Furry is a component of the CCM3-GCKIII signaling pathway

Abstract

Aim: Mutations in 3 genes encoding proteins of the Cerebral Cavernous Malformations (CCM) ternary complex cause autosomal dominant cerebral vascular disease. Targets of CCM complex regulation have been identified; however, the molecular mechanisms connecting CCM3 to these downstream effectors remain elusive. We aim to determine the mechanism of CCM3 action by using a Drosophila model to elucidate the signaling pathway downstream of CCM3. Previously, we showed that CCM3 and its binding partner, Germinal Center Kinase 3, are required in tracheal terminal cells to prevent tube morphogenesis defects. Further, we established that GCKIII phosphorylates and directly activates a downstream kinase, Tricornered (Drosophila STK38/38L ortholog). Here we aim to test whether Tricornered-associated scaffolding protein, Furry, is required for CCM3-GCKIII signaling.

Methods: We utilized the FRT-FLP system to generate genetic mosaic Drosophila larvae and adults. Mitotic recombination was induced in embryos (trachea) or larvae (wing disc). The animals were heterozygous for the gene of interest (ccm3 or furry), but after recombination, homozygous mutant daughter cells were produced. In addition, the GAL4-UAS system was used to express dominant negative GCKIII in wing disc cells. Mutant cells were analyzed by brightfield and/or fluorescent microscopy.

Results: We find that wing cells mutant for ccm3, or expressing dominant negative GCKIII, produce wing hair defects characteristic of mutations in tricornered and furry. Likewise, tracheal terminal cells mutant for furry produce tube dilation defects characteristic of cells mutant for ccm3 or GCKIII.

Conclusion: CCM3 and GCKIII act upstream of Furry-Tricornered, suggesting the conservation from yeast of a Hippo-like signaling pathway that regulates morphogenesis. We speculate that some combination of Furry/Furrylike and STK38/38L are therefore likely to act downstream of CCM3 in endothelial cells.

Keywords

CCM3 / GCKIII / NDR / STK38/38L / tube morphogenesis / planar cell polarity

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