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Shoc2 is targeted to late endosomes and required for Erk1/2 activation in EGF-stimulated cells.

Galperin E, Abdelmoti L, Sorkin A - PLoS ONE (2012)

Bottom Line: In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells.Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes.These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, Kentucky, United States of America. emilia.galperin@uky.edu

ABSTRACT
Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF) receptor (EGFR), we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S) mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.

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YFP-H-RAS (N17S) mutant inhibits Shoc2 recruitment to endosomes. A, HeLa cells were transfected with YFP-H-RAS (N17S), serum-starved and then incubated with 10 ng/ml EGF for 12 min at 37°C. Cells were then fixed, permeabilized and stained with anti- Shoc2 antibodies and secondary Cy3 donkey anti-rabbit. Insets show high magnification images of the regions of the cell indicated by the white rectangle. Cells expressing YFP-H-RAS (N17S) are outlined in the Shoc2 image. Scale bars, 10 µm. B, HeLa cells were transfected with the YFP-H-RAS (N17S) mutant and serum-starved. Cells were then incubated with 10 ng/ml EGF for 12 min at 37°C and lysed. The lysates were probed for Shoc2, RAS, phospho-ERK1/2 and total ERK1/2 by western blotting. C, Multiple images from the experiments exemplified in A were inspected, and the percentage of cells containing Shoc2 endosomes was calculated (+/−S.D.). The data are representative of 3 independent experiments, a vs. b, P<0.001 (one-way ANOVA test using SigmaStat 3.5 was used to determine differences).
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pone-0036469-g004: YFP-H-RAS (N17S) mutant inhibits Shoc2 recruitment to endosomes. A, HeLa cells were transfected with YFP-H-RAS (N17S), serum-starved and then incubated with 10 ng/ml EGF for 12 min at 37°C. Cells were then fixed, permeabilized and stained with anti- Shoc2 antibodies and secondary Cy3 donkey anti-rabbit. Insets show high magnification images of the regions of the cell indicated by the white rectangle. Cells expressing YFP-H-RAS (N17S) are outlined in the Shoc2 image. Scale bars, 10 µm. B, HeLa cells were transfected with the YFP-H-RAS (N17S) mutant and serum-starved. Cells were then incubated with 10 ng/ml EGF for 12 min at 37°C and lysed. The lysates were probed for Shoc2, RAS, phospho-ERK1/2 and total ERK1/2 by western blotting. C, Multiple images from the experiments exemplified in A were inspected, and the percentage of cells containing Shoc2 endosomes was calculated (+/−S.D.). The data are representative of 3 independent experiments, a vs. b, P<0.001 (one-way ANOVA test using SigmaStat 3.5 was used to determine differences).

Mentions: To dissect the mechanisms of Shoc2 targeting to endosomes we tested whether activity of RAS, the key interacting partner of Shoc2, is necessary for the endosomal recruitment of Shoc2-tRFP. To this end, HeLa cells were transfected with YFP-H-RAS (S17N), a GDP bound mutant of RAS. This mutant is thought to have a dominant negative effect on the activity of RAS by occupying GTP-exchange factors of RAS [23], thus inhibiting EGF-induced, RAS-mediated ERK1/2 activation (Figure 4B). Cells expressing and not expressing YFP-H-RAS (S17N) were treated with EGF at 37°C for 10 min and then stained with Shoc2 antibodies. Figure 4A and C show that recruitment of Shoc2 to endosomes was impaired in cells overexpressing YFP-H-RAS (S17N) (outlined cells in upper panel of Figure 4A). In cells that did not express or expressed low levels of YFP-H-RAS (S17N) recruitment of Shoc2 to endosomes was intact or slightly reduced. These results suggest that RAS activity is required for Shoc2 endosomal localization.


Shoc2 is targeted to late endosomes and required for Erk1/2 activation in EGF-stimulated cells.

Galperin E, Abdelmoti L, Sorkin A - PLoS ONE (2012)

YFP-H-RAS (N17S) mutant inhibits Shoc2 recruitment to endosomes. A, HeLa cells were transfected with YFP-H-RAS (N17S), serum-starved and then incubated with 10 ng/ml EGF for 12 min at 37°C. Cells were then fixed, permeabilized and stained with anti- Shoc2 antibodies and secondary Cy3 donkey anti-rabbit. Insets show high magnification images of the regions of the cell indicated by the white rectangle. Cells expressing YFP-H-RAS (N17S) are outlined in the Shoc2 image. Scale bars, 10 µm. B, HeLa cells were transfected with the YFP-H-RAS (N17S) mutant and serum-starved. Cells were then incubated with 10 ng/ml EGF for 12 min at 37°C and lysed. The lysates were probed for Shoc2, RAS, phospho-ERK1/2 and total ERK1/2 by western blotting. C, Multiple images from the experiments exemplified in A were inspected, and the percentage of cells containing Shoc2 endosomes was calculated (+/−S.D.). The data are representative of 3 independent experiments, a vs. b, P<0.001 (one-way ANOVA test using SigmaStat 3.5 was used to determine differences).
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pone-0036469-g004: YFP-H-RAS (N17S) mutant inhibits Shoc2 recruitment to endosomes. A, HeLa cells were transfected with YFP-H-RAS (N17S), serum-starved and then incubated with 10 ng/ml EGF for 12 min at 37°C. Cells were then fixed, permeabilized and stained with anti- Shoc2 antibodies and secondary Cy3 donkey anti-rabbit. Insets show high magnification images of the regions of the cell indicated by the white rectangle. Cells expressing YFP-H-RAS (N17S) are outlined in the Shoc2 image. Scale bars, 10 µm. B, HeLa cells were transfected with the YFP-H-RAS (N17S) mutant and serum-starved. Cells were then incubated with 10 ng/ml EGF for 12 min at 37°C and lysed. The lysates were probed for Shoc2, RAS, phospho-ERK1/2 and total ERK1/2 by western blotting. C, Multiple images from the experiments exemplified in A were inspected, and the percentage of cells containing Shoc2 endosomes was calculated (+/−S.D.). The data are representative of 3 independent experiments, a vs. b, P<0.001 (one-way ANOVA test using SigmaStat 3.5 was used to determine differences).
Mentions: To dissect the mechanisms of Shoc2 targeting to endosomes we tested whether activity of RAS, the key interacting partner of Shoc2, is necessary for the endosomal recruitment of Shoc2-tRFP. To this end, HeLa cells were transfected with YFP-H-RAS (S17N), a GDP bound mutant of RAS. This mutant is thought to have a dominant negative effect on the activity of RAS by occupying GTP-exchange factors of RAS [23], thus inhibiting EGF-induced, RAS-mediated ERK1/2 activation (Figure 4B). Cells expressing and not expressing YFP-H-RAS (S17N) were treated with EGF at 37°C for 10 min and then stained with Shoc2 antibodies. Figure 4A and C show that recruitment of Shoc2 to endosomes was impaired in cells overexpressing YFP-H-RAS (S17N) (outlined cells in upper panel of Figure 4A). In cells that did not express or expressed low levels of YFP-H-RAS (S17N) recruitment of Shoc2 to endosomes was intact or slightly reduced. These results suggest that RAS activity is required for Shoc2 endosomal localization.

Bottom Line: In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells.Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes.These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, Kentucky, United States of America. emilia.galperin@uky.edu

ABSTRACT
Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF) receptor (EGFR), we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S) mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.

Show MeSH
Related in: MedlinePlus