Signaling adaptor ShcD suppresses extracellular signal-regulated kinase (Erk) phosphorylation distal to the Ret and Trk neurotrophic receptors

Abstract

Proteins of the Src homology and collagen (Shc) family are typically involved in signal transduction events involving Ras/MAPK and PI3K/Akt pathways. In the nervous system, they function proximal to the neurotrophic factors that regulate cell survival, differentiation, and neuron-specific characteristics. The least characterized homolog, ShcD, is robustly expressed in the developing and mature nervous system, but its contributions to neural cell circuitry are largely uncharted. We now report that ShcD binds to active Ret, TrkA, and TrkB neurotrophic factor receptors predominantly via its phosphotyrosine-binding (PTB) domain. However, in contrast to the conventional Shc adaptors, ShcD suppresses distal phosphorylation of the Erk MAPK. Accordingly, genetic knock-out of mouse ShcD enhances Erk phosphorylation in the brain. In cultured cells, this capacity is tightly aligned to phosphorylation of ShcD CH1 region tyrosine motifs, which serve as docking platforms for signal transducers, such as Grb2. Erk suppression is relieved through independent mutagenesis of the PTB domain and the CH1 tyrosine residues, and successive substitution of these tyrosines breaks the interaction between ShcD and Grb2, thereby promoting TrkB-Grb2 association. Erk phosphorylation can also be restored in the presence of wild type ShcD through Grb2 overexpression. Conversely, mutation of the ShcD SH2 domain results in enhanced repression of Erk. Although the SH2 domain is a less common binding interface in Shc proteins, we demonstrate that it associates with the Ptpn11 (Shp2) phosphatase, which in turn regulates ShcD tyrosine phosphorylation. We therefore propose a model whereby ShcD competes with neurotrophic receptors for Grb2 binding and opposes activation of the MAPK cascade.

The role of the ShcD and RET interaction in neuroblastoma survival and migration

“…Furthermore, the confirmed enrolment of ShcD as a substrate for other receptors, such as TrkB [10] , could justify the ability of ShcD to activate the RAS/ERK pathway in SK-N-AS overexpressing the ShcD protein with no GDNF treatment. While this manuscript was in preparation a study by Wills and colleagues revealed that ShcD deactivates ERK and AKT signalling downstream of RET and TrkB, which certainly concurs with our finding [30] ; nevertheless, their data do not reveal the consequence of the inhibition.…”

The ShcD phosphotyrosine adaptor subverts canonical EGF receptor trafficking

“…The emerging picture thus positions ShcD not as a passive scaffold recruited to a stimulated receptor, but as an active signalling regulator that influences EGFR in various cellular networks. Indeed, the noncanonical nature of ShcD has been reinforced by our recent discovery that the adaptor suppresses ERK1/2 phosphorylation distal to neurotrophic receptors (Wills et al, 2017). Although signalling output was not examined in the present work, it is tempting to speculate that the EGFR mislocalization we describe herein could similarly disconnect the receptor from canonical pathways.…”

Localization of phosphotyrosine adaptor protein ShcD/SHC4 in the adult rat central nervous system

“…Intriguingly, ShcD is expressed similarly to ShcA in the SVZ, though ShcD demonstrates additional localization to cells lining the lateral ventricle. In the olfactory system, ShcD is found in an overlapping pattern with ShcC in the main olfactory bulb, and uniquely in the OE and glomeruli, and it has a distinct function in suppressing Erk activation downstream of TrkB [32]. Given the established role of TrkB in migration [33] and differentiation [34] of NPCs, along with its high expression in OE-derived NPCs [35], it is tempting to speculate that ShcD could play a novel role in growth factor-mediated signaling in NPCs.…”

“…Rabbit anti-ShcD (1:1000) was generated against the unique CH2 region. Its specificity has been confirmed by preadsorption with the original antigen prior to immunohistochemistry [8, 13], by immunoblot against lysates prepared from cells expressing other Shc proteins [8], and by immunoblot on brain lysates prepared from ShcD knockout mice [32].…”