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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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Localization of endogenous Shoc2. A, Serum-starved HeLa cells were treated (+EGF) or not (-EGF) with 10 ng/ml EGF for 12 min at 37°C. The cells were then fixed, permeabilized and stained with Shoc2 antibodies and secondary Cy3 donkey anti-rabbit antibodies. Images of HeLa cells before (NDCV) and after de-convolution (DCV) are shown. Deconvoluted Images for Cos1 cells are shown. Insets show high magnification images of the regions of the cell indicated by white rectangles. Scale bar, 10 µm. B, Serum-starved HeLa cells were incubated with 2 mg/ml Dextran-Alexa488™ for 2 hours and treated with EGF as in (A). Below, cells were transfected with either GFP-Rab5 or GFP-Rab7, treated as in (A) and then fixed, permeabilized and stained with antibodies to Shoc2 followed by secondary Cy3-conjugated donkey anti-rabbit antibodies. Insets show high magnification images of the regions of the cell indicated by white rectangles. 31.7±8.5% (SD) of Shoc2 immunoreactivity was colocalized with Dextran-Alexa488™. Scale bar, 10 µm. C, Serum-starved HeLa cells were treated as in (A), fixed, permeabilized and stained with the Shoc2 antibody combined with either EEA.1 or LAMP1 antibody. Secondary Cy3-conjugated donkey anti-rabbit and Alexa488-conjugated donkey anti-mouse antibodies were used. Insets show high magnification images of the regions of the cell indicated by white rectangles. Scale bars, 10 µm.
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pone-0036469-g002: Localization of endogenous Shoc2. A, Serum-starved HeLa cells were treated (+EGF) or not (-EGF) with 10 ng/ml EGF for 12 min at 37°C. The cells were then fixed, permeabilized and stained with Shoc2 antibodies and secondary Cy3 donkey anti-rabbit antibodies. Images of HeLa cells before (NDCV) and after de-convolution (DCV) are shown. Deconvoluted Images for Cos1 cells are shown. Insets show high magnification images of the regions of the cell indicated by white rectangles. Scale bar, 10 µm. B, Serum-starved HeLa cells were incubated with 2 mg/ml Dextran-Alexa488™ for 2 hours and treated with EGF as in (A). Below, cells were transfected with either GFP-Rab5 or GFP-Rab7, treated as in (A) and then fixed, permeabilized and stained with antibodies to Shoc2 followed by secondary Cy3-conjugated donkey anti-rabbit antibodies. Insets show high magnification images of the regions of the cell indicated by white rectangles. 31.7±8.5% (SD) of Shoc2 immunoreactivity was colocalized with Dextran-Alexa488™. Scale bar, 10 µm. C, Serum-starved HeLa cells were treated as in (A), fixed, permeabilized and stained with the Shoc2 antibody combined with either EEA.1 or LAMP1 antibody. Secondary Cy3-conjugated donkey anti-rabbit and Alexa488-conjugated donkey anti-mouse antibodies were used. Insets show high magnification images of the regions of the cell indicated by white rectangles. Scale bars, 10 µm.

Mentions: To confirm the finding of Shoc2 localization on endosomes, the distribution of endogenous Shoc2 was examined by immunofluorescence microscopy. In serum-starved HeLa or Cos1 (not shown) cells Shoc2 was found diffusely distributed throughout the cell (Figure 2A). EGF treatment resulted in accumulation of a pool of Shoc2 in intracellular vesicular compartments in both cell types (Figure 2A). The accumulation of endogenous Shoc2 in intracellular vesicles was more pronounced in HeLa cells, and therefore, majority of the following immunofluorescence experiments was carried in these cells. As in experiments in living cells, the subcellular distribution and shapes of individual Shoc2 containing structures were characteristic of endosomes or lysosomes. Hence, to examine the nature of Shoc2-containing compartments, the endosomal system of the cells was loaded with Dextran-A488™, and the cells were then treated with EGF. Significant co-localization of Shoc2 and Dextran-A488™ was observed in EGF-treated cells. Co-localization analysis of deconvoluted images revealed that approximately 32% of cellular Shoc2 was located in dextran-containing endosomes (Figure 2B), indicating that Shoc2 compartments are indeed of an endosomal origin. The same extent of colocalization of Shoc2-containing vesicles was observed with GFP-Rab7 positive endosomes, suggesting that Shoc2 translocate to a sub-population of late endosomes. Interestingly, Shoc2 immunoreactivity was clustered along the perimeter of large Rab7-endosomes (Figure 2B). Virtually no co-localization of Shoc2 with markers of early endosomes (EEA.1, Rab5) and lysosomes (LAMP1) was observed (Figure 2B and C), suggesting that endogenous Shoc2 does not translocate to these compartments. Thus, Shoc2 compartments could be a specialized population of late endosomes.


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)

Localization of endogenous Shoc2. A, Serum-starved HeLa cells were treated (+EGF) or not (-EGF) with 10 ng/ml EGF for 12 min at 37°C. The cells were then fixed, permeabilized and stained with Shoc2 antibodies and secondary Cy3 donkey anti-rabbit antibodies. Images of HeLa cells before (NDCV) and after de-convolution (DCV) are shown. Deconvoluted Images for Cos1 cells are shown. Insets show high magnification images of the regions of the cell indicated by white rectangles. Scale bar, 10 µm. B, Serum-starved HeLa cells were incubated with 2 mg/ml Dextran-Alexa488™ for 2 hours and treated with EGF as in (A). Below, cells were transfected with either GFP-Rab5 or GFP-Rab7, treated as in (A) and then fixed, permeabilized and stained with antibodies to Shoc2 followed by secondary Cy3-conjugated donkey anti-rabbit antibodies. Insets show high magnification images of the regions of the cell indicated by white rectangles. 31.7±8.5% (SD) of Shoc2 immunoreactivity was colocalized with Dextran-Alexa488™. Scale bar, 10 µm. C, Serum-starved HeLa cells were treated as in (A), fixed, permeabilized and stained with the Shoc2 antibody combined with either EEA.1 or LAMP1 antibody. Secondary Cy3-conjugated donkey anti-rabbit and Alexa488-conjugated donkey anti-mouse antibodies were used. Insets show high magnification images of the regions of the cell indicated by white rectangles. Scale bars, 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351432&req=5

pone-0036469-g002: Localization of endogenous Shoc2. A, Serum-starved HeLa cells were treated (+EGF) or not (-EGF) with 10 ng/ml EGF for 12 min at 37°C. The cells were then fixed, permeabilized and stained with Shoc2 antibodies and secondary Cy3 donkey anti-rabbit antibodies. Images of HeLa cells before (NDCV) and after de-convolution (DCV) are shown. Deconvoluted Images for Cos1 cells are shown. Insets show high magnification images of the regions of the cell indicated by white rectangles. Scale bar, 10 µm. B, Serum-starved HeLa cells were incubated with 2 mg/ml Dextran-Alexa488™ for 2 hours and treated with EGF as in (A). Below, cells were transfected with either GFP-Rab5 or GFP-Rab7, treated as in (A) and then fixed, permeabilized and stained with antibodies to Shoc2 followed by secondary Cy3-conjugated donkey anti-rabbit antibodies. Insets show high magnification images of the regions of the cell indicated by white rectangles. 31.7±8.5% (SD) of Shoc2 immunoreactivity was colocalized with Dextran-Alexa488™. Scale bar, 10 µm. C, Serum-starved HeLa cells were treated as in (A), fixed, permeabilized and stained with the Shoc2 antibody combined with either EEA.1 or LAMP1 antibody. Secondary Cy3-conjugated donkey anti-rabbit and Alexa488-conjugated donkey anti-mouse antibodies were used. Insets show high magnification images of the regions of the cell indicated by white rectangles. Scale bars, 10 µm.
Mentions: To confirm the finding of Shoc2 localization on endosomes, the distribution of endogenous Shoc2 was examined by immunofluorescence microscopy. In serum-starved HeLa or Cos1 (not shown) cells Shoc2 was found diffusely distributed throughout the cell (Figure 2A). EGF treatment resulted in accumulation of a pool of Shoc2 in intracellular vesicular compartments in both cell types (Figure 2A). The accumulation of endogenous Shoc2 in intracellular vesicles was more pronounced in HeLa cells, and therefore, majority of the following immunofluorescence experiments was carried in these cells. As in experiments in living cells, the subcellular distribution and shapes of individual Shoc2 containing structures were characteristic of endosomes or lysosomes. Hence, to examine the nature of Shoc2-containing compartments, the endosomal system of the cells was loaded with Dextran-A488™, and the cells were then treated with EGF. Significant co-localization of Shoc2 and Dextran-A488™ was observed in EGF-treated cells. Co-localization analysis of deconvoluted images revealed that approximately 32% of cellular Shoc2 was located in dextran-containing endosomes (Figure 2B), indicating that Shoc2 compartments are indeed of an endosomal origin. The same extent of colocalization of Shoc2-containing vesicles was observed with GFP-Rab7 positive endosomes, suggesting that Shoc2 translocate to a sub-population of late endosomes. Interestingly, Shoc2 immunoreactivity was clustered along the perimeter of large Rab7-endosomes (Figure 2B). Virtually no co-localization of Shoc2 with markers of early endosomes (EEA.1, Rab5) and lysosomes (LAMP1) was observed (Figure 2B and C), suggesting that endogenous Shoc2 does not translocate to these compartments. Thus, Shoc2 compartments could be a specialized population of late endosomes.

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