The Ccm3-GckIII signaling axis regulates Rab11-dependent recycling to the apical compartment

Abstract

Kinase cascades underlie many signaling pathways and are key regulators of development and morphogenesis. We have characterized a Hippo-like kinase cascade consisting of Thousand and One kinase (Tao), Germinal Center Kinase III (GckIII/Wheezy), and Tricornered (Trc) that plays an essential role in morphogenesis of tracheal terminal cell tubes in Drosophila. In this cascade, GckIII is the central kinase and is thought to act together with its binding partner, Cerebral Cavernous Malformations 3 (Ccm3). As suggested by its name, Drosophila Ccm3 is the ortholog of a human vascular disease gene. As such, defining the Ccm3 pathway is critical to understanding both normal development and disease. Here we generate and characterize a null allele of Ccm3 in Drosophila. We uncover a maternal contribution of Ccm3 to embryonic development, show that maternal/zygotic null embryos have defective multicellular tracheal tubes, and that tracheal terminal cells derived from zygotic clones that also lack maternal Ccm3, show fully penetrant tube dilation defects identical to those we previously described for other pathway genes. We show that wildtype Ccm3 colocalizes with p-GckIII during early embryogenesis, and that in larval terminal cells, is found in the nucleus as well as associated with the apical membrane. We further demonstrate that Mouse protein 25 (Mo25), known to encode a protein that binds and stabilizes GckIII proteins in the active conformation, is required to prevent Ccm3-like tube dilations, showing that Mo25 and Ccm3, together with Tao, are required to fully activate GckIII, which directly phosphorylates and activates Tricornered (Trc). We show that this Ccm3 signaling cassette operates in other epithelial tissues such as the wing, and in non-epithelial tissues such as motor neurons. Lastly, we define a role of Ccm3-GckIII signaling in the distribution of active Rab11, leading us to propose that persistent local Rab11 activity results in elevated recycling of membrane and apical determinants to the apical domain, and consequent dilation of tubes. We validate this model by showing that loss of Rab11 activity ameliorates the tube dilation defects of pathway mutants.