Distinct Binding Preferences Between Ras and Raf Family Members and the Impact on Oncogenic Ras Signaling

Overview

Journal Mol Cell

Publisher Cell Press

Specialty Cell Biology

Date 2019 Oct 14

PMID 31606273

Citations 51

Authors

Elizabeth M Terrell

David E Durrant

Daniel A Ritt

Nancy E Sealover

Erin Sheffels

Russell Spencer-Smith

Dominic Esposito

Yong Zhou

John F Hancock

Robert L Kortum

Deborah K Morrison

Affiliations

Soon will be listed here.

Abstract

The Ras GTPases are frequently mutated in human cancer, and, although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. Here, we examine the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. We find that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf/C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function.

Citing Articles

Different Ras isoforms regulate synaptic plasticity in opposite directions.

Lopez-Merino E, Fernandez-Rodrigo A, Jiang J, Gutierrez-Eisman S, Fernandez de Sevilla D, Fernandez-Medarde A EMBO J. 2025; .

PMID: 39984756 DOI: 10.1038/s44318-025-00390-8.

From bench to bedside: murine models of inherited and sporadic brain arteriovenous malformations.

Ricciardelli A, Genet G, Genet N, McClugage 3rd S, Kan P, Hirschi K Angiogenesis. 2025; 28(2):15.

PMID: 39899215 PMC: 11790818. DOI: 10.1007/s10456-024-09953-5.

Motif-guided identification of KRAS-interacting proteins.

Wu S, Gao X, Wu D, Liu L, Yao H, Meng X BMC Biol. 2024; 22(1):264.

PMID: 39563372 PMC: 11575137. DOI: 10.1186/s12915-024-02067-w.

Mediating kinase activity in Ras-mutant cancer: potential for an individualised approach?.

Healy F, Turner A, Marensi V, MacEwan D Front Pharmacol. 2024; 15:1441938.

PMID: 39372214 PMC: 11450236. DOI: 10.3389/fphar.2024.1441938.

Identification of an H-Ras nanocluster disrupting peptide.

Steffen C, Manoharan G, Pavic K, Yeste-Vazquez A, Knuuttila M, Arora N Commun Biol. 2024; 7(1):837.

PMID: 38982284 PMC: 11233548. DOI: 10.1038/s42003-024-06523-9.

References

Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D . RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer. 2011; 11(11):761-74. PMC: 3632399. DOI: 10.1038/nrc3106. View

Hu Y, Smyth G . ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods. 2009; 347(1-2):70-8. DOI: 10.1016/j.jim.2009.06.008. View

Fabian J, Vojtek A, Cooper J, Morrison D . A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function. Proc Natl Acad Sci U S A. 1994; 91(13):5982-6. PMC: 44121. DOI: 10.1073/pnas.91.13.5982. View

Inouye K, Mizutani S, Koide H, Kaziro Y . Formation of the Ras dimer is essential for Raf-1 activation. J Biol Chem. 2000; 275(6):3737-40. DOI: 10.1074/jbc.275.6.3737. View

Dougherty M, Muller J, Ritt D, Zhou M, Zhou X, Copeland T . Regulation of Raf-1 by direct feedback phosphorylation. Mol Cell. 2005; 17(2):215-24. DOI: 10.1016/j.molcel.2004.11.055. View

Fischer A, Hekman M, Kuhlmann J, Rubio I, Wiese S, Rapp U . B- and C-RAF display essential differences in their binding to Ras: the isotype-specific N terminus of B-RAF facilitates Ras binding. J Biol Chem. 2007; 282(36):26503-16. DOI: 10.1074/jbc.M607458200. View

Cox A, Der C . Ras history: The saga continues. Small GTPases. 2011; 1(1):2-27. PMC: 3109476. DOI: 10.4161/sgtp.1.1.12178. View

Sanjana N, Shalem O, Zhang F . Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014; 11(8):783-784. PMC: 4486245. DOI: 10.1038/nmeth.3047. View

Schubbert S, Shannon K, Bollag G . Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer. 2007; 7(4):295-308. DOI: 10.1038/nrc2109. View

10.

Ambrogio C, Kohler J, Zhou Z, Wang H, Paranal R, Li J . KRAS Dimerization Impacts MEK Inhibitor Sensitivity and Oncogenic Activity of Mutant KRAS. Cell. 2018; 172(4):857-868.e15. DOI: 10.1016/j.cell.2017.12.020. View

11.

Ritt D, Monson D, Specht S, Morrison D . Impact of feedback phosphorylation and Raf heterodimerization on normal and mutant B-Raf signaling. Mol Cell Biol. 2009; 30(3):806-19. PMC: 2812223. DOI: 10.1128/MCB.00569-09. View

12.

Hekman M, Hamm H, Villar A, Bader B, Kuhlmann J, Nickel J . Associations of B- and C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts. J Biol Chem. 2002; 277(27):24090-102. DOI: 10.1074/jbc.M200576200. View

13.

Lavoie H, Therrien M . Regulation of RAF protein kinases in ERK signalling. Nat Rev Mol Cell Biol. 2015; 16(5):281-98. DOI: 10.1038/nrm3979. View

14.

Boussemart L, Girault I, Malka-Mahieu H, Mateus C, Routier E, Rubington M . Secondary Tumors Arising in Patients Undergoing BRAF Inhibitor Therapy Exhibit Increased BRAF-CRAF Heterodimerization. Cancer Res. 2016; 76(6):1476-84. DOI: 10.1158/0008-5472.CAN-15-2900-T. View

15.

Hunter J, Manandhar A, Carrasco M, Gurbani D, Gondi S, Westover K . Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations. Mol Cancer Res. 2015; 13(9):1325-35. DOI: 10.1158/1541-7786.MCR-15-0203. View

16.

Sheffels E, Sealover N, Theard P, Kortum R . Anchorage-independent growth conditions reveal a differential SOS2 dependence for transformation and survival in -mutant cancer cells. Small GTPases. 2019; 12(1):67-78. PMC: 7781674. DOI: 10.1080/21541248.2019.1611168. View

17.

Zhou Y, Prakash P, Liang H, Cho K, Gorfe A, Hancock J . Lipid-Sorting Specificity Encoded in K-Ras Membrane Anchor Regulates Signal Output. Cell. 2017; 168(1-2):239-251.e16. PMC: 5653213. DOI: 10.1016/j.cell.2016.11.059. View

18.

Pfleger K, Eidne K . Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods. 2006; 3(3):165-74. DOI: 10.1038/nmeth841. View

19.

Hu J, Stites E, Yu H, Germino E, Meharena H, Stork P . Allosteric activation of functionally asymmetric RAF kinase dimers. Cell. 2013; 154(5):1036-1046. PMC: 3844432. DOI: 10.1016/j.cell.2013.07.046. View

20.

Waters A, Ozkan-Dagliyan I, Vaseva A, Fer N, Strathern L, Hobbs G . Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies. Sci Signal. 2017; 10(498). PMC: 5812265. DOI: 10.1126/scisignal.aao3332. View