Functional Studies of Shaggy/Glycogen Synthase Kinase 3 Phosphorylation Sites in Drosophila melanogaster

Abstract

Early studies of glycogen synthase kinase 3 (GSK-3) in mammalian systems focused on its pivotal role in glycogen metabolism and insulin-mediated signaling. It is now recognized that GSK-3 is central to a number of diverse signaling systems. Here, we show that the major form of the kinase Shaggy (Sgg), the GSK-3 fly ortholog, is negatively regulated during insulin-like/phosphatidylinositol 3-kinase (PI3K) signaling in vivo. Since genetic studies of Drosophila melanogaster had previously shown that Wingless (Wg) signaling also acts to antagonize Sgg, we investigate how the kinase might integrate, or else discriminate, signaling inputs by Wg and insulin. Using Drosophila cell line assays, we found, in contrast to previous reports, that Wg induces accumulation of its transducer Armadillo (Arm)/β-catenin without significant alteration of global Sgg-specific activity. In agreement with a previous study using human GSK-3β, Wg did not cause phosphorylation changes of the Ser9 or Tyr214 regulatory phosphorylated sites of Sgg. Conversely, as shown in mammalian systems, insulin-induced inhibition of Sgg-specific activity by phosphorylation at the N-terminal pseudosubstrate site (Ser9) did not induce Arm/β-catenin accumulation, showing selectivity in response to the different signaling pathways. Interestingly, a minigene bearing a Ser9-to-Ala change rescued mutant sgg without causing abnormal development, suggesting that the regulation of Sgg via the inhibitory pseudosubstrate domain is dispensable for many aspects of its function. Our studies of Drosophila show that Wg and insulin or PI3K pathways do not converge on Sgg but that they exhibit cross-regulatory interactions.

We thank A. Bauer, C. Akermann, and B. Heller for technical help, P. Eberling for peptide synthesis, N. Messaddeq and J.-L. Vonesch for assistance with confocal microscopy, and Y. Lutz for the generation of MAbs. We thank M J. Milner for providing cl-8 cells, F. van Leeuven, C. Samos, K. Willert, and R. Nusse for kindly sending Drosophila cell lines and Wg antibodies, D. M. Duncan for Dll antibodies, and S. Leevers for fly stocks. We thank M. Labouesse and anonymous reviewers for comments on an early version of the manuscript. M.B. is most grateful to N. Tapon and P. Léopold for encouragement and help during manuscript preparation.

Our work was supported by the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg, and a grant from the EEC network (CHRX-CTS940692 501258). D.P. was supported by a fellowship by the CNRS. A numeric acquisition system was obtained by a subvention from the French ARC to M.B. (no. 7389).