(H) Combined loss-of-function of and does not affect the levels of the in vivo Ex protein stability reporter. the transmembrane protein Crb, which in addition to its recognised role in polarity, also regulates tissue growth by modulating the Notch and Hpo pathways (Grzeschik et al., 2010; Chen et al., 2010; Hafezi et al., 2012; Ling et al., 2010; Robinson et al., 2010; Richardson and Pichaud, 2010). Via its FERM-binding motif (FBM), Crb recruits Ex to the apical membrane, where it can promote inhibition of DGKH Yki (Genevet and Tapon, 2011; Chen et al., 2010; Hafezi et al., 2012; Ling et al., 2010; Robinson et al., 2010; Su et al., 2017). However, besides activating Hpo signalling through Ex, Crb also stimulates Ex phosphorylation and turnover of Ex protein (Genevet and Tapon, 2011; Grzeschik et al., 2010; Chen et al., 2010; Ling et al., 2010; Robinson et al., 2010; Ribeiro et al., 2014; Laprise, 2011). We have previously shown that ubiquitylation and degradation of Ex downstream of Crb is usually mediated by the phospho-dependent SCFSlimb/-TrCP E3 ligase complex, which is also thought to regulate Ex levels independently of Crb (Ribeiro et al., 2014; Zhang et al., 2015). However, the identity of the kinase(s) that promote Ex degradation downstream of Crb is currently unknown. Here, we ON-01910 (rigosertib) identify the Casein Kinase 1 (CKI) family of protein kinases as regulators of Ex stability that function downstream of the polarity protein Crb. Depletion of CKI kinases suppresses Crb-induced Ex ON-01910 (rigosertib) phosphorylation, ubiquitylation and degradation. Interestingly, CKI kinases regulate Ex in a partially redundant manner, which suggests that regulation of Ex stability is a key step in the regulation of Yki function and the maintenance of tissue homeostasis. Results Crb promotes conversation of Ex?with Casein kinase 1 family proteins ON-01910 (rigosertib) We have previously shown that Crb regulates Ex protein stability in a -TrCP-dependent manner (Ribeiro et al., 2014). The Ex:Slimb (Slmb, -TrCP) conversation is mediated by a -TrCP consensus sequence immediately following the Ex N-terminal FERM domain (452TSGIVS457). In agreement with the fact that -TrCP targets substrates for ubiquitylation and degradation through the recognition of a phosphodegron, Ex is phosphorylated in S2 cells in the presence of ectopic Crb (full-length or the intracellular domain, Crbintra) (Ling et al., 2010; Robinson et al., 2010; Ribeiro et al., 2014). However, the kinase(s) involved in Ex degradation downstream of Crb are currently unknown. In our previous report, we used affinity purification coupled with mass spectrometry (AP-MS) to identify Slmb as an Ex interacting protein (Ribeiro et al., 2014). Upon re-analysis of our AP-MS data, we observed that Gilgamesh (Gish), the orthologue of Casein kinase 1, was specifically purified by an Ex truncation that fully recapitulates the Crb-mediated effect on Ex ON-01910 (rigosertib) stability (Ex1-468) (Figure 1A) (Ribeiro et al., 2014). Importantly, Gish peptides were detected in the Ex1-468 AP-MS only upon co-expression with wild-type (wt) Crb but not with a FERM-binding motif mutant version of Crb (FBM) that cannot bind Ex or promote its depletion (Figure 1A) (Ling et al., 2010; Robinson et al., 2010; Ribeiro et al., 2014). In contrast, Hpo interacted with Ex regardless of Crb presence, in agreement with previous reports (Figure 1A) (Yu et al., 2010). Open in a separate window Figure 1. Gish, the orthologue of CkI interacts with Ex in a Crb-dependent manner.(A) An AP-MS approach identified Gish as an Ex-interacting protein. Summary table with AP-MS results for Gish and Hpo. CG# denotes Flybase CG number, while unique and total denote the number of peptides detected in the MS analysis. (B) and (C) Crbintra promotes Ex:Gish binding in a FBM-dependent manner. Reciprocal co-IPs were performed with FLAG-tagged NTAN or Ex1-468 and HA-tagged Gish (B), or with FLAG-tagged NTAN or GishisoI and V5-tagged Ex1-468 CAAX (C), in the presence of Myc-tagged GFP, Crbintra or CrbFBM. The expression and presence of co-purified proteins were analysed by immunoblotting with the.