Abstract
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.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at http://mcb.asm.org/ .
ACKNOWLEDGMENTS
This work was supported by a Cancer Research UK studentship/project grant (C14503/A48) and a Royal Society Project grant. M.A.P. was a Wellcome Trust University Award holder when this work started.
We thank Bijal Sidapra for her contribution to Fig. and Tao Zhang for help in modeling the kinetics of Ci-75 formation. We thank Alexander Soloviev, David Strutt, Martin Zeidler, Liz Smythe, Carl Smythe, and James Briscoe for useful comments on the work and the manuscript.