14-3-3 family proteins regulate multiple signalling pathways. Understanding biological functions of 14-3-3 proteins has been limited by the functional redundancy of conserved isotypes. Here we provide evidence that 14-3-3 proteins regulate two interacting components of Tor signalling in Drosophila, translationally controlled tumour protein (Tctp) and Rheb GTPase. Single knockdown of 14-3-3ɛ or 14-3-3ζ isoform does not show obvious defects in organ development but causes synergistic genetic interaction with Tctp and Rheb to impair tissue growth. 14-3-3 proteins physically interact with Tctp and Rheb. Knockdown of both 14-3-3 isoforms abolishes the binding between Tctp and Rheb, disrupting organ development. Depletion of 14-3-3s also reduces the level of phosphorylated S6 kinase, phosphorylated Thor/4E-BP and cyclin E (CycE). Growth defects from knockdown of 14-3-3 and Tctp are suppressed by CycE overexpression. This study suggests a novel mechanism of Tor regulation mediated by 14-3-3 interaction with Tctp and Rheb.

Wingless (Wg)/Wnt signaling is fundamental in metazoan development. Armadillo (Arm)/β-catenin and Dishevelled (Dsh) are key components of Wnt signal transduction. Recent studies suggest that intracellular trafficking of Wnt signaling components is important, but underlying mechanisms are not well known. Here, we show that Klp64D, the Drosophila homolog of Kif3A kinesin II subunit, is required for Wg signaling by regulating Arm during wing development. Mutations in klp64D or RNAi cause wing notching and loss of Wg target gene expression. The wing notching phenotype by Klp64D knockdown is suppressed by activated Arm but not by Dsh, suggesting that Klp64D is required for Arm function. Furthermore, klp64D and arm mutants show synergistic genetic interaction. Consistent with this genetic interaction, Klp64D directly binds to the Arm repeat domain of Arm and can recruit Dsh in the presence of Arm. Overexpression of Klp64D mutated in the motor domain causes dominant wing notching, indicating the importance of the motor activity. Klp64D shows subcellular localization to intracellular vesicles overlapping with Arm and Dsh. In klp64D mutants, Arm is abnormally accumulated in vesicular structures including Golgi, suggesting that intracellular trafficking of Arm is affected. Human KIF3A can also bind β-catenin and rescue klp64D RNAi phenotypes. Taken together, we propose that Klp64D is essential for Wg signaling by trafficking of Arm via the formation of a conserved complex with Arm.

Wnt/Wg-signalling is critical signalling in all metazoans. Recent studies suggest that IFT-A proteins and Kinesin-2 modulate canonical Wnt/Wg-signalling independently of their ciliary role. Whether they function together in Wnt-signalling and their mechanistic role in the pathway remained unresolved. Here we demonstrate that Kinesin-2 and IFT-A proteins act as a complex during Drosophila Wg-signalling, affecting pathway activity in the same manner, interacting genetically and physically, and co-localizing with β-catenin, the mediator of Wnt/Wg-signalling on microtubules. Following pathway activation, Kinesin-2/IFT-A mutant cells exhibit high cytoplasmic β-catenin levels, yet fail to activate Wg-targets. In mutant tissues in both, Drosophila and mouse/MEFs, nuclear localization of β-catenin is markedly reduced. We demonstrate a conserved, motor-domain dependent function of the Kinesin-2/IFT-A complex in promoting nuclear translocation of β-catenin. We show that this is mediated by protecting β-catenin from a conserved cytoplasmic retention process, thus identifying a mechanism for Kinesin-2/IFT-A in Wnt-signalling that is independent of their ciliary role.

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