Previous Article | Next Article 
Molecular and Cellular Biology, March 2009, p. 1306-1320, Vol. 29, No. 5
0270-7306/09/$08.00+0 doi:10.1128/MCB.01271-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism
,
Alexey E. Granovsky,1,
Matthew C. Clark,1,2,
Dan McElheny,3,
Gary Heil,3
Jia Hong,1,2
Xuedong Liu,4
Youngchang Kim,5
Grazyna Joachimiak,5
Andrzej Joachimiak,5
Shohei Koide,3, and
Marsha Rich Rosner1,2,*
Ben May Department for Cancer Research,1 Department of Neurobiology, Pharmacology, and Physiology,2 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637,3 Department of Chemistry and Biochemistry, University of Colorado—Boulder, Boulder, Colorado 80309,4 Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, Illinois5
Received 11 August 2008/ Returned for modification 10 October 2008/ Accepted 15 December 2008
Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.
* Corresponding author. Mailing address: Gordon Center for Integrative Sciences, 929 East 57th Street, Chicago, IL 60637. Phone: (773) 702-0380. Fax: (773) 702-4476. E-mail: m-rosner{at}uchicago.edu
Published ahead of print on 22 December 2008.
Supplemental material for this article may be found at http://mcb.asm.org/.
M.C.C., A.E.G., and D.M. contributed equally to this work. S.K. and M.R.R. contributed equally as senior authors.
Molecular and Cellular Biology, March 2009, p. 1306-1320, Vol. 29, No. 5
0270-7306/09/$08.00+0 doi:10.1128/MCB.01271-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»