Identification of Domains Responsible for Ubiquitin-Dependent Degradation of dMyc by Glycogen Synthase Kinase 3β and Casein Kinase 1 Kinases

Abstract

In the present study, we report that ubiquitin-mediated degradation of dMyc, the Drosophila homologue of the human c-myc proto-oncogene, is regulated in vitro and in vivo by members of the casein kinase 1 (CK1) family and by glycogen synthase kinase 3β (GSK3β). Using Drosophila S2 cells, we demonstrate that CK1α promotes dMyc ubiquitination and degradation with a mechanism similar to the one mediated by GSK3β in vertebrates. Mutation of ck1α or -ε or sgg/gsk3β in Drosophila wing imaginal discs results in the accumulation of dMyc protein, suggesting a physiological role for these kinases in vivo. Analysis of the dMyc amino acid sequence reveals the presence of conserved domains containing potential phosphorylation sites for mitogen kinases, GSK3β, and members of the CK1 family. We demonstrate that mutations of specific residues within these phosphorylation domains regulate dMyc protein stability and confer resistance to degradation by CK1α and GSK3β kinases. Expression of the dMyc mutants in the compound eye of the adult fly results in a visible defect that is attributed to the effect of dMyc on growth, cell death, and inhibition of ommatidial differentiation.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank Shin-ichi Yanagawa and Kennet Moberg for plasmids; Daniel Fimiartz for confocal imaging; Jorge Morales for electron microscopy support; and Peter Gallant, Nathalie Methot, and Myriam Zecca for critical reading of the manuscript. We thank the Developmental Studies Hybridoma Bank under the auspices of NICHD for antibodies.

This study was supported by the National Center for Research Resources (NIH 5G12RR03060), by a Public Health Service grant from the NIH (5SC1DK085047-1) and the Research Foundation RF-CUNY (P.B.), and by the CaRisBo Foundation (S.R.)