Hedgehog (Hh) signaling is fundamental to many developmental processes in organisms as divergent as Drosophila melanogaster and humans. The Hh family of secreted proteins activates intracellular signaling cascades for a variety of cellular processes and plays an essential role in embryonic patterning and cell type specification. Vertebrate homologues of Hh are involved in patterning of limbs, lungs, hair follicles and other processes and misregulation of or mutations in Hh signaling genes lead to a number of developmental defects and disorders. Critical to Hh activation is a large protein complex termed the Hedgehog Signaling Complex (HSC). The HSC interprets the level of Hh activation by regulating the levels and activity of one of its components, the transcription factor Ci. Two other components of the HSC are the Ser/Thr protein kinase Fused (Fu) and the putative motor/scaffolding protein Costal-2 (Cos2). Cos2 has significant sequence identity to kinesin family motors, suggesting that Cos2 motility might be important for its function. It also appears to be enriched on microtubules in a manner that is attenuated when cells are exposed to Hh, again consistent with it being an Hh regulated kinesin family member. However, the lack of a key conserved catalytic sequence in Cos2 raises doubts as to whether it is a functional kinesin motor and it has been proposed that Cos2 functions solely as a scaffolding protein, binding and enriching various other Hh pathway components. In order to clarify the role Cos2 plays in Hh signaling, we will perform a detailed mechanistic analysis of its activity. Our preliminary results show that the Cos2 motor domain has both ATPase and GTPase activities. The proposed studies will explore in detail the Cos2 catalytic cycle, as well as characterizing the Cos2 protein interactome using structural and mechanistic approaches to investigate the interaction of Cos2 with microtubules and other binding partners. As a step towards achieving these goals, we have developed a bacterial-based system for the high level expression of a eukaryotic-like nonpolymerizing tubulin dimer. The high-resolution crystal structures to be obtained here will provide key insight into the mode of action of Cos2 in Hh signaling and may lead to novel therapeutic targets for regulation of Hedgehog signaling.

Public Health Relevance

The Hedgehog (Hh) family of secreted proteins activates intracellular signaling cascades for a variety of cellular processes, plays an essential role in development, and disruption of the Hh pathway leads to an increasingly large number of different human pathologies. Costal-2 (Cos2) is a distant relative of cellular motor proteins, and is essential for proper signaling in the Hh pathway. This proposal seeks to characterize Cos2 in order to figure out exactly how it operates, a critical first step in designing rational preventative and curative strategies against diseases related to malfunctions in the Hh signaling pathway.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Gindhart, Joseph G
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Dartmouth College
Schools of Arts and Sciences
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Cochran, Jared C; Thompson, Morgan E; Kull, F Jon (2013) Metal switch-controlled myosin II from Dictyostelium discoideum supports closure of nucleotide pocket during ATP binding coupled to detachment from actin filaments. J Biol Chem 288:28312-23
Kull, F Jon; Endow, Sharyn A (2013) Force generation by kinesin and myosin cytoskeletal motor proteins. J Cell Sci 126:9-19
Waitzman, Joshua S; Larson, Adam G; Cochran, Jared C et al. (2011) The loop 5 element structurally and kinetically coordinates dimers of the human kinesin-5, Eg5. Biophys J 101:2760-9
Cochran, Jared C; Zhao, Yu Cheng; Wilcox, Dean E et al. (2011) A metal switch for controlling the activity of molecular motor proteins. Nat Struct Mol Biol 19:122-7