Sequential actions of myotubularin lipid phosphatases regulate endosomal PI(3)P and growth factor receptor trafficking

Abstract

Two different human diseases, X-linked myotubular myopathy and Charcot-Marie-Tooth disease, result from mutant MTM1 or MTMR2 lipid phosphatases. Although events involved in endosomal PI(3)P and PI(3,5)P(2) synthesis are well established and pivotal in receptor signaling and degradation, enzymes involved in phosphoinositide degradation and their roles in trafficking are incompletely characterized. Here, we dissect the functions of the MTM1 and MTMR2 myotubularins and establish how they contribute to endosomal PI(3)P homeostasis. By mimicking loss of function in disease through siRNA-mediated depletion of the myotubularins, excess PI(3)P accumulates on early (MTM1) and late (MTMR2) endosomes. Surprisingly, the increased PI(3)P blocks the egress of epidermal growth factor receptors from early or late endosomes, suggesting that the accumulation of signaling receptors in distinct endosomes may contribute to the unique disease etiologies when MTM1 or MTMR2 are mutant. We further demonstrate that direct myotubularin binding to the type III PI 3-kinase complex hVps34/hVps15 leads to phosphatase inactivation. The lipid kinase-phosphatase interaction also precludes interaction of the PI 3-kinase with Rab GTPase activators. Thus, unique molecular complexes control kinase and phosphatase activation and locally regulate PI(3)P on discrete endosome populations, thereby providing a molecular rationale for related human myo- and neuropathies.

Figure 1.

MTM1 or MTMR2 depletion increases cellular PI(3)P levels. A431 cells were transfected with MTM1 or MTMR2 siRNA. GAPDH siRNA-transfected samples served as negative controls. (A) Cells were metabolically labeled with myo-[2-³H]inositol. Individual GroPs were resolved by HPLC. Chromatogram shows percentage of the summed radioactivity from all GroP species (total ³H radioactivity) as a function of elution time for each indicated sample. Individual GroP species are quantified by area integration and normalized to controls (bar graph, mean ± SEM, n = 3–4, unpaired t test, two-tailed **p < 0.01, *p < 0.05). (B) PI(3)P were extracted and detected with an Echelon mass strip assay. Left panel, the PI(3)P strip template; middle panel, a representative PI(3)P strip with triangles highlighting controls, standards, or samples and an immunoblot for actin in each sample to confirm equal amounts of cells used for PI(3)P isolation. A standard curve shows the chemiluminescent signal intensity as a function of pmol of spotted PI(3)P standard. Right bar graph shows PI(3)P amounts in each sample (mean ± SEM, n = 3–4, unpaired t test: MTM1 209, two-tailed *p < 0.05; MTMR2 19, one-tailed *p < 0.05). (C) A431 cells were cotransfected with Cy3-labeled MTM1 or MTMR2 siRNA (red) and GFP-2xFYVE^Hrs (green). Cy3-labeled GAPDH siRNA (red) or mock-transfected samples served as controls. Endogenous EEA1 (blue) and Rab7 (blue) were detected with immunofluorescence staining. Samples were scanned at identical settings on confocal microscope. Fluorescence intensity of cellular PI(3)P levels (total membrane bound GFP-2xFYVE^Hrs), early endosomal (EE) PI(3)P levels (GFP-2xFYVE^Hrs colocalizing with EEA1) and late endosomal (LE) PI(3)P levels (GFP-2xFYVE^Hrs colocalizing with Rab7) in each sample is the average pixel intensity in the selected cell that is above the threshold quantified using Slidebook 4.1 image analysis software. Increase in EE PI(3)P of 86% (mean ± SEM, n = 10, unpaired t test, two-tailed **p < 0.01) in MTM1 siRNA-transfected cells versus GAPDH control. Left panel, cellular PI(3)P and LE PI(3)P in MTM1 siRNA-transfected cells do not change relative to GAPDH control (mean ± SEM, n = 10, unpaired t test, two-tailed p = 0.36, 0.38). Increase in cellular PI(3)P of 17% (mean ± SEM, n = 20, unpaired t test, two-tailed ***p < 0.0001) and in LE PI(3)P of 19% (mean ± SEM, n = 12, unpaired t test, two-tailed *p < 0.02) in MTMR2 siRNA-transfected cells versus mock control. Right panel, EE PI(3)P levels remains unchanged in the presence of MTMR2 siRNA compared with mock control (mean ± SEM, n = 20, unpaired t test, two-tailed p = 0.53).

Figure 2.

MTMR2 localizes to Rab7-positive late endosomes and is not detected on Rab5-positive early endosomes. BHK cells transiently transfected with Flag-MTMR2wt or Flag-MTMR2D320A (catalytically inactive mutant) were detected with immunofluorescence staining for Flag-MTMR2 (red) and the cotransfected endosomal markers, Rab5 (green) and Rab7 (green). Rab5Q79L (constitutively active mutant) facilitates analysis of recruitment to enlarged Rab5-positive early endosomes. Colocalization is yellow in color merge. Enlarged views of marked areas (insets) demonstrate the absence of any colocalization. Images were collected using 40× objective and scan zoom 3.0. Bar, 10 μm.

Figure 3.

EGFR degradation after ligand stimulation is impaired in SCC-12F cells with myotubularin depletion. SCC-12F cells were transfected with MTM1 or MTMR2 siRNA. GAPDH siRNA-transfected samples served as negative controls. At 48 h after transfection, cells were stimulated with EGF for indicated time. (A) Total protein (10 μg/lane) was resolved by SDS-PAGE. Western blot shows EGFR and actin in each sample (top panel). The ratio of EGFR to actin was quantified with chemiluminescent signal on immunoblots by densitometry. Bar graph shows basal EGFR/actin in unstimulated cells normalized to negative control (mean ± SEM, n = 4, unpaired t test, two-tailed p > 0.05). Line graph shows percent of basal EGFR/actin degraded as a function of time after ligand stimulation (mean ± SEM, n = 4, unpaired t test, one-tailed *p < 0.05). The details of additional statistical evaluations are given in Supplementary Methods. (B) Immunofluorescence staining shows EGFR localization (green) and EEA1 (red) in the indicated siRNA-transfected samples without ligand stimulation (no EGF) or with EGF stimulation for 15 min. Images were collected using 40× objective and scan zoom 1.8. Bar, 10 μm. (C) SCC-12F cells were transfected with Cy3-labeled MTM1 or MTMR2 siRNA. Cy3-labeled GAPDH siRNA-transfected samples served as negative controls. Cells were serum starved for 2 h and left unstimulated or stimulated with EGF up to 10 min. Cell surface EGFR was labeled with an anti-EGFR antibody conjugated to fluorescein and 20,000 cells were quantified by flow cytometry. Bar graph shows cell surface fluorescence intensity in unstimulated cells normalized to negative control and line graph shows percent decrease of cell surface fluorescence intensity as a function of time up to 10-min EGF treatment (mean ± SEM, n = 2).

Figure 4.

EGFR degradation after ligand stimulation is impaired in A431 cells with myotubularin depletion. (A) A431 cells were transfected with MTM1 or MTMR2 siRNA. GAPDH siRNA-transfected samples served as negative controls. At 48 h after transfection, cells were stimulated with EGF for indicated time. Total protein (2.5 μg/lane) was resolved by SDS-PAGE. Western blot shows EGFR and actin in each sample (top panel). The ratio of EGFR to actin was quantified with chemiluminescent signal on immunoblots by densitometry. Bar graph shows basal EGFR/actin in unstimulated cells normalized to negative control (mean ± SEM, n = 3, unpaired t test, two-tailed **p < 0.01). Line graph shows percent of basal EGFR/actin degraded as a function of time after ligand stimulation (mean ± SEM, n = 3–8, unpaired t test, MTM1 209: 2 h one-tailed *p < 0.05; 3 h two-tailed *p < 0.05; 4 h two-tailed *p < 0.05; MTMR2 19: 3 h two-tailed *p < 0.01; 4 h two-tailed *p < 0.01). The details of additional statistical evaluations are given in Supplementary Methods. (B) A431 cells were transfected with Cy3-labeled MTMR2 siRNA (red) for 48 h. The micrograph shows EGFR localization (green) in MTMR2 siRNA-transfected (filled arrows) versus untransfected cells (open arrows). Images were collected using 40× objective and scan zoom 1.8. Bar, 10 μm. A431 cells were transfected with Cy3-labeled MTM1 or MTMR2 siRNA. Cy3-labeled GAPDH siRNA-transfected samples served as negative controls. At 48 h after transfection, cell surface EGFR was labeled with an anti-EGFR antibody conjugated to fluorescein, and 20,000 cells were quantified by flow cytometry for each sample. Bar graph shows cell surface fluorescence intensity normalized to negative control (mean ± SEM, n = 3, unpaired t test, two-tailed *p < 0.05). (C) A431 cells were transfected with Cy3-labeled MTM1 or MTMR2 siRNA. Mock-transfected samples served as negative controls. Cells were serum starved overnight and left unstimulated or stimulated with EGF for 3 h. Cell surface EGFR was labeled with an anti-EGFR antibody conjugated to fluorescein, and 20,000 cells were quantified by flow cytometry. Plotted are percentages of total cells analyzed (% of max) as a function of cell surface fluorescence intensity for indicated samples. The numbers inside each graph denote the percent decrease in cell surface fluorescence after 3 h EGF treatment compared with no EGF treatment.

Figure 5.

MTM1 and MTMR2 function differentially in endosomal sorting of EGFR. A431 cells were transfected with MTM1 or MTMR2 siRNA. Mock-transfected samples served as negative controls. At 48 h after transfection, cells were prepared for immunofluorescence staining without ligand stimulation (no EGF) or with EGF stimulation for 3 h. EGFR (green) was costained with transferrin receptor (TfR, red), EEA1 (red), or Rab7 (red) in indicated samples. (A) Mock-transfected cells, (B) MTM1 siRNA-transfected cells, (C) MTMR2 siRNA-transfected cells. Images were collected using 40× objective and scan zooms (A) 3.5, (B and C) 1.0. Bars, 10 μm.

Figure 6.

The WD40 domain of hVps15 mediates exclusive binding of MTMR2 or Rab7 to the hVps34/hVps15 PI 3-kinase complex. (A) BHK cells overexpressing Flag-MTMR2wt or Flag-MTMR2D320A (catalytically inactive mutant), hVps34 and Rab7 were immunostained. Images of Flag-MTMR2 (red), hVps34 (green), and Rab7 (blue) are shown in gray-scale and color merge. Color merge shows colocalization (white) of MTMR2 with hVps34 on Rab7-positive late endosomes. Images were collected using 40× objective and scan zoom 1.0. Bar, 10 μm. (B) BHK cells overexpressing Flag-MTMR2, hVps34 and V5-hVps15 were processed for immunoprecipitation (IP) with an anti-Flag mAb. Western blot shows coprecipitated hVps34 or hVps15 (left panel) and expression levels of individual protein in whole cell lysates (WCL) for each sample with indicated input amounts (right panel). MTMR2 associates with the hVps34/hVps15 complex. (C) BHK cells overexpressing Rab7, Flag-MTMR2 and V5-hVps15 were processed for IP with anti-Rab7 or anti-Flag antibodies, respectively. Western blot shows coprecipitated hVps15 (left panel) and WCL (middle panel). MTMR2 and Rab7 form a mutually exclusive complex with hVps15. GST-MTMR2, 60 ng, immobilized on glutathione-Sepharose beads was incubated with identical aliquots of V5-hVps15–containing cell lysates. After washing, increasing concentrations of Rab7-containing cell lysates (0, 1×, 10×) were added for incubation. Western blot shows coprecipitated proteins on beads and proteins in the supernatant (right panel). Binding of MTMR2 or Rab7 to hVps15 is competitive. (D) BHK cells overexpressing full-length V5-hVps15 (wt) or domain deletion mutants (Δ) (indicated in top panel) and Flag-MTMR2 were processed for IP with an anti-Flag mAb. Western blot shows coprecipitated hVps15 (left panel) and WCL (middle panel). Right graph shows the ratio of coprecipitated hVps15 to MTMR2 in each sample quantified with chemiluminescent signal on immunoblots by densitometry (mean ± SEM, n = 4; unpaired t test, two-tailed *p < 0.05, **p < 0.01). hVps15 WD40Δ exhibits diminished complex formation with MTMR2. (E) Equimolar amounts of GST-MTMR2 immobilized on glutathione-Sepharose beads were incubated with in vitro–synthesized, ³⁵S-l-methionine–labeled, full-length, hVps15 domain deletion mutants or individual hVps15 domains. Binding of the hVps15 proteins to GST-MTMR2 was quantified by phosphoimage analysis and binding was normalized relative to binding to GST (nonspecific binding equal to 1), which served as the negative control (mean ± SEM, n = 2, unpaired t test, two-tailed *p < 0.05). hVps15 WD40Δ exhibits diminished complex formation with MTMR2, but binding is reconstituted with a HEAT/WD40 domain. Representative binding data are shown in Supplementary Figure S6 online. (F) BHK cells overexpressing Flag-MTMR2wt or Flag-MTMR2 coli domain deletion mutant (CoilΔ) (indicated in top panel) with V5-hVps15 were processed for IP with an anti-Flag mAb. Western blot shows coprecipitated hVps15 (bottom panel). The MTMR2 coil domain is dispensable for binding to hVps15. (G) Equimolar amounts of full-length GST-MTMR2, GST-PH, GST-PTP, GST-Coil, or GST proteins were purified and confirmed with Coomassie staining (top panel). The GST fusion proteins immobilized on glutathione-Sepharose beads were incubated with equal amount V5-hVps15–containing cell lysates. Western blot shows coprecipitated hVps15 (bottom panel). Both the PH and PTP domains of MTMR2 bind to hVps15 independently.

Figure 7.

Binding of hVps15 inhibits MTMR2 PI(3)P phosphatase activity. (A) BHK cells overexpressing Flag-MTMR2wt or Flag-MTMR2D320A (catalytically inactive mutant) and V5-hVps15 were processed for immunoprecipitation (IP) with anti-Flag and anti-V5 antibodies, respectively. Western blot shows MTMR2 or hVps15 in the precipitated complex (bottom panel). The precipitated hVps15/MTMR2 complex was monitored for PI(3)P phosphatase activity with NBD6-PI(3)P substrate. Reaction products together with NBD6-PI and NBD6-PI(3)P standards were resolved by TLC with migration as indicated (top panel). The hVps15/MTMR2 complex IP by hVps15 lacks phosphatase activity. (B) The minimum amount of GST-MTMR2 required to convert 1.5 μg NBD6-PI(3)P into NBD6-PI within 15 min in the phosphatase assay was titrated to be 60 ng. GST, 60 ng, served as controls (top panel). Western blot shows the titrated GST-MTMR2 in the assay (bottom panel). (C) GST-MTMR2 (60 ng) immobilized on glutathione-Sepharose beads was incubated with increasing concentrations of V5-hVps15-containing or hVps34/V5-hVps15–containing cell lysates. Wortmannin was added in indicated samples. Phosphatase reaction products were resolved by TLC with the migration of PI and PI(3)P as indicated (top panel). Western blot shows the amounts of each protein in the phosphatase assay (bottom panel). Binding of hVps15 inactivates MTMR2 in a dose-dependent manner. The presence of the hVps34 does not shift the equilibrium in favor of PI(3)P production nor does the presence of wortmannin increase the production of PI.

Figure 8.

Model for coordinate regulation of PI(3)P homeostasis and endocytosis through the interplay between the hVps34/hVps15 lipid kinase complex and myotubularin phosphatases. (A) The hVps34/hVps15 lipid kinase complex is recruited or activated on endosomes by activated Rab GTPases and generates a localized burst of PI(3)P. Membrane recruitment of the myotubularins or their clustering in PI(3)P domains initiates PI(3)P degradation. Interaction of the myotubularin with the hVps34/hVps15 lipid kinase complex on endosomes simultaneously displaces the Rab GTPase required for hVps34/hVps15 activation and occludes the phosphatase domain, causing inactivation of the myotubularin. (B) Endosomes may undergo waves of PI(3)P synthesis and degradation in response to cycles of hVps34/hVps15 activation and inactivation. Such a mechanism allows the acute recruitment and release or activation and inactivation of trafficking machinery components for temporal regulation of membrane transport.