The role of WWP-family E3 ubiquitin ligases in neuronal development and function

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Ubiquitination is a posttranslational modification catalyzed by the sequential action of three classes of enzymes, E1, E2 enzymes, and E3 Ub ligases. Substrate specificity of ubiquitination is mainly determined by the E3 ligases, of which some 600 different isoforms are encoded in the human genome. Substrate proteins can be conjugated either with the monomeric form of Ub (monoubiquitination) or with a tandem poly-Ub chain (polyubiquitination). Monoubiquitination affects multiple protein characteristics and controls, for instance, endocytosis of receptors, DNA repair, or virus budding. The consequence of polyubiquitination depends on the type of the poly-Ub chain. To form a poly-Ub chain, the C-terminal glycine residue of one Ub is conjugated to a lysine residue on the surface of the donor Ub (K6, K11, K27, K29, K33, K48, or K63). K48-linked poly-Ub chains are recognized by the proteasome, where the target protein is degraded, while K63-linked poly-Ub chains are involved in the proteasome-independent regulation of protein functions. Interestingly, K63-linked poly-Ub chains are almost as abundant as K48-linked chains in the mammalian brain, indicating that K-63 linked chain-dependent regulation of cellular processes may be as important as Ub mediated proteasomal degradation. WWP1 and WWP2 (WWP1/2) are members of the Nedd4 superfamily of E3 ligases. Nedd4 family E3 ligases regulate the expression, localization, or function of substrates mainly in a proteasome-independent manner. Consistently, WWP1/2 as well as other Nedd4 superfamily E3 ligases preferentially conjugate mono-Ub or K63-linked poly-Ub chains rather than K48-linked poly-Ub chains. Although the molecular machinery involved in the regulation of monoubiquitination or K63-linked polyubiquitination in cultured non-neuronal cells has been explored to a substantial degree, our understanding of such 'non-proteasomal' roles of ubiquitination in nerve cell development and function in vivo is very rudimentary. We reported recently that Nedd4-1 and Nedd4-2 play key roles in neurite branching (Kawabe et al., Neuron 65, 358, 2010; Hsia et al., Proc. Natl. Acad. Sci. U.S.A. 111, 13205, 2014). Another Nedd4 superfamily member, Smurf1, is crucial for axon acquisition. In contrast, the roles of WWP1/2 in developing and mature neurons are essentially unknown. We have started to address this issue systematically, performed a preliminary characterization of WWP1/2 double KO mice, and discovered several prominent phenotypic changes in axon formation, migration, and synapse formation in double KO neurons. In the project proposed here, we will study the mechanisms through which WWP1/2 regulate axon formation, neuronal migration, and synapse formation, by identifying and characterizing the substrates of WWP1/2 and by determining how WWP E3 ligases are regulated via upstream signaling pathways.