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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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Endogenous Shoc2 localizes with active EGFR. A, Serum-starved Cos1 cells were treated with 10 ng/ml of EGF for 12 min at 37°C, fixed, permeabilized and stained with Shoc2 and EGFR (Ab528) antibodies followed by secondary Alexa548 donkey anti-rabbit and Alexa488 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. B, High magnification images of the regions similar to those presented in (A) with examples of co-localization of Shoc2 and EGFR. Filter channels used for imaging of living cells as in (A) insets. Scale bars, 5 µm.
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pone-0036469-g003: Endogenous Shoc2 localizes with active EGFR. A, Serum-starved Cos1 cells were treated with 10 ng/ml of EGF for 12 min at 37°C, fixed, permeabilized and stained with Shoc2 and EGFR (Ab528) antibodies followed by secondary Alexa548 donkey anti-rabbit and Alexa488 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. B, High magnification images of the regions similar to those presented in (A) with examples of co-localization of Shoc2 and EGFR. Filter channels used for imaging of living cells as in (A) insets. Scale bars, 5 µm.

Mentions: To examine whether Shoc2 accumulated in endosomes containing internalized EGFR, we used COS-1 cells that express a relatively high level of endogenous EGFR and have large endosomes, which facilitates light microscopic analysis. Cos1 cells were stimulated with EGF, fixed and co-stained with EGFR and Shoc2 antibodies. While most of Shoc2 endosomes did not contain EGFR, a pool of Shoc2 was co-localized with endosomal EGFR (Figure 3A). As in the case of Rab7 endosomes in HeLa cells (Figure 2B), clusters of Shoc2 were often located in the membrane of large endosomes (likely multi-vesicular bodies) containing EGFR (Figure 3A and B). Taken together, the data in Figures 1, 2, and 3 suggest that a subset of multi-vesicular bodies and late endosomes is the main site of Shoc2 localization in EGF-stimulated cells.


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)

Endogenous Shoc2 localizes with active EGFR. A, Serum-starved Cos1 cells were treated with 10 ng/ml of EGF for 12 min at 37°C, fixed, permeabilized and stained with Shoc2 and EGFR (Ab528) antibodies followed by secondary Alexa548 donkey anti-rabbit and Alexa488 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. B, High magnification images of the regions similar to those presented in (A) with examples of co-localization of Shoc2 and EGFR. Filter channels used for imaging of living cells as in (A) insets. Scale bars, 5 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351432&req=5

pone-0036469-g003: Endogenous Shoc2 localizes with active EGFR. A, Serum-starved Cos1 cells were treated with 10 ng/ml of EGF for 12 min at 37°C, fixed, permeabilized and stained with Shoc2 and EGFR (Ab528) antibodies followed by secondary Alexa548 donkey anti-rabbit and Alexa488 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. B, High magnification images of the regions similar to those presented in (A) with examples of co-localization of Shoc2 and EGFR. Filter channels used for imaging of living cells as in (A) insets. Scale bars, 5 µm.
Mentions: To examine whether Shoc2 accumulated in endosomes containing internalized EGFR, we used COS-1 cells that express a relatively high level of endogenous EGFR and have large endosomes, which facilitates light microscopic analysis. Cos1 cells were stimulated with EGF, fixed and co-stained with EGFR and Shoc2 antibodies. While most of Shoc2 endosomes did not contain EGFR, a pool of Shoc2 was co-localized with endosomal EGFR (Figure 3A). As in the case of Rab7 endosomes in HeLa cells (Figure 2B), clusters of Shoc2 were often located in the membrane of large endosomes (likely multi-vesicular bodies) containing EGFR (Figure 3A and B). Taken together, the data in Figures 1, 2, and 3 suggest that a subset of multi-vesicular bodies and late endosomes is the main site of Shoc2 localization in EGF-stimulated cells.

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