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Molecular and Cellular Biology, December 2004, p. 10397-10405, Vol. 24, No. 23
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.23.10397-10405.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Manuel Ascano Jr.1,2 and David J. Robbins1,3*

Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover,1 Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire,3 Department of Molecular Genetics, Graduate Program, University of Cincinnati Medical Center, Cincinnati, Ohio2

Received 21 July 2004/ Returned for modification 3 September 2004/ Accepted 14 September 2004

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 ({Delta}Fu) and its carboxyl-terminal domain (Fu-tail). We show that {Delta}Fu 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 {Delta}Fu to Cos2. We demonstrate that {Delta}Fu 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.

* Corresponding author. Mailing address: Dartmouth Medical School, Department of Pharmacology and Toxicology, 10 North College St., 7650 Remsen Hall, Hanover, NH 03755. Phone: (603) 650-1716. Fax: (603) 650-1129. E-mail: david.j.robbins{at}dartmouth.edu.

Molecular and Cellular Biology, December 2004, p. 10397-10405, Vol. 24, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.23.10397-10405.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Farzan, S. F., Stegman, M. A., Ogden, S. K., Ascano, M. Jr, Black, K. E., Tacchelly, O., Robbins, D. J. (2009). A Quantification of Pathway Components Supports a Novel Model of Hedgehog Signal Transduction. J. Biol. Chem. 284: 28874-28884 [Abstract] [Full Text]  
  • Thakur, M., Chaba, R., Mondal, A. K., Chakraborti, P. K. (2008). Interdomain Interaction Reconstitutes the Functionality of PknA, a Eukaryotic Type Ser/Thr Kinase from Mycobacterium tuberculosis. J. Biol. Chem. 283: 8023-8033 [Abstract] [Full Text]  
  • Ogden, S. K., Casso, D. J., Ascano, M. Jr., Yore, M. M., Kornberg, T. B., Robbins, D. J. (2006). Smoothened Regulates Activator and Repressor Functions of Hedgehog Signaling via Two Distinct Mechanisms. J. Biol. Chem. 281: 7237-7243 [Abstract] [Full Text]  
  • Chen, M.-H., Gao, N., Kawakami, T., Chuang, P.-T. (2005). Mice Deficient in the Fused Homolog Do Not Exhibit Phenotypes Indicative of Perturbed Hedgehog Signaling during Embryonic Development. Mol. Cell. Biol. 25: 7042-7053 [Abstract] [Full Text]  


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