Phosphorylation by double-time/CKIε and CKIα targets Cubitus Interruptus for Slimb/β-TRCP-mediated proteolytic processing

Hedgehog (Hh) proteins govern animal development by regulating the Gli/ Ci family of transcription factors. In Drosophila, Hh signaling blocks proteolytic processing of full-length Ci to generate a truncated repressor form. Ci processing requires sequential phosphorylation by PKA, GSK3, and a casein kinase I (CKI) family member(s). Here we show that Double-time (DBT)/CKIε and CKIα act in conjunction to promote Ci processing. CKI phosphorylates Ci at three clusters of serine residues primed by PKA and GSK3 phosphorylation. CKI phosphorylation of Ci confers binding to the F-box protein Slimb/β-TRCP, the substrate recognition component of the SCF^{Slimb/β-TRCP} ubiquitin ligase required for Ci processing. CKI phosphorylation sites act cooperatively to promote Ci processing in vivo. Substitution of Ci phosphorylation clusters with a canonical Slimb/β-TRCP recognition motif in β-catenin renders Slimb/β-TRCP binding and Ci processing independent of CKI. We propose that phosphorylation of Ci by CKI creates multiple Slimb/β-TRCP binding sites that act cooperatively to recruit SCF^{Slimb/β-TRCP}.