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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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Shoc2 S2G mutant is co-localized with EGF, Rab5 and H-RAS. A, Cos1-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-H-RAS. Serum-starved cells were incubated with 10 ng/ml EGF-Alexa647 for 12 min at 37°C. Insets show high-magnification images of the regions of the cell indicated by white rectangles. Scale bar, 10 µm. B, High-magnification images of the regions similar to those that are presented in A-insets shown to highlight colocalization of the Shoc2 S2G mutant with EGF and H-RAS. C, Cos-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-Rab5. Serum-starved cells were treated with EGF-Alexa647 as in A. Insets show high-magnification images of the regions of the cell indicated by white rectangle. Scale bar, 10 µm. D, Cos-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-Rab7. Serum-starved cells were treated with EGF-Alexa647 for 30 min. Insets show high-magnification images of the regions of the cell indicated by white rectangle. Scale bar, 10 µm.
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pone-0036469-g008: Shoc2 S2G mutant is co-localized with EGF, Rab5 and H-RAS. A, Cos1-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-H-RAS. Serum-starved cells were incubated with 10 ng/ml EGF-Alexa647 for 12 min at 37°C. Insets show high-magnification images of the regions of the cell indicated by white rectangles. Scale bar, 10 µm. B, High-magnification images of the regions similar to those that are presented in A-insets shown to highlight colocalization of the Shoc2 S2G mutant with EGF and H-RAS. C, Cos-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-Rab5. Serum-starved cells were treated with EGF-Alexa647 as in A. Insets show high-magnification images of the regions of the cell indicated by white rectangle. Scale bar, 10 µm. D, Cos-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-Rab7. Serum-starved cells were treated with EGF-Alexa647 for 30 min. Insets show high-magnification images of the regions of the cell indicated by white rectangle. Scale bar, 10 µm.

Mentions: Analysis of the subcellular localization of Shoc2-tRFP* (S2G) using live-cell fluorescence microscopy demonstrated that in serum-starved Cos1-LVI cells Shoc2-tRFP* (S2G) is located mainly in the plasma membrane (data not shown). It is likely that stable association of Shoc2-tRFP* (S2G) with the membrane is mediated by its N-myristoylation and interactions of the cluster of positively-charged amino acids in the amino-terminus of Shoc2 (residues 5–60). After treatment with EGF the mutant was accumulated in endosome-like compartments (Figure 8A, Movie S2). In contrast to endosomes containing wild-type Shoc2 (Figures 1, 2, and 3), the S2G mutant was highly co-localized with EGF-Alexa647 and YFP-H-RAS in endosomes (Figure 8A and B). Moreover, Shoc2-tRFP* (S2G) mutant was found to be well co-localized with endosomes containing CFP-Rab5, which was especially evident on “donut-shape” profiles of large endosomes (Figure 8C). This pattern of localization in the plasma membrane and endosomes is reminiscent of the subcellular distribution of K-Ras that is modified by farnesylation (a fatty-acid modification similar to myristoylation) and has a positively-charged region in the proximity to the farnesylation site [27], [28]. Most important, unlike wild-type Shoc2, Shoc2-tRFP* (S2G) was not significantly co-localized with CFP-Rab7 (Figure 8D). These data demonstrated that upon EGFR activation, Shoc2-tRFP* (S2G) is recruited to early endosomal compartments and that Shoc2 located on the plasma membrane and early endosomes is incapable of promoting ERK1/2 activation under conditions of cell stimulation with low EGF concentrations.


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)

Shoc2 S2G mutant is co-localized with EGF, Rab5 and H-RAS. A, Cos1-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-H-RAS. Serum-starved cells were incubated with 10 ng/ml EGF-Alexa647 for 12 min at 37°C. Insets show high-magnification images of the regions of the cell indicated by white rectangles. Scale bar, 10 µm. B, High-magnification images of the regions similar to those that are presented in A-insets shown to highlight colocalization of the Shoc2 S2G mutant with EGF and H-RAS. C, Cos-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-Rab5. Serum-starved cells were treated with EGF-Alexa647 as in A. Insets show high-magnification images of the regions of the cell indicated by white rectangle. Scale bar, 10 µm. D, Cos-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-Rab7. Serum-starved cells were treated with EGF-Alexa647 for 30 min. Insets show high-magnification images of the regions of the cell indicated by white rectangle. Scale bar, 10 µm.
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pone-0036469-g008: Shoc2 S2G mutant is co-localized with EGF, Rab5 and H-RAS. A, Cos1-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-H-RAS. Serum-starved cells were incubated with 10 ng/ml EGF-Alexa647 for 12 min at 37°C. Insets show high-magnification images of the regions of the cell indicated by white rectangles. Scale bar, 10 µm. B, High-magnification images of the regions similar to those that are presented in A-insets shown to highlight colocalization of the Shoc2 S2G mutant with EGF and H-RAS. C, Cos-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-Rab5. Serum-starved cells were treated with EGF-Alexa647 as in A. Insets show high-magnification images of the regions of the cell indicated by white rectangle. Scale bar, 10 µm. D, Cos-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-Rab7. Serum-starved cells were treated with EGF-Alexa647 for 30 min. Insets show high-magnification images of the regions of the cell indicated by white rectangle. Scale bar, 10 µm.
Mentions: Analysis of the subcellular localization of Shoc2-tRFP* (S2G) using live-cell fluorescence microscopy demonstrated that in serum-starved Cos1-LVI cells Shoc2-tRFP* (S2G) is located mainly in the plasma membrane (data not shown). It is likely that stable association of Shoc2-tRFP* (S2G) with the membrane is mediated by its N-myristoylation and interactions of the cluster of positively-charged amino acids in the amino-terminus of Shoc2 (residues 5–60). After treatment with EGF the mutant was accumulated in endosome-like compartments (Figure 8A, Movie S2). In contrast to endosomes containing wild-type Shoc2 (Figures 1, 2, and 3), the S2G mutant was highly co-localized with EGF-Alexa647 and YFP-H-RAS in endosomes (Figure 8A and B). Moreover, Shoc2-tRFP* (S2G) mutant was found to be well co-localized with endosomes containing CFP-Rab5, which was especially evident on “donut-shape” profiles of large endosomes (Figure 8C). This pattern of localization in the plasma membrane and endosomes is reminiscent of the subcellular distribution of K-Ras that is modified by farnesylation (a fatty-acid modification similar to myristoylation) and has a positively-charged region in the proximity to the farnesylation site [27], [28]. Most important, unlike wild-type Shoc2, Shoc2-tRFP* (S2G) was not significantly co-localized with CFP-Rab7 (Figure 8D). These data demonstrated that upon EGFR activation, Shoc2-tRFP* (S2G) is recruited to early endosomal compartments and that Shoc2 located on the plasma membrane and early endosomes is incapable of promoting ERK1/2 activation under conditions of cell stimulation with low EGF concentrations.

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