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.

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.

Here we compare the amount and the morphology of Au nanostructures electrodeposited from a solution containing 2.5 Â 10 -4 M AuCl 4 and 0.1 M cetyltrimethylammonium bromide (CTAB) onto nonseeded and Au-nanoparticle (NP)-seeded mercaptopropyltrimethoxysilane (MPTMS)-functionalized glass/indium tin oxide (glass/ITO) electrodes as a function of the electrode potential and deposition time. The method is similar to the previously reported seed-mediated chemical synthesis of Au nanorods (NRs) in solution and on surfaces, except that we replace the chemical reducing agent (ascorbic acid) with the electrochemical potential. The deposition can be classified into three different potential ranges on the nonseeded and seeded electrodes on the basis of the amount of Au deposited and the morphology of the deposited nanostructures. On the nonseeded glass/ITO/MPTMS electrode, at potentials ranging from -0.30 to -0.20 V, there are a significant number of Au deposits on the surface with mainly branched morphology. At deposition potentials ranging from -0.10 to 0.27 V, there is very little deposition of Au but the few deposits also have a branched morphology. At 0.27 V and higher, there is no Au deposition on the glass/ITO/MPTMS electrode. Because Au seeds catalyze Au deposition, the three potential ranges, the amount of Au, and the morphologies are quite different on the glass/ITO/MPTMS/Au NP seed electrodes compared to those on the nonseeded glass/ITO/MPTMS electrodes. There is a significant amount of Au (more than on the nonseeded electrode) on the surface over a wider range of potentials from -0.30 to 0.27 V, and they have spherical morphology. From 0.30 to 0.35 V, less Au deposits on the electrode and there are 5-15% Au NRs on the surface in addition to spherical NPs. Above 0.35 V, there is no Au deposition on the glass/ITO/MPTMS/Au seed electrode. For depositions within the potential range of 0.30 to 0.35 V on glass/ITO/MPTMS/Au seed electrodes, the size and shape distributions of the Au nanostructures, including NRs, are similar to those previously synthesized by chemical seed-mediated growth in solution and directly on nonconductive surfaces. The yield, length, and aspect ratio of the Au NRs depend on the deposition time; the average length ranges from about 100 to 400 nm for times of 30 to 120 min. The electrochemical seed-mediated growth of Au is optimal from 0.30 to 0.35 V versus Ag/AgCl under our conditions, which could be useful for enhancing the signal in sensing strategies that employ Au NPs as optical or electrochemical tags.

The ERK1/2 signaling pathway is critical in organismal development and tissue morphogenesis. Deregulation of this pathway leads to congenital abnormalities with severe developmental dysmorphisms. The core ERK1/2 cascade relies on scaffold proteins such as Shoc2 to guide and fine-tune its signals. Mutations in shoc2 lead to the development of the pathology termed Noonan-like Syndrome with Loose Anagen Hair (NSLAH). However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear. Here we show that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin-specific protease USP7. We identify that in the Shoc2 module USP7 functions as a molecular “switch” that controls the E3 ligase HUWE1 and the HUWE1-induced regulatory feedback loop. We also demonstrate that disruption of Shoc2-USP7 binding leads to aberrant activation of the Shoc2-ERK1/2 axis. Importantly, our studies reveal a possible role for USP7 in the pathogenic mechanisms underlying NSLAH extending our understanding of how ubiquitin-specific proteases regulate intracellular signaling.

Polyphenolic compounds including a number of natural products such as resveratrol, curcumin, catechin derivatives, and nordihydroguaiaretic acid have effects on the assembly of Aβ fibrils and oligomers as well as on fibril morphology. Based on a lead structure obtained from a screen of a small molecule diversity library, simple benzoic acid derivatives distinguished by the number and position of hydroxyls on the aromatic ring displayed different abilities to dissociate pre-formed biotinyl-Aβ(1–42) oligomers. The 2, 3-, 2, 5-, and 3, 4- dihydroxybenzoic acid (DHBA) isomers were active oligomer dissociators. The remaining DHBA isomers and the monohydroxy and unsubstituted benzoic acids were inactive and did not compete with the active compounds to block oligomer dissociation. None of the compounds blocked oligomer assembly, indicating that they do not interact with monomeric Aβ to shift the oligomer-monomer equilibrium. Dissociating activity was not associated with quinone redox cycling capacity of the compounds. Gallic acid (3, 4, 5-trihydroxybenzoic acid) stabilized biotinyl-Aβ(1–42) oligomers against intrinsic dissociation and blocked the effects of the active dissociators, independent of the concentration of dissociator. A model for the mechanism of action of the DHBA dissociators proposes that these compounds destabilize oligomer structure promoting progressive monomer dissociation rather than fissioning oligomers into smaller, but still macromolecular species. Gallic acid blocks dissociation by stabilizing oligomers against this process.