Characterization of Novel Drosophila Rho Kinase Substrates & their role in Non-Canonical Wnt/Planer Cell Polarity Signaling

Abstract

Polarity across the plane of an epithelium is a fundamental phenomenon required for the formation of complex tissues. This phenomenon is known as Planar Cell Polarity (PCP) and is controlled by the non-canonical Wnt/Fz-PCP signaling pathway. A genome-wide Rho kinase substrate screen was performed to identify new components of the pathway. Herein, I describe the identification and characterization of two novel Rok substrates: Combover/CG10732 and Maf-S.;Using mass spectrometry, I identified sites of Rok phosphorylation on Combover and site-directed mutagenesis has confirmed these are bona-fide sites of Rok phosphorylation. A combover mutant allele, generated by homologous recombination, was homozygous viable and failed to phenocopy the initial multiple hair cell (MHC) RNAi phenotype. However, overexpression of the Combover isoforms caused a strong MHC phenotype. Furthermore, the removal of a gene dose of rok or members of the planer cell polarity effector gene family enhanced the MHC phenotype. Protein interaction studies revealed Multiple wing hairs (Mwh) as a Cmb interacting protein. Additionally, combination double mutant interaction studies with two different mwh alleles and cmbKO displayed a suppression of the MHC phenotype. This indicated the contribution of cmb to wing hair formation. Cmb localization studies in pupal wing discs shows the protein accumulates at the apical membrane, where the PCP effectors are known to be active. This data is consistent with a model in which Mwh antagonizes Cmb, a novel Rok Substrate, during wing hair initiation and formation.;Using in vitro Rok assays and mass spectra analysis, five sites of Rok phosphorylation on Maf-S were identified. Homozygotes of a null allele of maf-S failed to survive past the L2 stage. Clonal analysis in the Drosophila eye showed no discernible phenotype in maf-S clones. Additionally, no change in eye architecture was noted in maf-S/jun clones, when compared to jun clones. Genetic interaction studies with sevenless<disheveled and sevenless<frizzled showed that the maf-S null allele and a maf-S deficiency were able to suppress the effects of aberrant PCP signaling. Current data suggests that Maf-S is a newly identified Rok substrate that may play a role in the transcriptional branch of the Planar Cell Polarity Signaling Pathway.