An Intramolecular Association between Two Domains of the Protein Kinase Fused Is Necessary for Hedgehog Signaling

Abstract

The protein kinase Fused (Fu) is an integral member of the Hedgehog (Hh) signaling pathway. Although genetic studies demonstrate that Fu is required for the regulation of the Hh pathway, the mechanistic role that it plays remains largely unknown. Given our difficulty in developing an in vitro kinase assay for Fu, we reasoned that the catalytic activity of Fu might be highly regulated. Several mechanisms are known to regulate protein kinases, including self-association in either an intra- or an intermolecular fashion. Here, we provide evidence that Hh regulates Fu through intramolecular association between its kinase domain (ΔFu) and its carboxyl-terminal domain (Fu-tail). We show that ΔFu and Fu-tail can interact in trans, with or without the kinesin-related protein Costal 2 (Cos2). However, since the majority of Fu is found associated with Cos2 in vivo, we hypothesized that Fu-tail, which binds Cos2 directly, would be able to tether ΔFu to Cos2. We demonstrate that ΔFu colocalizes with Cos2 in the presence of Fu-tail and that this colocalization occurs on a subset of membrane vesicles previously characterized to be important for Hh signal transduction. Additionally, expression of Fu-tail in fu mutant flies that normally express only the kinase domain rescues the fu wing phenotype. Therefore, reestablishing the association between these two domains of Fu in trans is sufficient to restore Hh signal transduction in vivo. In such a manner we validate our hypothesis, demonstrating that Fu self-associates and is functional in an Hh-dependent manner. Our results here enhance our understanding of one of the least characterized, yet critical, components of Hh signal transduction.

We thank the members of the Robbins laboratory, Y. Sanchez (University of Cincinnati), and R. Craig (Dartmouth Medical School) for their helpful discussions and/or critical review of the manuscript. We thank the members of the Cartwright laboratory (University of Cincinnati) and the Lin laboratory (University of Cincinnati) for their assistance in our transgenic Drosophila experiments. We thank K. Nybakken (Harvard) for providing the Fu-tail transgenic Drosophila and for useful discussions. We also thank A. Lavanway (Dartmouth College) and K. Orndorff (Dartmouth-Hitchcock Medical Center) for expert assistance in confocal microscopy and colocalization analysis.

This work was supported by National Institutes of Health grant CA82628 (to D.J.R.), NCI training grant 5T32 ES07250 (to M.A.), and the Albert J. Ryan Foundation (to M.A.).