by co-immunoprecipitation (co-IP) (Fig 1A). This is a specific interaction, as among the three LAPTM family members, PIPKIγi5 specifically associated with LAPTM4B (Fig 1B). The LAPTM4B interaction is also specific for PIPKIγi5, but not other PIPKIγ isoforms (Fig 1C). To test whether the kinase activity of PIPKIγi5 modulates its LAPTM4B interaction, a D316A kinase dead mutant (PIPKIγi5KD) was used in co-IP assays. As shown in Fig 1D, PIPKIγi5KD had diminished LAPTM4B association, indicating that phosphoinositide generation is required. PIPKIγi5 modulates EGF-stimulated EGFR lysosomal trafficking, but the PIPKIγi5–LAPTM4B interaction was not regulated by EGF stimulation (Fig 1D). Figure 1. PIPKIγi5 specifically interacts with the endosomal transmembrane protein LAPTM4B Endogenous PIPKIγi5 and LAPTM4B were immunoprecipitated from the whole-cell lysates of MDA-MB-231 cells followed by immunoblotting to examine the co-immunoprecipitated PIPKIγi5 and LAPTM4B. PIPKIγi5 specifically interacts with LAPTM4B, but not LAPTM4A or LAPTM5. Top: schematic diagram of all three LAPTM members. Bottom: each Flag-tagged LAPTM protein was immunoprecipitated from HEK293 cells cotransfected with Myc-tagged PIPKIγi5 and empty vector or Flag-tagged LAPTM, and the co-immunoprecipitated PIPKIγi5 was examined by immunoblotting. LAPTM4B selectively associates with PIPKIγi5. Top: schematic diagram of four PIPKI isoforms. Bottom: Myc-tagged PIPKIγi5 was immunoprecipitated from HEK293 cells expressing Flag-tagged LAPTM4B and each isoform of PIPKI, followed by immunoblotting to examine the co-immunoprecipitated LAPTM4B. Kinase activity of PIPKIγi5 is required for LAPTM4B association. HEK293 cells expressing LAPTM4B and wild-type (WT) or kinase-dead (KD) PIPKIγi5 were starved overnight, stimulated or not with 100 ng/ml EGF for 15 min, and harvested for immunoprecipitation with anti-myc. The co-immunoprecipitated LAPTM4B was detected by immunoblotting. Endogenous LAPTM4B is targeted to both early and late endosomes. MDA-MB-231 cells were fixed and costained for endogenous LAPTM4B (red) and EEA1 or LAMP1 (green). Boxes are selected regions for magnified view. Note: non-specific nuclear staining by the LAPTM4B anti-sera. Scale bar: 10 μm. Quantification of LAPTM4B colocalization with EEA1 and LAMP1 (mean + SD; n ≥ 4). Schematic diagram for LAPTM4B endosomal localization based on quantification in (F). LAPTM4B partially colocalizes with PIPKIγi5. MDA-MB-231 cells expressing Myc-tagged PIPKIγi5 were stained with LAPTM4B anti-sera (red), anti-Myc (green), and DAPI. Box is selected region for magnified view. Scale bar: 10 μm. MDA-MB-231 cells stably expressing Flag-LAPTM4B were stained with LAPTM4B anti-sera followed by silver-enhanced immuno-electron microscopy. The early and late MVEs were defined by the number of intraluminal vesicles. N, nucleus; M, mitochondria; MVE, multi-vesicular endosome; and PM, plasma membrane. Scale bars: 2 μm (left); 200 nm (middle and right). Data information: Data are representative for at least four independent experiments. IP, immunoprecipitate; WCL, whole-cell lysates. Source data are available online for this figure. Source Data for Figure 1 [embj201489425-sup-0008-sourcedata_Fig1.pdf] Download figure Download PowerPoint Ectopically expressed LAPTM4B localizes to late endosomes and lysosomes (Milkereit & Rotin, 2011; Vergarajauregui et al, 2011). Consistently, we observed that HA-tagged LAPTM4B was primarily colocalized with late endosome/lysosome markers CD63 and LAMP1 and a partial overlap with early endosome marker EEA1 (Supplementary Fig S1A). To ascertain the subcellular localization of the endogenous protein, rabbit polyclonal LAPTM4B anti-sera were used to stain cells. The anti-sera stained endogenous LAPTM4B with significant colocalization with both LAMP1 and EEA1 (Fig 1E and F), indicating a wide distribution of LAPTM4B through the endosomal system (Fig 1G). The specificity of the LAPTM4B anti-sera staining was validated by LAPTM4B knockdown that eliminated the endosomal but not the nuclear staining (Supplementary Fig S1B and C). LAPTM4B knockdown did not change LAPTM5 staining (Supplementary Fig S1B), indicating that both the LAPTM4B siRNA and anti-sera are specific. To determine whether LAPTM4B and PIPKIγi5 colocalize in cells, Myc-tagged PIPKIγi5 was expressed and costained with endogenous LAPTM4B. As shown in Fig 1H, PIPKIγi5 was localized to subdomains of LAPTM4B-positive endosomes. Loss of LAPTM4B did not prevent endosomal targeting of PIPKIγi5 (Supplementary Fig S1C), consistent with additional PIPKIγi5 targeting factors at endosomes. As LAPTM4B is a transmembrane protein at endosomes, we examined whether LAPTM4B is targeted to both endosomal limiting membrane and intraluminal vesicles by silver-enhanced immuno-electron microscopy (immuno-EM) that detects the subendosomal localization of LAPTM4B. As shown in Fig 1I, LAPTM4B specifically accumulated at MVEs, on both the limiting membrane and intraluminal vesicles. Early MVEs have fewer ILVs, and LAPTM4B was primarily at the limiting membrane (Fig 1I, right). These data support that LAPTM4B is initially sorted to the limiting membrane of MVEs and then partially sorted onto ILVs as the MVE matures. This is consistent with a partial colocalization between LAPTM4B and PIPKIγi5 at endosome surfaces. LAPTM4B inhibits EGF-stimulated EGFR degradation PIPKIγi5 plays a key role in ESCRT-mediated EGFR ILV sorting and lysosomal degradation (Sun et al, 2013b). As LAPTM4B interacts with PIPKIγi5 (Fig 1), we explored whether LAPTM4B also regulates EGF-stimulated EGFR degradation. Endogenous LAPTM4B expression was knocked down by siRNA in MDA-MB-231 cells. Strikingly, the degradation rate of EGFR was significantly enhanced after LAPTM4B knockdown (Fig 2A and B). After 1 h of EGF stimulation, the EGFR levels in control cells were not significantly reduced, but half of the EGFR was degraded in LAPTM4B knockdown cells (Fig 2A and B). Accelerated EGFR degradation after LAPTM4B knockdown also reduced EGFR and AKT signaling (Fig 2A, C and D). Knockdown of LAPTM4B in A431 cells (Supplementary Fig S2A) resulted in even more dramatic acceleration of EGFR degradation (Supplementary Fig S2B, CQ- and C), indicating that this was not a cell type-specific result. Pretreatment with the lysosomal inhibitor chloroquine fully blocked EGFR degradation in MDA-MB-231 and A431 cells pretreated with either control or LAPTM4B siRNA (Supplementary Fig S2B, CQ+ and D), indicating that the EGFR degradation in LAPTM4B knockdown cells remains lysosomal mediated. A distinct siRNA (siLAPTM4B#2) also efficiently knocked down LAPTM4B expression (Supplementary Fig S2E) and accelerated EGFR degradation (Supplementary Fig S2F and G). Figure 2. LAPTM4B knockdown accelerates EGF-stimulated EGFR degradation A. MDA-MB-231 cells transfected with control or LAPTM4B siRNA were starved and stimulated with 100 ng/ml EGF for indicated time periods, followed by whole-cell lysate harvest for immunoblotting analysis of EGFR levels. B–D. Quantification of the levels of EGFR (B), pEGFR (Y1068)(C), and pAKT (S473)(D) from the analysis in (A) (mean ± SD; n = 3). E. Control or LAPTM4B siRNA-transfected MDA-MB-231 cells were starved, pulsed with 25 ng/ml Alexa-555-EGF for 3 min, washed, and chased for indicated time periods followed by fixation, DAPI staining, and fluorescence microscopy. Scale bar: 10 μm. F. Quantification of the relative amounts of Alexa-555-EGF internalized in the indicated conditions (mean + SD, n = 3). G. Quantification of the Alexa-555-EGF degradation in control and LAPTM4B knockdown cells in (E) (mean ± SD, n = 3). H. Control or LAPTM4B-overexpressing MDA-MB-231 cells were starved and then stimulated with 100 ng/ml EGF for 1–4 h. EGFR degradation and signaling were analyzed by Western blot. Specific antibodies recognizing pEGFR (Y1068) and pAKT (S473) were used. I–K. Quantification for the levels of EGFR (I) and pEGFR (J) normalized to actin and pAKT (K) normalized to AKT in control or LAPTM4B-overexpressing cells (mean ± SD, n = 3). Data information: Data are representative for at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one-tailed t-test. Source data are available online for this figure. Source Data for Figure 2 [embj201489425-sup-0009-sourcedata_Fig2.pdf] Download figure Download PowerPoint To further confirm that loss of LAPTM4B accelerates EGF-stimulated EGFR degradation, a pulse-chase experiment using Alexa-555-EGF was performed to analyze EGF degradation in control or LAPTM4B knockdown cells by fluorescence microscopy (Fig 2E). After a brief pulse with a lower concentration of Alexa-555-EGF, only a small pool (~10%) of total EGFR is EGF-bound and internalized (Fig 2F). Though similar amounts of EGF were initially internalized in control and LAPTM4B knockdown cells, the loss of EGF was more rapid in cells lacking LAPTM4B (Fig 2E and G). The combined results confirm that LAPTM4B inhibits EGF-stimulated EGFR degradation and enhances EGFR signaling. LAPTM4B is overexpressed in many human cancers (Kasper et al, 2005; Li et al, 2010b). Therefore, we investigated whether ectopic expression of LAPTM4B could inhibit EGFR degradation. As shown in Fig 2H–K, overexpression of LAPTM4B strongly inhibited EGF-stimulated EGFR degradation, resulting in greatly enhanced and prolonged activation of EGFR and AKT. LAPTM4B inhibits EGFR trafficking through late endosomes LAPTM4B is an endosomal protein suggesting that it may inhibit EGFR degradation by modulating EGFR endosomal trafficking. Cells were stimulated with EGF, and EGFR was costained with EEA1 and LAMP1, respectively. Trafficking of EGFR through these compartments was analyzed by quantifying the colocalization of EGFR with EEA1 or LAMP1. After 15 min, the majority of EGFR accumulated at EEA1 compartments in both control and LAPTM4B knockdown cells (Fig 3A and B), signifying that internalization and trafficking to the early endosome were not affected. After 2 h, EGFR colocalized well with LAMP1 in control cells but surprisingly not in LAPTM4B knockdown cells where EGFR showed more colocalization with EEA1 [Fig 3A–D, chloroquine (−)]. It is important to note that EGFR degradation is more rapid in LAPTM4B knockdown cells compared to control cells (Fig 2); the decreased EGFR colocalization with LAMP1 may result from accelerated lysosomal delivery and degradation of EGFR in knockdown cells, but not a block of EGFR trafficking at the early endosome. To confirm this possibility, the EGFR trafficking assay was performed in cells pretreated with lysosomal inhibitor chloroquine to block EGFR degradation. As shown in Fig 3A–D, chloroquine pretreatment rescued EGFR colocalization with LAMP1 in LAPTM4B knockdown cells and decreased EGFR colocalization with EEA1. These combined results indicate that EGFR is delivered faster into lysosomes for degradation upon loss of LAPTM4B. Figure 3. LAPTM4B inhibits EGF-stimulated EGFR endosomal sorting A–D. Control or LAPTM4B siRNA-transfected MDA-MB-231 cells were starved, pretreated or not with chloroquine for 2 h, stimulated with 100 ng/ml EGF for 15 min, washed, and chased for indicated time periods before fixation for costaining of EGFR (red) with EEA1 (A, green) or LAMP1 (C, green). Quantification of the average percentages of EGFR signals colocalized with EEA1 (B) and LAMP1 (D) at indicated time points; mean + SD; n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, one-tailed t-test. E. MDA-MB-231 cells were starved, stimulated with 100 ng/ml EGF for 15 or 120 min, fixed, and costained for EGFR and LAPTM4B, followed by fluorescence microscopy. Cells with higher and lower LAPTM4B expression were marked with arrows and arrowheads, respectively. F. The amounts of total EGFR staining in individual cells in (E) were plotted against LAPTM4B levels at 15 min and 120 min, respectively. Note: for LAPTM4B quantification, the non-specific nuclear staining was not included. Trend lines and Pearson's correlation coefficients are shown. AU, arbitrary unit. Data information: Data are representative for three independent experiments. Boxes are selected regions for magnified view. Scale bars: 10 μm. Download figure Download PowerPoint In cells, LAPTM4B is stochastically expressed with some cells having high and others low LAPTM4B levels (Fig 3E, see arrows). To determine whether EGF-stimulated EGFR trafficking is slowed in LAPTM4B-positive late endosomes, cells were stimulated with EGF for 15 min or 120 min and then fixed and costained for endogenous LAPTM4B and EGFR. After 15 min of EGF stimulation, all cells had similar amounts of EGFR staining at endosomes with partial colocalization with LAPTM4B (Fig 3E, top). After 120 min of EGF stimulation, significantly more EGFR was detected in cells with higher LAPTM4B expression (arrows), and the remaining EGFR colocalized with LAPTM4B (Fig 3E, bottom). These results indicate that EGF-stimulated EGFR trafficking is inhibited in LAPTM4B-positive endosomes. The scatter plot of the EGFR versus LAPTM4B levels in each cell at 15 min and 120 min, respectively, was shown in Fig 3F; no significant correlation between EGFR and LAPTM4B levels at 15 min was detected, but a positive correlation was observed at 120 min, consistent with the model that enhanced LAPTM4B expression inhibits EGFR degradation. LAPTM4B inhibits EGF-stimulated EGFR intraluminal sorting LAPTM4B may enhance EGFR signaling by inhibiting EGF-stimulated EGFR intraluminal sorting in LAPTM4B-positive endosomes. For this, intraluminal sorting of Alexa-555-EGF into enlarged endosomes induced by expression of constitutively active Rab5Q79L was quantified (Stenmark et al, 1994; Simonsen et al, 1998; Hanafusa et al, 2011). The results demonstrate diminished intraluminal sorting of EGF occurred at endosomes with higher LAPTM4B staining (Fig 4A). Quantification of EGF intraluminal sorting revealed a significant inverse relationship between the EGF intraluminal sorting and LAPTM4B levels at individual endosomes (Fig 4B), suggesting that LAPTM4B inhibits intraluminal sorting of EGF at LAPTM4B-positive endosomes. Figure 4. LAPTM4B inhibits EGF-stimulated intraluminal sorting of EGFR MDA-MB-231 cells expressing GFP-Rab5Q79L were starved and stimulated with 100 ng/ml Alexa-555-EGF for 90 min followed by fixation and immunostaining for LAPTM4B (blue). The percentages of luminal Alexa-555-EGF in individual endosomes were plotted against endosomal LAPTM4B levels. Trend lines and Pearson's correlation coefficients are shown. Data are representative for three independent experiments. Control or LAPTM4B knockdown MDA-MB-231 cells were starved overnight, cell surface EGFR was labeled with immuno-gold on ice. Cells were then stimulated with EGF for 1 h at 37°C and fixed for the EM study. Scale bar, 200 nm. See Materials and Methods for details. Relative amounts of immuno-gold-labeled EGFR in the MVE lumen versus MVE limiting membrane were quantified. Over 80 endosomes for each siRNA treatment from three independent experiments were used for quantification (mean + SD; ***P < 0.001, one-tailed t-test). MDA-MB-231 cells were transfected with GFP-Rab5Q79L and Flag-LAPTM4B, starved, and stimulated with Alexa-555-EGF for 90 min and followed by intraluminal sorting analysis. See Materials and Methods for details. Quantification of EGF localization in LAPTM4B-positive and LAPTM4B-negative endosomes in (E) (mean + SD; n = 3; ***P < 0.0007, one-tailed t-test). Data information: Boxes are selected regions for magnified view. Scale bars (A, E): 10 μm. Download figure Download PowerPoint To confirm the role for LAPTM4B in EGFR intraluminal sorting, an EM approach was used. Serum-starved cells were stimulated with 100 ng/ml EGF for 1 h, and the intraluminal sorting of immuno-gold labeled EGFR was quantified. Knockdown of LAPTM4B significantly increases EGF-stimulated intraluminal sorting of EGFR from ~50% in control cells to ~75% in knockdown cells (Fig 4C and D). When overexpressed, LAPTM4B displayed a non-uniform distribution among endosomes, and consistently, intraluminal sorting of Alexa-555-EGF was strongly inhibited in LAPTM4B-positive endosomes (Fig 4E and F). Together, these results demonstrate that LAPTM4B blocks EGF-stimulated EGFR intraluminal sorting in LAPTM4B-positive endosomes. As LAPTM4B inhibits EGFR intraluminal sorting, the EGFR accumulated on the endosomal surface could be recycled (Sorkin et al, 1991). To assess this, EGFR recycling assay was performed in control and LAPTM4B knockdown cells (Sigismund et al, 2008). Surprisingly, knockdown of LAPTM4B did not change EGF-stimulated EGFR recycling (Supplementary Fig S3). These results indicate that LAPTM4B-promoted EGFR signaling comes from active EGFR at the endosome as LAPTM4B blocks EGFR intraluminal sorting at LAPTM4B-positive endosomes without enhancing EGFR recycling. PtdIns(4,5)P2 regulates LAPTM4B interaction with PIPKIγi5 To examine how the LAPTM4B–PIPKIγi5 interaction may modulate EGFR trafficking and degradation, the interaction was further characterized. LAPTM4B is a unique member of the LAPTM family as it has an additional N-terminal extension (amino acids 1–91) (Shao et al, 2003). It has been shown that the pro-survival functions of human LAPTM4B require its N-terminal extension (Shao et al, 2003). Deletion of the LAPTM4B N-terminus abolished the interaction with PIPKIγi5 (Fig 5A), and the LAPTM4B N-terminus (LAPTM4B-N) directly interacted with PIPKIγi5 C-terminus in GST pull-down assay (Fig 5B). Further, co-IP experiments using LAPTM4B truncation mutants revealed that amino acids 1–40 were not critical for PIPKIγi5 interaction (Fig 5C), indicating that amino acids 41–91 were required. This region contains a polybasic motif (PBM) with a cluster of basic arginine residues (Fig 5D). The cytoplasmic PBMs in ion channels and transporters have been shown to bind Ptdlns(4,5)P2, which is essential for their functions (Suh & Hille, 2005; Huang, 2007). To analyze whether the LAPTM4B-PBM binds phosphoinositides, the LAPTM4B-N with or without PBM mutation (6RQ and 8RQ, Fig 5D) was expressed and purified from E. coli and assayed for phosphoinositide binding using PIP strips (Fig 5E). Wild-type LAPTM4B-N bound multiple phosphoinositides including PtdIns(4,5)P2, while the 6RQ and 8RQ mutants lost all phosphoinositide binding ability (Fig 5F), indicating that the LAPTM4B-PBM is capable of binding to phosphoinositides. Figure 5. Phosphoinositide regulates LAPTM4B int
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