Phosphorylation of the C-Raf N Region Promotes Raf Dimerization

ABSTRACT

The activation of Raf kinases by the small GTPase Ras requires two major sets of phosphorylations. One set lies within the activation loop, and the other lies within the N-terminal acidic region (N region). In the most abundant isoform of Raf, C-Raf, N-region phosphorylations occur on serine 338 (S338) and tyrosine 341 (Y341) and are thought to provide allosteric activation of the Raf dimer. We show that the phosphorylations of these N-region sites does not require C-Raf dimerization, but rather, they precede dimerization. One of these phosphorylations (phospho-Y341) is required for C-Raf dimerization, and this action can be replicated by phosphomimetic mutants both in vivo and in vitro. The role of the phosphorylation of Y341 in promoting Raf dimerization is distinct from its well-known function in facilitating S338 phosphorylation. In Ras mutant pancreatic cancer cell lines, the phosphorylation and dimerization of C-Raf are basally elevated. Dimerization is thought to contribute to their elevated growth rate through their activation of the mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase [ERK]) signaling cascade. Blocking the tyrosine phosphorylation of C-Raf with Src family inhibitors blocks growth, basal dimerization, and ERK activation in these cells. We suggest that the kinases mediating C-Raf Y341 phosphorylation are potential candidate drug targets in selected Ras-dependent cancers.

KEYWORDS:

• C-Raf
• B-Raf
• Ras
• dimerization
• phosphorylation
• extracellular signal-regulated kinase (ERK)

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ACKNOWLEDGMENTS

We are grateful to Jeffrey Tyner (OHSU) for specific kinase inhibitors and Rosie Sears (OHSU) for selected pancreatic cell lines. We thank Jon Savage and Ujwal Shinde (OHSU) for help in CD studies.

These studies were supported in part by NIH grants R01 DK090309 and R21 CA191392-01 and pilot funds from the OHSU Knight Cancer Institute and the Brenden-Colson Center for Pancreatic Care, OHSU. P.J.S.S. received funding from the Million Dollar Bike Ride grant program (MDBR-17-128), Orphan Disease Center, University of Pennsylvania.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

We have no competing interests.

P.J.S.S. and M.T. designed the study. M.T. collected and analyzed data. P.J.S.S. and M.T. wrote and prepared the manuscript. T.J.D. and Y.L. collected and analyzed data from multiple figures. Y.K. collected and analyzed data from Fig. 4.