Role of Armadillo repeat 2 and kinesin-II motor subunit Klp64D for wingless signaling in Drosophila.

Armadillo (Arm) is crucial for transducing Wingless (Wg) signaling. Previously, we have shown that Klp64D, a motor subunit of Drosophila kinesin-II, interacts with Arm for Wg signaling. Molecular basis for this interaction has remained unknown. Here we identify a critical Arm repeat (AR) required for binding Klp64D and Wg signaling. Arm/[Formula: see text]-catenin family proteins contain a conserved domain of 12 Arm repeats (ARs). Five of these ARs can interact with Klp64D, but only the second AR (AR2) binds to the cargo/tail domain of Klp64D. Overexpression of AR2 in wing imaginal disc is sufficient to cause notched wing margin. This phenotype by AR2 is enhanced or suppressed by reducing or increasing Klp64D expression, respectively. AR2 overexpression inhibits Wg signaling activity in TopFlash assay, consistent with its dominant-negative effects on Klp64D-dependent Wg signaling. Overexpression of the Klp64D cargo domain also results in dominant-negative wing notching. Genetic rescue data indicate that both AR2 and Klp64D cargo regions are required for the function of Arm and Klp64D, respectively. AR2 overexpression leads to an accumulation of Arm with GM130 Golgi marker in Klp64D knockdown. This study suggests that Wg signaling for wing development is regulated by specific interaction between AR2 and the cargo domain of Klp64D.

Crumbs, Galla and Xpd are required for Kinesin-5 regulation in mitosis and organ growth in Drosophila.

Xeroderma Pigmentosum D (XPD, also known as ERCC2) is a multi-functional protein involved in transcription, DNA repair and chromosome segregation. In Drosophila, Xpd interacts with Crumbs (Crb) and Galla to regulate mitosis during embryogenesis. It is unknown how these proteins are linked to mitosis. Here, we show that Crb, Galla-2 and Xpd regulate nuclear division in the syncytial embryo by interacting with Klp61F, the Drosophila mitotic Kinesin-5 associated with bipolar spindles. Crb, Galla-2 and Xpd physically interact with Klp61F and colocalize to mitotic spindles. Knockdown of any of these proteins results in similar mitotic defects. These phenotypes are restored by overexpression of Klp61F, suggesting that Klp61F is a major effector. Mitotic defects of galla-2 RNAi are suppressed by Xpd overexpression but not vice versa. Depletion of Crb, Galla-2 or Xpd results in a reduction of Klp61F levels. Reducing proteasome function restores Klp61F levels and suppresses mitotic defects caused by knockdown of Crb, Galla-2 or Xpd. Furthermore, eye growth is regulated by Xpd and Klp61F. Hence, we propose that Crb, Galla-2 and Xpd interact to maintain the level of Klp61F during mitosis and organ growth.

Ebi modulates wing growth by ubiquitin-dependent downregulation of Crumbs in Drosophila.

Notch signaling at the dorsoventral (DV) boundary is essential for patterning and growth of wings in Drosophila The WD40 domain protein Ebi has been implicated in the regulation of Notch signaling at the DV boundary. Here we show that Ebi regulates wing growth by antagonizing the function of the transmembrane protein Crumbs (Crb). Ebi physically binds to the extracellular domain of Crb (Crbext), and this interaction is specifically mediated by WD40 repeats 7-8 of Ebi and a laminin G domain of Crbext Wing notching resulting from reduced levels of Ebi is suppressed by decreasing the Crb function. Consistent with this antagonistic genetic relationship, Ebi knockdown in the DV boundary elevates the Crb protein level. Furthermore, we show that Ebi is required for downregulation of Crb by ubiquitylation. Taken together, we propose that the interplay of Crb expression in the DV boundary and ubiquitin-dependent Crb downregulation by Ebi provides a mechanism for the maintenance of Notch signaling during wing development.

14-3-3 proteins regulate Tctp-Rheb interaction for organ growth in Drosophila.

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.

Kinesin-II recruits Armadillo and Dishevelled for Wingless signaling in Drosophila.

Wingless (Wg)/Wnt signaling is fundamental in metazoan development. Armadillo (Arm)/ß-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 ß-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.