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A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity

Abstract

cRaf-1 is a mitogen-activated protein kinase that is the main effector recruited by GTP-bound Ras in order to activate the MAP kinase pathway1. Inactive Raf is found in the cytosol in a complex with Hsp90, Hsp50 (Cdc37)2,3 and the 14-3-3 proteins4. GTP-bound Ras binds Raf and is necessary but not sufficient for the stable activation of Raf that occurs in response to serum, epidermal growth factor, platelet-derived growth factor or insulin5,6,7,8. These agents cause a two- to threefold increase in overall phosphorylation of Raf on serine/threonine residues8,9, and treatment of cRaf-1 with protein (serine/threonine) phosphatases can deactivate it, at least partially10. The role of 14-3-3 proteins in the regulation of Raf's kinase activity is uncertain4,11 and is investigated here. Active Raf can be almost completely deactivated in vitro by displacement of 14-3-3 using synthetic phosphopeptides. Deactivation can be substantially reversed by addition of purified recombinant bacterial 14-3-3; however, Raf must have been previously activated in vivo to be reactivated by 14-3-3 in vitro. The ability of 14-3-3 to support Raf activity is dependent on phosphorylation of serine residues on Raf and on the integrity of the 14-3-3 dimer; mutant monomeric forms of 14-3-3, although able to bind Raf in vivo, do not enable Raf to be activated in vivo or restore Raf activity after displacement of 14-3-3 in vitro. The 14-3-3 protein is not required to induce dimerization of Raf. We propose that dimeric 14-3-3 is needed both to maintain Raf in an inactive state in the absence of GTP-bound Ras and to stabilize an active conformation of Raf produced during activation in vivo.

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Figure 1: The binding of 14-3-3 to Raf is required for Raf kinase activity.
Figure 2: Raf inactivated by phosphopeptide in vitro can be reactivated by recombinant 14-3-3.
Figure 3: The binding of 14-3-3 to Raf is necessary for Raf activity but not for Raf oligomerization.
Figure 4: Dimerization-deficient mutants of 14-3-3 bind Raf but do not support Raf activity in vivo or in vitro.

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Acknowledgements

The authors were supported by a grant from Eli Lilly, Inc. We thank A. Khatri for peptide synthesis and purification, and J. Prendable for preparation of the manuscript.

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Correspondence to Joseph Avruch.

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Tzivion, G., Luo, Z. & Avruch, J. A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature 394, 88–92 (1998). https://doi.org/10.1038/27938

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