Membrane interactions of the globular domain and the hypervariable region of KRAS4b define its unique diffusion behavior
- Frederick National Laboratory for Cancer Research, United States
- Lawrence Livermore National Laboratory, United States
- University of California, San Francisco, United States
Abstract
The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here we characterize RAS in live human and mouse cells using single molecule tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein's globular domain. To understand the altered mobility of an oncogenic KRAS4b we used complementary experimental and molecular dynamic simulation approaches to reveal a detailed mechanism.
Data availability
For the molecular dynamic simulations, trajectories and inputs have been provided on the webpage at https://bbs.llnl.gov/KRAS4b-simulation-data.html.For the images, we will access a suitable repository, and make the data freely available.
Article and author information
Author details
Felice C Lightstone
Funding
National Cancer Institute (NIH Contract HHSN261200800001E)
- De Chen
- Prabhakar R Gudla
- John Columbus
- Karen Worthy
- Megan Rigby
- Suman Mukhopadhyay
- Katie Powell
- William Burgan
- Vanessa Wall
- Dominic Esposito
- Dhirendra Simanshu
- Dwight V Nissley
- Thomas Turbyville
U.S. Department of Energy (Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. LLNL-JRNL-771099-DRAFT)
- Yue Yang
- Felice C Lightstone
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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Membrane interactions of the globular domain and the hypervariable region of KRAS4b define its unique diffusion behavior
eLife 9:e47654.
https://doi.org/10.7554/eLife.47654
Further reading
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- Cancer Biology
Why does a normal cell possibly harboring genetic mutations in oncogene or tumor suppressor genes becomes malignant and develops a tumor is a subject of intense debate. Various theories have been proposed but their experimental test has been hampered by the unpredictable and improbable malignant transformation of single cells. Here, using an optogenetic approach we permanently turn on an oncogene (KRASG12V) in a single cell of a zebrafish brain that, only in synergy with the transient co-activation of a reprogramming factor (VENTX/NANOG/OCT4), undergoes a deterministic malignant transition and robustly and reproducibly develops within 6 days into a full-blown tumor. The controlled way in which a single cell can thus be manipulated to give rise to cancer lends support to the ‘ground state theory of cancer initiation’ through ‘short-range dispersal’ of the first malignant cells preceding tumor growth.
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- Cancer Biology
Most human pancreatic ductal adenocarcinoma (PDAC) are not infiltrated with cytotoxic T cells and are highly resistant to immunotherapy. Over 90% of PDAC have oncogenic KRAS mutations, and phosphoinositide 3-kinases (PI3Ks) are direct effectors of KRAS. Our previous study demonstrated that ablation of Pik3ca in KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cells induced host T cells to infiltrate and completely eliminate the tumors in a syngeneic orthotopic implantation mouse model. Now, we show that implantation of Pik3ca−/− KPC (named αKO) cancer cells induces clonal enrichment of cytotoxic T cells infiltrating the pancreatic tumors. To identify potential molecules that can regulate the activity of these anti-tumor T cells, we conducted an in vivo genome-wide gene-deletion screen using αKO cells implanted in the mouse pancreas. The result shows that deletion of propionyl-CoA carboxylase subunit B gene (Pccb) in αKO cells (named p-αKO) leads to immune evasion, tumor progression, and death of host mice. Surprisingly, p-αKO tumors are still infiltrated with clonally enriched CD8+ T cells but they are inactive against tumor cells. However, blockade of PD-L1/PD1 interaction reactivated these clonally enriched T cells infiltrating p-αKO tumors, leading to slower tumor progression and improve survival of host mice. These results indicate that Pccb can modulate the activity of cytotoxic T cells infiltrating some pancreatic cancers and this understanding may lead to improvement in immunotherapy for this difficult-to-treat cancer.
