RESUMO
The Shoc2 scaffold protein is crucial in transmitting signals within the Epidermal Growth Factor Receptor (EGFR)-mediated Extracellular signal-Regulated Kinase (ERK1/2) pathway. While the significance of Shoc2 in this pathway is well-established, the precise mechanisms through which Shoc2 governs signal transmission remain to be fully elucidated. Hereditary variants in Shoc2 are responsible for Noonan Syndrome with Loose anagen Hair (NSLH). However, due to the absence of known enzymatic activity in Shoc2, directly assessing how these variants affect its function is challenging. ERK1/2 phosphorylation is used as a primary parameter of Shoc2 function, but the impact of Shoc2 mutants on the pathway activation is unclear. This study investigates how the NSLH-associated Shoc2 variants influence EGFR signals in the context of the ERK1/2 and AKT downstream signaling pathways. We show that when the ERK1/2 pathway is a primary signaling pathway activated downstream of EGFR, Shoc2 variants cannot upregulate ERK1/2 phosphorylation to the level of the WT Shoc2. Yet, when the AKT and ERK1/2 pathways were activated, in cells expressing Shoc2 variants, ERK1/2 phosphorylation was higher than in cells expressing WT Shoc2. In cells expressing the Shoc2 NSLH mutants, we found that the AKT signaling pathway triggers the PAK activation, followed by phosphorylation of Raf-1/MEK1/2 and activation of the ERK1/2 signaling axis. Hence, our studies reveal a previously unrecognized feedback regulation downstream of the EGFR and provide additional evidence for the role of Shoc2 as a "gatekeeper" in controlling the selection of downstream effectors within the EGFR signaling network.
RESUMO
The extracellular signal-regulated kinase (ERK1/2) pathway is essential in embryonic development. The scaffold protein Shoc2 is a critical modulator of ERK1/2 signals, and mutations in the shoc2 gene lead to the human developmental disease known as Noonan-like syndrome with loose anagen hair (NSLH). The loss of Shoc2 and the shoc2 NSLH-causing mutations affect the tissues of neural crest (NC) origin. In this study, we utilized the zebrafish model to dissect the role of Shoc2-ERK1/2 signals in the development of NC. These studies established that the loss of Shoc2 significantly altered the expression of transcription factors regulating the specification and differentiation of NC cells. Using comparative transcriptome analysis of NC-derived cells from shoc2 CRISPR/Cas9 mutant larvae, we found that Shoc2-mediated signals regulate gene programs at several levels, including expression of genes coding for the proteins of extracellular matrix (ECM) and ECM regulators. Together, our results demonstrate that Shoc2 is an essential regulator of NC development. This study also indicates that disbalance in the turnover of the ECM may lead to the abnormalities found in NSLH patients.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Crista Neural , Animais , Humanos , Matriz Extracelular , Sistema de Sinalização das MAP Quinases/fisiologia , Síndrome de Noonan/genética , Peixe-Zebra/genética , Síndrome dos Cabelos Anágenos FrouxosRESUMO
The ERK1/2 (also known as MAPK3 and MAPK1, respectively) 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 reveal 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, thereby extending our understanding of how ubiquitin-specific proteases regulate intracellular signaling.
Assuntos
Síndrome dos Cabelos Anágenos Frouxos , Sistema de Sinalização das MAP Quinases , Síndrome de Noonan , Peptidase 7 Específica de Ubiquitina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/genética , Transdução de Sinais , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Peptidase 7 Específica de Ubiquitina/genéticaRESUMO
The Shoc2 scaffold protein is crucial in transmitting signals within the Epidermal Growth Factor Receptor (EGFR)-mediated Extracellular signal-regulated Kinase (ERK1/2) pathway. While the significance of Shoc2 in this pathway is well-established, the precise mechanisms through which Shoc2 governs signal transmission remain to be fully elucidated. Hereditary mutations in Shoc2 are responsible for Noonan Syndrome with Loose anagen Hair (NSLH). However, due to the absence of known enzymatic activity in Shoc2, directly assessing how these mutations affect its function is challenging. ERK1/2 phosphorylation is used as a primary parameter of Shoc2 function, but the impact of Shoc2 mutants on the pathway activation is unclear. This study investigates how the NSLH-associated Shoc2 variants influence EGFR signals in the context of the ERK1/2 and AKT downstream signaling pathways. We show that when the ERK1/2 pathway is a primary signaling pathway activated downstream of EGFR, Shoc2 variants cannot upregulate ERK1/2 phosphorylation to the level of the WT Shoc2. Yet, when the AKT and ERK1/2 pathways were activated, in cells expressing Shoc2 variants, ERK1/2 phosphorylation was higher than in cells expressing WT Shoc2. We found that, in cells expressing the Shoc2 NSLH mutants, the AKT signaling pathway triggers the PAK activation, followed by phosphorylation and Raf-1/MEK1/2 /ERK1/2 signaling axis activation. Hence, our studies reveal a previously unrecognized feedback regulation downstream of the EGFR and provide evidence for the Shoc2 role as a "gatekeeper" in controlling the selection of downstream effectors within the EGFR signaling network.
RESUMO
Polyphenolic compounds including a number of natural products such as resveratrol, curcumin, catechin derivatives, and nordihydroguaiaretic acid have effects on the assembly of Aß 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 preformed biotinyl-Aß(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 Aß 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-Aß(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.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Ácido Benzoico/química , Catecóis/química , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Biotinilação , Dopamina/análogos & derivados , Dopamina/química , Glutaral/química , Humanos , Hidroxibenzoatos , Isomerismo , Fragmentos de Peptídeos/metabolismo , Proteólise , SolubilidadeRESUMO
Here we compare the amount and the morphology of Au nanostructures electrodeposited from a solution containing 2.5 x 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.
RESUMO
Glial cell-line derived neurotrophic factor (GDNF) has demonstrated robust effects on dopamine (DA) neuron function and survival. A post-translational processing model of the human GDNF proprotein theorizes the formation of smaller, amidated peptide(s) from the proregion that exhibit neurobiological function, including an 11-amino-acid peptide named dopamine neuron stimulating peptide-11 (DNSP-11). A single treatment of DNSP-11 was delivered to the substantia nigra in the rat to investigate effects on DA-neuron function. Four weeks after treatment, potassium (K+) and D-amphetamine evoked DA release were studied in the striatum using microdialysis. There were no significant changes in DA-release after DNSP-11 treatment determined by microdialysis. Dopamine release was further examined in discrete regions of the striatum using high-speed chronoamperometry at 1-, 2-, and 4-weeks after DNSP-11 treatment. Two weeks after DNSP-11 treatment, potassium-evoked DA release was increased in specific subregions of the striatum. However, spontaneous locomotor activity was unchanged by DNSP-11 treatment. In addition, we show that a single treatment of DNSP-11 in the MN9D dopaminergic neuronal cell line results in phosphorylation of ERK1/2, which suggests a novel cellular mechanism responsible for increases in DA function.
Assuntos
Dopamina/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Linhagem Celular/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/química , Fosforilação/efeitos dos fármacos , Ratos Endogâmicos F344 , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Estriado Ventral/efeitos dos fármacos , Estriado Ventral/metabolismoRESUMO
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.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Sequência Conservada , Fator de Crescimento Epidérmico/farmacologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ligação Proteica , Domínios Proteicos/fisiologia , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Interferência de RNA , Alinhamento de Sequência , Transdução de Sinais/efeitos dos fármacosRESUMO
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.