Format

Send to

Choose Destination
J Mol Biol. 2020 Feb 14;432(4):1199-1215. doi: 10.1016/j.jmb.2020.01.002. Epub 2020 Jan 10.

Oncogenic K-Ras4B Dimerization Enhances Downstream Mitogen-activated Protein Kinase Signaling.

Author information

1
Departments of Chemical and Biological Engineering, Research Center for Translational Medicine, Koc University, Istanbul 34450, Turkey.
2
Departments of Molecular Biology and Genetics, Koc University, Istanbul 34450, Turkey.
3
Computational Structural Biology Section, Frederick National Laboratory for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
4
Computational Structural Biology Section, Frederick National Laboratory for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA; Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
5
Departments of Chemical and Biological Engineering, Research Center for Translational Medicine, Koc University, Istanbul 34450, Turkey; Departments of Molecular Biology and Genetics, Koc University, Istanbul 34450, Turkey.
6
Departments of Computer Engineering, Research Center for Translational Medicine, Koc University, Istanbul 34450, Turkey. Electronic address: agursoy@ku.edu.tr.
7
Departments of Chemical and Biological Engineering, Research Center for Translational Medicine, Koc University, Istanbul 34450, Turkey. Electronic address: okeskin@ku.edu.tr.

Abstract

Ras recruits and activates effectors that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, Raf's activation requires dimerization, which can be facilitated by Ras dimerization. Previously, we showed that active K-Ras4B dimerizes in silico and in vitro through two major interfaces: (i) β-interface, mapped to Switch I and effector-binding regions, (ii) α-interface at the allosteric lobe. Here, we chose constitutively active K-Ras4B as our control and two double mutants (K101D and R102E; and R41E and K42D) in the α- and β-interfaces. Two of the mutations are from The Cancer Genome Atlas (TCGA) and the Catalogue Of Somatic Mutations In Cancer (COSMIC) data sets. R41 and R102 are found in several adenocarcinomas in Ras isoforms. We performed site-directed mutagenesis, cellular localization experiments, and molecular dynamics (MD) simulations to assess the impact of the mutations on K-Ras4B dimerization and function. α-interface K101D/R102E double mutations reduced dimerization but only slightly reduced downstream phosphorylated extracellular signal-regulated kinase (ERK) (pERK) levels. While β-interface R41E/K42D double mutations did not interfere with dimerization, they almost completely blocked K-Ras4B-mediated ERK phosphorylation. Both double mutations increased downstream phosphorylated Akt (pAkt) levels in cells. Changes in pERK and pAkt levels altered ERK- and Akt-regulated gene expressions, such as EGR1, JUN, and BCL2L11. These results underscore the role of the α-interface in K-Ras4B homodimerization and the β-surface in effector binding. MD simulations highlight that the membrane and hypervariable region (HVR) interact with both α- and β-interfaces of K-Ras4B mutants, respectively, inhibiting homodimerization and probably effector binding. Mutations at both interfaces interfered with mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase signaling but in different forms and extents. We conclude that dimerization is not necessary but enhances downstream MAPK signaling.

KEYWORDS:

K-Ras4B dimerization; K-Ras4B mutation; MAPK signaling; interface mutants; membrane localization

PMID:
31931009
DOI:
10.1016/j.jmb.2020.01.002

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center