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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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CHC siRNA inhibits Shoc2 recruitment to endosomes. A, HeLa cells were transfected with CHC or non-targeting (NT2) siRNAs and serum-starved. The cells were incubated with 2 ng/ml EGF and 5 µg/ml Tfr-TR for 10 min at 37°C, and fixed. Insets show high magnification images of the regions of the cell indicated by the white rectangle. Scale bar, 10 µm. B, 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. C, HeLa cells from the experiment in (A) were lysed and probed for CHC, and total ERK1/2 (loading control).
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pone-0036469-g005: CHC siRNA inhibits Shoc2 recruitment to endosomes. A, HeLa cells were transfected with CHC or non-targeting (NT2) siRNAs and serum-starved. The cells were incubated with 2 ng/ml EGF and 5 µg/ml Tfr-TR for 10 min at 37°C, and fixed. Insets show high magnification images of the regions of the cell indicated by the white rectangle. Scale bar, 10 µm. B, 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. C, HeLa cells from the experiment in (A) were lysed and probed for CHC, and total ERK1/2 (loading control).

Mentions: To investigate the role of clathrin-dependent endocytosis in Shoc2-tRFP targeting to endosomes, clathrin heavy chain (CHC) was depleted by siRNA. Knock-down of CHC with this duplex was previously demonstrated to efficiently block the endocytosis of EGF and transferrin [24]. The cells were stimulated with a low concentration of EGF (2 ng/ml), conditions favoring internalization of EGF-receptor complexes predominantly via clathrin coated pits. The blockade of endocytosis of transferrin labeled with Alexa488 (Tfn-A488) was used as control for the efficiency of CHC depletion in individual cells. As shown in Figure 5, CHC siRNA dramatically reduced targeting of Shoc2-tRFP to endosomes (Figure 5A and B). These data demonstrated that clathrin-dependent processes are involved in EGF-induced Shoc2-tRFP recruitment to 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)

CHC siRNA inhibits Shoc2 recruitment to endosomes. A, HeLa cells were transfected with CHC or non-targeting (NT2) siRNAs and serum-starved. The cells were incubated with 2 ng/ml EGF and 5 µg/ml Tfr-TR for 10 min at 37°C, and fixed. Insets show high magnification images of the regions of the cell indicated by the white rectangle. Scale bar, 10 µm. B, 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. C, HeLa cells from the experiment in (A) were lysed and probed for CHC, and total ERK1/2 (loading control).
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getmorefigures.php?uid=PMC3351432&req=5

pone-0036469-g005: CHC siRNA inhibits Shoc2 recruitment to endosomes. A, HeLa cells were transfected with CHC or non-targeting (NT2) siRNAs and serum-starved. The cells were incubated with 2 ng/ml EGF and 5 µg/ml Tfr-TR for 10 min at 37°C, and fixed. Insets show high magnification images of the regions of the cell indicated by the white rectangle. Scale bar, 10 µm. B, 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. C, HeLa cells from the experiment in (A) were lysed and probed for CHC, and total ERK1/2 (loading control).
Mentions: To investigate the role of clathrin-dependent endocytosis in Shoc2-tRFP targeting to endosomes, clathrin heavy chain (CHC) was depleted by siRNA. Knock-down of CHC with this duplex was previously demonstrated to efficiently block the endocytosis of EGF and transferrin [24]. The cells were stimulated with a low concentration of EGF (2 ng/ml), conditions favoring internalization of EGF-receptor complexes predominantly via clathrin coated pits. The blockade of endocytosis of transferrin labeled with Alexa488 (Tfn-A488) was used as control for the efficiency of CHC depletion in individual cells. As shown in Figure 5, CHC siRNA dramatically reduced targeting of Shoc2-tRFP to endosomes (Figure 5A and B). These data demonstrated that clathrin-dependent processes are involved in EGF-induced Shoc2-tRFP recruitment to 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