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J. Biol. Chem., Vol. 281, Issue 8, 5084-5093, February 24, 2006

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Biochemical Characterization of the Diaphanous Autoregulatory Interaction in the Formin Homology Protein FHOD1^*

André Schönichen, Michael Alexander, Judith E. Gasteier, Fanny E. Cuesta, Oliver T. Fackler, and Matthias Geyer¹

From the Max Planck Institute for Molecular Physiology, Department of Physical Biochemistry, D-44227 Dortmund, Germany and the Department of Virology, University of Heidelberg, D-69120 Heidelberg, Germany

Diaphanous related formins (DRFs) are cytoskeleton remodeling proteins that mediate specific upstream GTPase signals to regulate cellular processes such as cytokinesis, cell polarity, and organelle motility. Previous work on the Rho-interacting DRF mDia has established that the biological activity of DRFs is regulated by an autoinhibitory interaction of a C-terminal diaphanous autoregulatory domain (DAD) with the DRF N terminus. This autoinhibition is released upon competitive binding of an activated GTPase to the N terminus of the DRF. Analyzing autoregulation of the Rac1-interacting DRF FHOD1, we utilized in vitro binding studies to identify a 60-amino acid DAD at the protein C terminus that recognizes an N-terminal formin homology (FH) 3 domain. Importantly, the FH3 domain of FHOD1 does not overlap with the proposed Rac1-binding domain. The FHOD1 DAD was found to contain one functional hydrophobic autoregulatory motif, while a previously uncharacterized basic cluster that is conserved in all DRF family DADs also contributed to the FH3-DAD interaction. Simultaneous mutation of both motifs efficiently released autoinhibition of FHOD1 in NIH3T3 cells resulting in the formation of actin stress fibers and increased serum response element transcription. A second putative hydrophobic autoregulatory motif N-terminal of the DAD belongs to a unique FHOD subdomain of yet undefined function. NMR structural analysis and size exclusion chromatography experiments revealed that the FHOD1 DAD is intrinsically unstructured with a tendency for a helical conformation in the hydrophobic autoregulation motif. Together, these data suggest that in FHOD1, DAD acts as signal sequence for binding to the well folded and monomeric FH3 domain and imply an activation mechanism that differs from competitive binding of Rac1 and DAD to one interaction site.

Received for publication, August 22, 2005 , and in revised form, November 17, 2005.

^* This work was supported by a fellowship from the Chica and Heinz Schaller Stiftung (to O. T. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Max-Planck-Institut für Molekulare Physiologie, Abt. Physikalische Biochemie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany. Tel.: 49-231-133-2366; Fax: 49-231-133-2399; E-mail: matthias.geyer{at}mpi-dortmund.mpg.de.

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