The extracellular signal-related kinase 1 and 2 (ERK1/2) pathway is a highly conserved signaling cascade with numerous essential functions in development. The scaffold protein Shoc2 amplifies the activity of the ERK1/2 pathway and is an essential modulator of a variety of signaling inputs. Germline mutations in Shoc2 are associated with the human developmental disease known as the Noonan-like syndrome with loose anagen hair. Clinical manifestations of this disease include congenital heart defects, developmental delays, distinctive facial abnormalities, reduced growth and cognitive deficits along with hair anomalies. The many molecular details of pathogenesis of the Noonan-like syndrome and related developmental disorders, cumulatively called RASopathies, remain poorly understood. Mouse knockouts for Shoc2 are embryonic lethal, emphasizing the need for additional animal models to study the role of Shoc2 in embryonic development. Here, we characterize a zebrafish shoc2 mutant, and show that Shoc2 is essential for development, and that its loss is detrimental for the development of the neural crest and for hematopoiesis. The zebrafish model of the Noonan-like syndrome described here provides a novel system for the study of structure-function analyses and for genetic screens in a tractable vertebrate system.

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.

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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.

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, i.e. RASopathies. 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) RASopathy. However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear. We found that Shoc2 assembles an elegant multi‐component complex that incorporates several proteins of the ubiquitin system. To fine‐tune the amplitude of ERK1/2 signal transmitted via the complex, Shoc2 tethers the E3 ligase HUWE1, the (AAA+) ATPases, PSMC5 and VCP/p97, and the deubiquitinating enzyme, USP7. All of these enzymes are integral to the intricate feedback mechanism. Our recent studies demonstrated that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin‐specific protease USP7. We identified 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 found that disruption of Shoc2‐USP7 binding leads to aberrant activation of the Shoc2‐ERK1/2 axis. The zebrafish vertebrate model was then used to show that Shoc2 congenital mutations affecting Shoc2 interaction with USP7 lead to aberrant Shoc2 ubiquitination and signal transmission. Thus, our studies reveal a role for USP7 in the pathogenic mechanisms underlying NSLAH extending our understanding of how ubiquitin‐specific proteases regulate intracellular signaling. In summary, our studies are the first to demonstrate that the Shoc2 scaffold employs multi‐protein enzymatic machinery to govern the amplitude of Shoc2‐ERK1/2 signals. We also uncover novel molecular mechanisms underlying the pathogenesis of Noonan‐like syndrome with loose anagen hair. Overall, these studies significantly advance our understanding of the mechanisms by which non‐enzymatic scaffolds regulate the specificity and dynamics of the ERK1/2 signaling networks.