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A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK

Nature volume 472, pages 366–369 (2011)Cite this article

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Abstract

In metazoans, the Ras–Raf–MEK (mitogen-activated protein-kinase kinase)–ERK (extracellular signal-regulated kinase) signalling pathway relays extracellular stimuli to elicit changes in cellular function and gene expression. Aberrant activation of this pathway through oncogenic mutations is responsible for a large proportion of human cancer. Kinase suppressor of Ras (KSR)1,2,3 functions as an essential scaffolding protein to coordinate the assembly of Raf–MEK–ERK complexes4,5. Here we integrate structural and biochemical studies to understand how KSR promotes stimulatory Raf phosphorylation of MEK (refs 6, 7). We show, from the crystal structure of the kinase domain of human KSR2 (KSR2(KD)) in complex with rabbit MEK1, that interactions between KSR2(KD) and MEK1 are mediated by their respective activation segments and C-lobe αG helices. Analogous to BRAF (refs 8, 9), KSR2 self-associates through a side-to-side interface involving Arg 718, a residue identified in a genetic screen as a suppressor of Ras signalling1,2,3. ATP is bound to the KSR2(KD) catalytic site, and we demonstrate KSR2 kinase activity towards MEK1 by in vitro assays and chemical genetics. In the KSR2(KD)–MEK1 complex, the activation segments of both kinases are mutually constrained, and KSR2 adopts an inactive conformation. BRAF allosterically stimulates the kinase activity of KSR2, which is dependent on formation of a side-to-side KSR2–BRAF heterodimer. Furthermore, KSR2–BRAF heterodimerization results in an increase of BRAF-induced MEK phosphorylation via the KSR2-mediated relay of a signal from BRAF to release the activation segment of MEK for phosphorylation. We propose that KSR interacts with a regulatory Raf molecule in cis to induce a conformational switch of MEK, facilitating MEK’s phosphorylation by a separate catalytic Raf molecule in trans.

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Figure 1: Structure of the KSR2(KD)–MEK1 heterodimer.
Figure 2: KSR2 is a protein kinase.
Figure 3: KSR2 and BRAF homodimerize through a conserved side-to-side interface, but generate different quaternary structures.
Figure 4: Stimulation of MEK phosphorylation by an allosteric transition of KSR mediated by BRAF binding to the Arg 718 side-to-side interface.

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Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the RCSB Protein Data Bank with accession numbers 2y4i and 2y4isf, respectively.

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Acknowledgements

This work was supported by a Cancer Research UK grant to D.B., ICR studentships to D.F.B. and W.C.H.C. and HHMI grant to K.M.S. We thank staff at the ESRF for help with data collection and K. Wood and V. Good for help with protein production and Z. Zhang for assistance with cloning. Mass spectrometry was made possible by NIH grants NCRR RR015804 and NCRR RR001614. The MEK1/p50Cdc37 baculovirus was a gift from C. Vaughan. We thank Cell Signaling Technologies for help with phosphospecific MEK antibodies.

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Author notes

  1. Damian F. Brennan and Arvin C. Dar: These authors contributed equally to this work.

Authors and Affiliations

  1. Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK ,

    Damian F. Brennan, William C. H. Chao & David Barford

  2. Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, 94107, California, USA

    Arvin C. Dar, Nicholas T. Hertz & Kevan M. Shokat

  3. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, 94107, California, USA

    Alma L. Burlingame

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Contributions

D.F.B. determined and analysed the MEK1–KSR2 structure; A.C.D. conducted biochemical analysis of Raf–KSR–MEK phosphorylation and inhibitor studies; N.T.H. carried out phosphoproteomics mass spectrometry studies; W.C.H.C. helped with protein production; A.L.B. analysed mass spectrometry data; K.M.S. designed and analysed experiments relating to the Raf–KSR–MEK phosphorylation and inhibitor studies; and D.B. designed experiments and analysed data.

Corresponding authors

Correspondence to Kevan M. Shokat or David Barford.

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The authors declare no competing financial interests.

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Brennan, D., Dar, A., Hertz, N. et al. A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK. Nature 472, 366–369 (2011). https://doi.org/10.1038/nature09860

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