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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 is required for ERK1/2 activation by EGF in Cos1 cells. A, Parental Cos1 and Cos1 cells stably expressing Shoc2-shRNA (Cos1-LV1) were serum-starved and treated with 0.2 ng/ml EGF for indicated times at 37°C. The lysates were probed for EGFR, Raf-1, Shoc2, activated ERK1/2 (pERK1/2), activated MEK1/2 (pMEK1/2), total ERK1/2 (ERK1/2) and MEK1/2 (MEK1/2). B, Parental Cos1 and Cos1-LV1 cells were serum-starved and treated or not (0) with increasing concentrations of EGF (0.1, 0.2, 0,5, 1, 2 ng/ml) for 12 min at 37°C. The lysates were probed for Shoc2, activated ERK1/2 (pERK1/2), total ERK1/2 and GAPDH (loading control).
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pone-0036469-g006: Shoc2 is required for ERK1/2 activation by EGF in Cos1 cells. A, Parental Cos1 and Cos1 cells stably expressing Shoc2-shRNA (Cos1-LV1) were serum-starved and treated with 0.2 ng/ml EGF for indicated times at 37°C. The lysates were probed for EGFR, Raf-1, Shoc2, activated ERK1/2 (pERK1/2), activated MEK1/2 (pMEK1/2), total ERK1/2 (ERK1/2) and MEK1/2 (MEK1/2). B, Parental Cos1 and Cos1-LV1 cells were serum-starved and treated or not (0) with increasing concentrations of EGF (0.1, 0.2, 0,5, 1, 2 ng/ml) for 12 min at 37°C. The lysates were probed for Shoc2, activated ERK1/2 (pERK1/2), total ERK1/2 and GAPDH (loading control).

Mentions: Heterologous overexpression of the components of the ERK activation pathway and, in particular, scaffold proteins, in the presence of their endogenous counterparts often leads to the formation of non-specific complexes and sequesters binding partners from specific protein-protein interactions [9]. Therefore, to perform structure-function analysis of the Shoc2 role in EGFR signaling to ERK1/2, we used an RNAi approach. First, the most efficient siRNA sequence among 4 siRNA duplexes targeting Shoc2 (duplex #1) was identified (data not shown). Depletion of Shoc2 using this siRNA duplex significantly reduced ERK1/2 activity in EGF-treated cells (Figure S2). The effect of Shoc2 knockdown was most evident when the cells were stimulated with a low (0.2 ng/ml) concentration of EGF (Figure S2). Next, Cos1 cells with constitutive knock-down of Shoc2 (Cos1-LV1) were generated using lentiviruses carrying shRNA that was prepared based on the siRNA duplex #1 sequence. In order to prevent clonal variations due to the different sites of viral genome incorporation, a pool population of shRNA-expressing Cos-LV1 cells was used in subsequent experiments. Figure 6A shows that constitutive depletion of Shoc2 protein resulted in the dramatic decrease in the extent of phosphorylation of MEK1/2 and ERK1/2 upon EGFR activation. As expected, the effect of Shoc2 knockdown was most evident when the cells were stimulated with low (0.1–0.5 ng/ml) concentrations of EGF (Figure 6B). Such EGF concentrations are detected in human plasma and most tissues where EGFR is accessible to EGF [25], [26]. Transient expression of the Shoc2-tRFP mutant, in which 6 “silent” mutations were introduced to render it to be resistant to duplex #1 without changing its amino acid sequence (Shoc2-tRFP*), in Cos-LV1 cells has rescued EGF-induced ERK1/2 phosphorylation (Figure 6A). The ERK1/2 phosphorylation signal in these cells was lower than in parental COS1 cells, presumably, due to the fact that not all COS-LV1 cells expressed Shoc2-tRFP*.


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 is required for ERK1/2 activation by EGF in Cos1 cells. A, Parental Cos1 and Cos1 cells stably expressing Shoc2-shRNA (Cos1-LV1) were serum-starved and treated with 0.2 ng/ml EGF for indicated times at 37°C. The lysates were probed for EGFR, Raf-1, Shoc2, activated ERK1/2 (pERK1/2), activated MEK1/2 (pMEK1/2), total ERK1/2 (ERK1/2) and MEK1/2 (MEK1/2). B, Parental Cos1 and Cos1-LV1 cells were serum-starved and treated or not (0) with increasing concentrations of EGF (0.1, 0.2, 0,5, 1, 2 ng/ml) for 12 min at 37°C. The lysates were probed for Shoc2, activated ERK1/2 (pERK1/2), total ERK1/2 and GAPDH (loading control).
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pone-0036469-g006: Shoc2 is required for ERK1/2 activation by EGF in Cos1 cells. A, Parental Cos1 and Cos1 cells stably expressing Shoc2-shRNA (Cos1-LV1) were serum-starved and treated with 0.2 ng/ml EGF for indicated times at 37°C. The lysates were probed for EGFR, Raf-1, Shoc2, activated ERK1/2 (pERK1/2), activated MEK1/2 (pMEK1/2), total ERK1/2 (ERK1/2) and MEK1/2 (MEK1/2). B, Parental Cos1 and Cos1-LV1 cells were serum-starved and treated or not (0) with increasing concentrations of EGF (0.1, 0.2, 0,5, 1, 2 ng/ml) for 12 min at 37°C. The lysates were probed for Shoc2, activated ERK1/2 (pERK1/2), total ERK1/2 and GAPDH (loading control).
Mentions: Heterologous overexpression of the components of the ERK activation pathway and, in particular, scaffold proteins, in the presence of their endogenous counterparts often leads to the formation of non-specific complexes and sequesters binding partners from specific protein-protein interactions [9]. Therefore, to perform structure-function analysis of the Shoc2 role in EGFR signaling to ERK1/2, we used an RNAi approach. First, the most efficient siRNA sequence among 4 siRNA duplexes targeting Shoc2 (duplex #1) was identified (data not shown). Depletion of Shoc2 using this siRNA duplex significantly reduced ERK1/2 activity in EGF-treated cells (Figure S2). The effect of Shoc2 knockdown was most evident when the cells were stimulated with a low (0.2 ng/ml) concentration of EGF (Figure S2). Next, Cos1 cells with constitutive knock-down of Shoc2 (Cos1-LV1) were generated using lentiviruses carrying shRNA that was prepared based on the siRNA duplex #1 sequence. In order to prevent clonal variations due to the different sites of viral genome incorporation, a pool population of shRNA-expressing Cos-LV1 cells was used in subsequent experiments. Figure 6A shows that constitutive depletion of Shoc2 protein resulted in the dramatic decrease in the extent of phosphorylation of MEK1/2 and ERK1/2 upon EGFR activation. As expected, the effect of Shoc2 knockdown was most evident when the cells were stimulated with low (0.1–0.5 ng/ml) concentrations of EGF (Figure 6B). Such EGF concentrations are detected in human plasma and most tissues where EGFR is accessible to EGF [25], [26]. Transient expression of the Shoc2-tRFP mutant, in which 6 “silent” mutations were introduced to render it to be resistant to duplex #1 without changing its amino acid sequence (Shoc2-tRFP*), in Cos-LV1 cells has rescued EGF-induced ERK1/2 phosphorylation (Figure 6A). The ERK1/2 phosphorylation signal in these cells was lower than in parental COS1 cells, presumably, due to the fact that not all COS-LV1 cells expressed Shoc2-tRFP*.

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