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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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Wild-type Shoc2 but not Shoc2 (S2G) mutant rescues Shoc2 knockdown. A, Cos-LV1 cells were transiently transfected with full-length Shoc2-tRFP or Shoc2-tRFP (S2G) mutant. Cells were serum-starved and treated with 0.2 ng/ml EGF for indicated times at 37°C. The lysates were probed by western blotting for activated ERK1/2 (pERK1/2) and total ERK1/2 (ERK1/2). Low magnification images of Shoc2-tRFP* and Shoc2-tRFP* (S2G) presented to highlight expression efficiency of these proteins in Cos-LV1 cells. B, Multiple blots from the experiments exemplified in A were analyzed. Bars represent the mean values (±S.E., n = 3) of phosphorylated ERK1/2 activity normalized to total ERK in arbitrary units (pERK/ERK), a vs. b, P<0.05 (one-way ANOVA test using SigmaStat 3.5 was used to determine differences in phosphorylated ERK1/2 activity).
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pone-0036469-g007: Wild-type Shoc2 but not Shoc2 (S2G) mutant rescues Shoc2 knockdown. A, Cos-LV1 cells were transiently transfected with full-length Shoc2-tRFP or Shoc2-tRFP (S2G) mutant. Cells were serum-starved and treated with 0.2 ng/ml EGF for indicated times at 37°C. The lysates were probed by western blotting for activated ERK1/2 (pERK1/2) and total ERK1/2 (ERK1/2). Low magnification images of Shoc2-tRFP* and Shoc2-tRFP* (S2G) presented to highlight expression efficiency of these proteins in Cos-LV1 cells. B, Multiple blots from the experiments exemplified in A were analyzed. Bars represent the mean values (±S.E., n = 3) of phosphorylated ERK1/2 activity normalized to total ERK in arbitrary units (pERK/ERK), a vs. b, P<0.05 (one-way ANOVA test using SigmaStat 3.5 was used to determine differences in phosphorylated ERK1/2 activity).

Mentions: It has been proposed that overexpression of the Shoc2 mutant with the S2G substitution (serine 2 is mutated to glycine), found in Noonan-like syndrome patients, results in N-myristoylation and targeting of this Shoc2 mutant to the plasma membrane, and enhances EGFR-dependent ERK1/2 activity [22]. To test the effect of S2G mutation in the background of cells lacking endogenous Shoc2, the Shoc2-tRFP* (S2G) mutant was transiently expressed in Cos-LV1 cells (Figure 7A and B). At the expression levels that were maximally achievable for these constructs in Cos-LV1 cells, the Shoc2-tRFP* (S2G) mutant was unable to restore the ERK1/2 activity above the basal level of EGF-induced ERK1/2 activity in Shoc2-depleted cells (Figure 7A and B). These data suggest that the S2G mutation is inhibitory to the Shoc2 function in ERK activation.


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)

Wild-type Shoc2 but not Shoc2 (S2G) mutant rescues Shoc2 knockdown. A, Cos-LV1 cells were transiently transfected with full-length Shoc2-tRFP or Shoc2-tRFP (S2G) mutant. Cells were serum-starved and treated with 0.2 ng/ml EGF for indicated times at 37°C. The lysates were probed by western blotting for activated ERK1/2 (pERK1/2) and total ERK1/2 (ERK1/2). Low magnification images of Shoc2-tRFP* and Shoc2-tRFP* (S2G) presented to highlight expression efficiency of these proteins in Cos-LV1 cells. B, Multiple blots from the experiments exemplified in A were analyzed. Bars represent the mean values (±S.E., n = 3) of phosphorylated ERK1/2 activity normalized to total ERK in arbitrary units (pERK/ERK), a vs. b, P<0.05 (one-way ANOVA test using SigmaStat 3.5 was used to determine differences in phosphorylated ERK1/2 activity).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351432&req=5

pone-0036469-g007: Wild-type Shoc2 but not Shoc2 (S2G) mutant rescues Shoc2 knockdown. A, Cos-LV1 cells were transiently transfected with full-length Shoc2-tRFP or Shoc2-tRFP (S2G) mutant. Cells were serum-starved and treated with 0.2 ng/ml EGF for indicated times at 37°C. The lysates were probed by western blotting for activated ERK1/2 (pERK1/2) and total ERK1/2 (ERK1/2). Low magnification images of Shoc2-tRFP* and Shoc2-tRFP* (S2G) presented to highlight expression efficiency of these proteins in Cos-LV1 cells. B, Multiple blots from the experiments exemplified in A were analyzed. Bars represent the mean values (±S.E., n = 3) of phosphorylated ERK1/2 activity normalized to total ERK in arbitrary units (pERK/ERK), a vs. b, P<0.05 (one-way ANOVA test using SigmaStat 3.5 was used to determine differences in phosphorylated ERK1/2 activity).
Mentions: It has been proposed that overexpression of the Shoc2 mutant with the S2G substitution (serine 2 is mutated to glycine), found in Noonan-like syndrome patients, results in N-myristoylation and targeting of this Shoc2 mutant to the plasma membrane, and enhances EGFR-dependent ERK1/2 activity [22]. To test the effect of S2G mutation in the background of cells lacking endogenous Shoc2, the Shoc2-tRFP* (S2G) mutant was transiently expressed in Cos-LV1 cells (Figure 7A and B). At the expression levels that were maximally achievable for these constructs in Cos-LV1 cells, the Shoc2-tRFP* (S2G) mutant was unable to restore the ERK1/2 activity above the basal level of EGF-induced ERK1/2 activity in Shoc2-depleted cells (Figure 7A and B). These data suggest that the S2G mutation is inhibitory to the Shoc2 function in ERK activation.

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