A unique protection signal in Cubitus interruptus prevents its complete proteasomal degradation.

The limited proteolysis of Cubitus interruptus (Ci), the transcription factor for the developmentally and medically important Hedgehog (Hh) signaling pathway, triggers a critical switch between transcriptional repressor and activator forms. Ci repressor is formed when the C terminus of full-length Ci is degraded by the ubiquitin-proteasome pathway, an unusual reaction since the proteasome typically completely degrades its substrates. We show that several regions of Ci are required for generation of the repressor form: the zinc finger DNA binding domain, a single lysine residue (K750) near the degradation end point, and a 163-amino-acid region at the C terminus. Unlike other proteins that are partially degraded by the proteasome, dimerization is not a key feature of Ci processing. Using a pulse-chase assay in cultured Drosophila cells, we distinguish between regions required for initiation of degradation and those required for the protection of the Ci N terminus from degradation. We present a model whereby the zinc finger region and K750 together form a unique protection signal that prevents the complete degradation of Ci by the proteasome.

JDRC 8: Cell biology and development.

Many exciting new discoveries in the fields of cell biology and development were presented at JDRC8, including the molecular characterization of long-studied phenomena such as cell competition, the discovery of long-sought-after molecules such as nutrient sensors in higher organisms, and insights into the role of two of the 135 short ORFs in the Drosophila genome. This review will cover diverse topics roughly divided into four areas: cell biology and signaling, the cytoskeleton, transcription and cell fate determination and metabolism.

Planar polarity is positively regulated by casein kinase Iepsilon in Drosophila.

Members of the casein kinase I (CKI) family have been implicated in regulating canonical Wnt/Wingless (Wg) signaling by phosphorylating multiple pathway components. Overexpression of CKI in vertebrate embryos activates Wg signaling, and one target is thought to be the cytoplasmic effector Dishevelled (Dsh), which is an in vitro target of CKI phosphorylation. Phosphorylation of Dsh by CKI has also been suggested to switch its activity from noncanonical to canonical Wingless signaling. However, in vivo loss-of-function experiments have failed to identify a clear role for CKI in positive regulation of Wg signaling. By examining hypomorphic mutations of the Drosophila CKIepsilon homolog discs overgrown (dco)/double-time, we now show that it is an essential component of the noncanonical/planar cell polarity pathway. Genetic interactions indicate that dco acts positively in planar polarity signaling, demonstrating that it does not act as a switch between canonical and noncanonical pathways. Mutations in dco result in a reduced level of Dishevelled phosphorylation in vivo. Furthermore, in these mutants, Dishevelled fails to adopt its characteristic asymmetric subcellular localisation at the distal end of pupal wing cells, and the site of hair outgrowth is disrupted. Finally, we also find that dco function in polarity is partially redundant with CKIalpha.

CKI, there's more than one: casein kinase I family members in Wnt and Hedgehog signaling.

Multiple members of the casein kinase I family of serine/threonine protein kinases are involved in positive and negative roles in Wnt and Hedgehog signaling. Here I review these roles, including recent results on casein kinase I (CKI) phosphorylation and activation of LRP6, and CKI phosphorylation of Ci and mediation of Ci-Slimb/beta-TrCP binding.

Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1.

The secreted signaling molecule Hedgehog regulates gene expression in target cells in part by preventing proteolysis of the full-length Cubitus interruptus (Ci-155) transcriptional activator to the Ci-75 repressor form. Ci-155 proteolysis depends on phosphorylation at three sites by Protein Kinase A (PKA). We show that these phosphoserines prime further phosphorylation at adjacent Glycogen Synthase Kinase 3 (GSK3) and Casein Kinase I (CK1) sites. Alteration of the GSK3 or CK1 sites prevents Ci-155 proteolysis and activates Ci in the absence of Hedgehog. Ci-155 proteolysis is also inhibited if cells lack activity of the Drosophila GSK3, Shaggy, previously implicated in Wingless signaling. Conversely, Ci-155 levels are reduced in Hedgehog-responding cells by overexpression of PKA and the Drosophila CK1, Double-time, a regulator of circadian rhythms.