CNK and HYP form a discrete dimer by their SAM domains to mediate RAF kinase signaling

doi:10.1073/pnas.0709705105

CNK and HYP form a discrete dimer by their SAM domains to mediate RAF kinase signaling

*Centre for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, ON, Canada M5G 1X5;
^†Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8;
^‡Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal CP 6128, Montréal, QC, Canada H3C 3J7;
^§Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853;
^¶NE-CAT, Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439; and
^‖Département de pathologie et de biologie cellulaire, Université de Montréal, 6128 Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7

Edited by Anthony J. Pawson, University of Toronto, Toronto, ON, Canada, and approved December 29, 2007 (received for review October 12, 2007)

Abstract

RAF kinase functions in the mitogen-activated protein kinase (MAPK) pathway to transmit growth signals to the downstream kinases MEK and ERK. Activation of RAF catalytic activity is facilitated by a regulatory complex comprising the proteins CNK (Connector enhancer of KSR), HYP (Hyphen), and KSR (Kinase Suppressor of Ras). The sterile α-motif (SAM) domain found in both CNK and HYP plays an essential role in complex formation. Here, we have determined the x-ray crystal structure of the SAM domain of CNK in complex with the SAM domain of HYP. The structure reveals a single-junction SAM domain dimer of 1:1 stoichiometry in which the binding mode is a variation of polymeric SAM domain interactions. Through in vitro and in vivo mutational analyses, we show that the specific mode of dimerization revealed by the crystal structure is essential for RAF signaling and facilitates the recruitment of KSR to form the CNK/HYP/KSR regulatory complex. We present two docking-site models to account for how SAM domain dimerization might influence the formation of a higher-order CNK/HYP/KSR complex.

Footnotes

**To whom correspondence may be addressed. E-mail: marc.therrien{at}umontreal.ca or sicheri{at}mshri.on.ca
Author contributions: T.R. and M.S. contributed equally to this work; T.R., M.S., M. Therrien, and F.S. designed research; T.R., M.S., and I.K. performed research; and T.R., M.S., M. Therrien, and F.S. analyzed data; M. Tyers contributed new reagents/analytic tools; and T.R., M. Therrien, and F.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3BS5 and 3BS7).
This article contains supporting information online at www.pnas.org/cgi/content/full/0709705105/DC1.