Functional Integration of the Conserved Domains of Shoc2 Scaffold

Shoc2 is a positive regulator of signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Shoc2 is also proposed to interact with RAS and Raf-1 in order to accelerate ERK1/2 activity. To understand the mechanisms by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor receptor (EGFR), we dissected the role of Shoc2 structural domains in binding to its signaling partners and its role in regulating ERK1/2 activity. Shoc2 is comprised of two main domains: the 21 leucine rich repeats (LRRs) core and the N-terminal non-LRR domain. We demonstrated that the N-terminal domain mediates Shoc2 binding to both M-Ras and Raf-1, while the C-terminal part of Shoc2 contains a late endosomal targeting motif. We found that M-Ras binding to Shoc2 is independent of its GTPase activity. While overexpression of Shoc2 did not change kinetics of ERK1/2 activity, both the N-terminal and the LRR-core domain were able to rescue ERK1/2 activity in cells depleted of Shoc2, suggesting that these Shoc2 domains are involved in modulating ERK1/2 activity

Shoc2 Is Targeted to Late Endosomes and Required for Erk1/2 Activation in EGF-Stimulated Cells

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

YFP-H-RAS (N17S) mutant inhibits Shoc2 recruitment to endosomes.

<p><b>A</b>, HeLa cells were transfected with YFP-H-RAS (N17S), serum-starved and then incubated with 10 ng/ml EGF for 12 min at 37°C. Cells were then fixed, permeabilized and stained with anti- Shoc2 antibodies and secondary Cy3 donkey anti-rabbit. Insets show high magnification images of the regions of the cell indicated by the white rectangle. Cells expressing YFP-H-RAS (N17S) are outlined in the Shoc2 image. Scale bars, 10 µm. <b>B</b>, HeLa cells were transfected with the YFP-H-RAS (N17S) mutant and serum-starved. Cells were then incubated with 10 ng/ml EGF for 12 min at 37°C and lysed. The lysates were probed for Shoc2, RAS, phospho-ERK1/2 and total ERK1/2 by western blotting. <b>C</b>, Multiple images from the experiments exemplified in <b><i>A</i></b> were inspected, and the percentage of cells containing Shoc2 endosomes was calculated (+/−S.D.). The data are representative of 3 independent experiments, a vs. b, P<0.001 (one-way ANOVA test using SigmaStat 3.5 was used to determine differences).</p

Endogenous Shoc2 localizes with active EGFR.

<p><b>A</b>, 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>B</b>, High magnification images of the regions similar to those presented in <i>(</i><b><i>A</i></b><i>)</i> with examples of co-localization of Shoc2 and EGFR. Filter channels used for imaging of living cells as in <i>(</i><b><i>A</i></b><i>)</i> insets. Scale bars, 5 µm.</p

Shoc2 S2G mutant is co-localized with EGF, Rab5 and H-RAS.

<p><b>A</b>, 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>B</b>, High-magnification images of the regions similar to those that are presented in <b>A</b>-insets shown to highlight colocalization of the Shoc2 S2G mutant with EGF and H-RAS. <b>C</b>, Cos-LV1 cells were transiently transfected with Shoc2-tRFP* (S2G) mutant and CFP-Rab5. Serum-starved cells were treated with EGF-Alexa647 as in <b><i>A</i></b>. Insets show high-magnification images of the regions of the cell indicated by white rectangle. Scale bar, 10 µm. <b>D</b>, 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.</p

Localization of Shoc2-tRFP and characterization of Shoc2-tRFP containing compartments.

<p><b>A</b>, Cos1 cells were transfected with Shoc2-tRFP and imaged live before EGF treatment. Shoc2-tRFP is located in the cytosol and nucleus. Scale bar, 10 µm. <b>B</b>, Shoc2-tRFP and CFP-Rab7 were transiently expressed in Cos1 cells. Cells were serum-starved for 16 h and then treated with 10 ng/ml EGF 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. A panel below shows multiple high-magnification images with examples of co-localization of Shoc2-tRFP with CFP-Rab7. Scale bar, 5 µm<b>. C</b>, Shoc2-tRFP and GFP-Rab5 were transiently expressed in Cos1 cells. Cells were serum-starved for 16 hr. Cells were then treated with 10 ng/ml EGF 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.</p

CHC siRNA inhibits Shoc2 recruitment to endosomes.

<p><b>A</b>, 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>B</b>, Multiple images from the experiments exemplified in <i>(</i><b><i>A</i></b><i>)</i> were inspected, and the percentage of cells containing Shoc2 endosomes was calculated (+/−S.D.). The data are representative of 3 independent experiments. <b>C</b>, HeLa cells from the experiment in <i>(</i><b><i>A</i></b><i>)</i> were lysed and probed for CHC, and total ERK1/2 (loading control).</p

Shoc2 is required for ERK1/2 activation by EGF in Cos1 cells.

<p><b>A</b>, 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>B</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).</p

Wild-type Shoc2 but not Shoc2 (S2G) mutant rescues Shoc2 knockdown.

<p><b>A</b>, 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>B</b>, Multiple blots from the experiments exemplified in <b><i>A</i></b> were analyzed. Bars represent the mean values (±S.E., <i>n</i> = 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).</p

Functional Integration of the Conserved Domains of Shoc2 Scaffold

<div><p>Shoc2 is a positive regulator of signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Shoc2 is also proposed to interact with RAS and Raf-1 in order to accelerate ERK1/2 activity. To understand the mechanisms by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor receptor (EGFR), we dissected the role of Shoc2 structural domains in binding to its signaling partners and its role in regulating ERK1/2 activity. Shoc2 is comprised of two main domains: the 21 leucine rich repeats (LRRs) core and the N-terminal non-LRR domain. We demonstrated that the N-terminal domain mediates Shoc2 binding to both M-Ras and Raf-1, while the C-terminal part of Shoc2 contains a late endosomal targeting motif. We found that M-Ras binding to Shoc2 is independent of its GTPase activity. While overexpression of Shoc2 did not change kinetics of ERK1/2 activity, both the N-terminal and the LRR-core domain were able to rescue ERK1/2 activity in cells depleted of Shoc2, suggesting that these Shoc2 domains are involved in modulating ERK1/2 activity.</p></div