CHMP6 and VPS4A mediate the recycling of Ras to the plasma membrane to promote growth factor signaling - PubMed (original) (raw)

CHMP6 and VPS4A mediate the recycling of Ras to the plasma membrane to promote growth factor signaling

Z-Y Zheng et al. Oncogene. 2012.

Abstract

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III (Endosomal Sorting Complex Required for Transport-III), were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding loop, and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound to N-Ras but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked epidermal growth factor receptor (EGFR) recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

CHMP6 and VPS4A are novel H-Ras binding proteins. (a) HT1080 cells expressing Yn-H-Ras(12V) and either Yc-tagged CHMP6 or VPS4A, together with CFP-tagged GalT, Rab5A, Rab7A, or Rab11A, which mark Golgi, early (E.) endosomes, late (L.) endosomes/MVBs, or recycling (R.) endosomes, respectively, were analyzed by confocal microscopy. CFP and the reconstituted YFP signals were pseudo-colored red and green. Dotted lines mark the cell boundaries. Insets are scaled up images to better show co-localization between the CFP and YFP signals. (b) FLAG-tagged CHMP6 or VPS4A were co-expressed with indicated H-Ras proteins in 293 cells. Immunoprecipitation was performed using either a pan-reactive Ras antibody or an antibody against the FLAG tag. Immunoprecipitated proteins as well as the total lysate inputs were analyzed by Western blots using indicated antibodies. (c) YFP-VPS4A was coexpressed with CFP-tagged H-Ras(12V)-8RK, H-Ras(61L), or Ub-H-Ras in COS-1 cells. These cells were examined by laser confocal microscopy. Regions of interest are scaled up in the inset to better show co-localization between CFP and YFP signals, as well as FRET. A heat-map is included to show the relative FRET efficiency from low (violet) to high (red). The quantified FRET efficiencies are shown at the bottom. The number of cells (n) examined were: 15 (H-Ras(12V)-8RK), 11 (H-Ras(61L)), and 12 (Ub-H-Ras). **P<0.01. (d) Cell lysates were immunoprecipitated with indicated Ras isoform-specific antibodies, and the resulting samples were similarly analyzed by Western blot using indicated antibodies.

Figure 2

Figure 2

Proper CHMP6 and VPS4A levels are important for H-Ras-induced cell transformation. (a) CHMP6 or VPS4A in NIH3T3 cells stably expressing H-Ras(61L) was silenced by shRNA. cDNA clones of CHMP6 and VPS4A refractory to shRNAs were also co-expressed in some samples. One the left, cell lysates were examined by Western blots using indicated antibodies and GAPDH is the loading control. The numbers below indicate protein levels relative to the non-silenced controls. On the right, the same cells were seeded into soft agar in triplicate and allowed to grow for 21 days before being stained with MTT. (b) NIH3T3 cells expressing SV40 T-antigen were infected with indicated shRNAs. Cells were cultured in quadruplicate (n=4) for 14 days before being fixed and stained with crystal violet. (c) CHMP6 or VPS4A in HT1080 and CAPAN-1 cells was silenced by shRNA. The cells were then seeded into soft agar in triplicate (n=3) and stained with MTT after 21 days. (d) NIH3T3 cells stably expressing H-Ras(61L) were infected with retroviruses to express CHMP6 or VPS4A before being seeded in soft agar in triplicate (n=3). Dominant negative Cdc42(17N) was used as a positive control for blocking Ras functions (Cheng et al., 2011). Colonies were counted 21 days later after MTT staining. All experiments were repeated at least one more time and similar results were obtained. *P<0.05, **P<0.01.

Figure 3

Figure 3

CHMP6 and VPS4A are important for Ras association and mobility on the membranes. (a) HT1080 cells expressing either H-Ras or H-Ras(12V) and the indicated shRNAs were harvested and fractionated by sucrose gradient ultracentrifugation. The fractions were examined for indicated proteins by Western blots (left). Na+/K+ ATPase and caveolin-1 are the markers for bulk membrane and lipid rafts, respectively. The sums of Ras proteins in fractions 11–13 over that in all fractions were analyzed and shown on the right. n = 3 separate experiments. The Ras protein levels in the non-silenced cells were set to 100%. *P<0.05, **P<0.01. (b) HT1080 cells were infected to express Dendra2 tagged H-Ras, as well as an shRNA against either CHMP6 or VPS4A. UV-activated Dendra2-H-Ras at the PM (dotted lines) was photobleached, and the fluorescence recovery in the same region was recorded over time (left). Recovered fluorescence, corrected for both spontaneous photobleaching of an unbleached cell in the same field and for background fluorescence, relative to that before photobleaching (100%) was plotted over time (right). The number of cells examined (n) were: 8 (non-bleached control), 13 (non-silencing shRNA), 10, (CHMP6 silenced), and 14 (VPS4A silenced).

Figure 4

Figure 4

Ras, CHMP6 and VPS4A control EGFR recycling. HT1080 cells carrying indicated shRNAs were stimulated with Alexa647-conjugated EGF in cycloheximide-containing medium. At indicated time points, cells were fixed and examined by confocal microscopy (left). Dotted lines were placed just outside the cell boundaries to show that there was no detectable signal at the PM. The red fluorescence at PM vs. total red fluorescence in the cell 30 min after EGF addition was quantified and shown on the right. The number of cells examined (n) were: 23 (non-silencing shRNA), 20 (N-RAS silenced), 7 (CHMP6 silenced), and 9 (VPS4A silenced). **P<0.01.

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