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Structural determinants controlling 14-3-3 recruitment to the endocytic adaptor Numb and dissociation of the Numb·?-adaptin complex.

ABSTRACT: Traffic of cargo across membranes helps establish, maintain, and reorganize distinct cellular compartments and is fundamental to many metabolic processes. The cargo-selective endocytic adaptor Numb participates in clathrin-dependent endocytosis by attaching cargoes to the clathrin adaptor ?-adaptin. The phosphorylation of Numb at Ser265 and Ser284 recruits the regulatory protein 14-3-3, accompanied by the dissociation of Numb from ?-adaptin and Numb's translocation from the cortical membrane to the cytosol. However, the molecular mechanisms underlying the Numb-?-adaptin interaction and its regulation by Numb phosphorylation and 14-3-3 recruitment remain poorly understood. Here, biochemical and structural analyses of the Numb·14-3-3 complex revealed that Numb phosphorylation at both Ser265 and Ser284 is required for Numb's efficient interaction with 14-3-3. We also discovered that an RQFRF motif surrounding Ser265 in Numb functions together with the canonical C-terminal DPF motif, required for Numb's interaction with ?-adaptin, to form a stable complex with ?-adaptin. Of note, we provide evidence that the phosphorylation-induced binding of 14-3-3 to Numb directly competes with the binding of ?-adaptin to Numb. Our findings suggest a potential mechanism governing the dynamic assembly of Numb with ?-adaptin or 14-3-3. This dual-site recognition of Numb by ?-adaptin may have implications for other ?-adaptin targets. We propose that the newly identified ?-adaptin-binding site surrounding Ser265 in Numb functions as a triggering mechanism for the dynamic dissociation of the Numb·?-adaptin complex.

SUBMITTER: Chen X

PROVIDER: S-EPMC5857998 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Structural determinants controlling 14-3-3 recruitment to the endocytic adaptor Numb and dissociation of the Numb·α-adaptin complex.

Chen Xing X Liu Ziheng Z Shan Zelin Z Yao Weiyi W Gu Aihong A Wen Wenyu W

The Journal of biological chemistry 20180130 11

Traffic of cargo across membranes helps establish, maintain, and reorganize distinct cellular compartments and is fundamental to many metabolic processes. The cargo-selective endocytic adaptor Numb participates in clathrin-dependent endocytosis by attaching cargoes to the clathrin adaptor α-adaptin. The phosphorylation of Numb at Ser<sup>265</sup> and Ser<sup>284</sup> recruits the regulatory protein 14-3-3, accompanied by the dissociation of Numb from α-adaptin and Numb's translocation from the ...[more]

PMID: 29382713

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Project description:Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of β-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of β-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive β-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.

Dataset Information

Structural determinants controlling 14-3-3 recruitment to the endocytic adaptor Numb and dissociation of the Numb·?-adaptin complex.

Publications

Structural determinants controlling 14-3-3 recruitment to the endocytic adaptor Numb and dissociation of the Numb·α-adaptin complex.

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