Of the three ras genes, kras is most frequently mutated in human cancer. Ras proteins are highly homologous but differ extensively in their C-terminal hypervariable regions that direct post-translational modifications (e.g. farnesylation) and membrane targeting. We discovered a post-translational modification unique to K-Ras: protein kinase C (PKC) mediated phosphorylation. We found that phosphorylation of K-Ras at serine 181 partially neutralized the adjacent polybasic stretch of amino acids and thereby activated a farnesyl-electrostatic switch that resulted in release of K-Ras from the plasma membrane and association with intracellular membranes, including the endoplasmic reticulum (ER), Golgi apparatus and the outer mitochondrial membrane. Most intriguing, K-Ras translocation to internal membranes was associated with cell death. Bryostatin 1, a potent PKC agonist, showed anti-tumor activity that was dependent on K-Ras serine 181. In the first cycle of this grant we proposed to expand upon these discoveries with three Aims: 1) Regulation of the farnesyl-electrostatic switch, 2) Mechanisms of phospho-K-Ras mediated apoptosis and 3) The Role of C-terminal phosphorylation of K-Ras in mouse tumor models. Much progress has been made on each aim. Most exciting are our discoveries that K-Ras signals for cell death from the cytoplasmic face of the ER, that Bcl-XL is required for phospho-K-Ras mediated cell death, and that phospho-K-Ras forms a trimolecular complex with Bcl-XL and the IP3 receptor (IP3R) and regulates the calcium channel activity. We have also been successful in constructing a double knock-in mouse that harbors a conditional oncogenic K-Ras allele that lacks the phosphorylation site at amino acid 181 (LSL-K-Ras12D181A). In this competing renewal application we propose to continue our studies with three aims:
Aim 1 : Regulation of the IP3 Receptor (IP3R) by phospho-K-Ras. We will characterize both structurally (protein-protein interactions) and functionally (electrophysiology) the molecular interactions between phospho-K-Ras, Bcl-XL and IP3R. We will ascertain if phospho-K-Ras alters mitochondrial calcium homeostasis. We will determine if IP3R, calpain and autophagy are required for phospho-K-Ras mediated cell death.
Aim 2 : Analysis of K-Ras Phosphorylation at Serine 181 in vivo. We will use our newly created LSL-K-Ras12D181A mice in two Cre- driven tumor models to test the hypothesis that phosphorylation at serine 181 negatively regulates K-Ras oncogenicity and we will use the same models to show that the efficacy of bryostatin 1 depends on phosphorylation of serine 181.
Aim 3 : Analysis of K-Ras Phosphorylation at Serine 181 in Human Tumor Cells. We will correlate susceptibility of human tumor cells lines with K-Ras mutation status and generate isogenic lines of human tumor cells with and without a phosphorylation site at position 181. We anticipate that our mechanistic studies of the cell biology of phospho-K-Ras along with our in vivo and human tumor cell analyses will reveal unique features of this important oncogene that can be exploited in developing anti-cancer drugs.

Public Health Relevance

Oncogenes are genes that cause cancer. K-Ras is the most important human oncogene. We discovered that K-Ras can be modified by the addition of a phosphate group and that this modification inhibits its cancer-promoting activity. We propose to study the cell biology and physiology of K-Ras phosphorylation to better understand how to exploit this process to develop anti-cancer drugs.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Cancer Molecular Pathobiology Study Section (CAMP)
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Watson, Joanna M
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New York University
Internal Medicine/Medicine
Schools of Medicine
New York
United States
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Fehrenbacher, Nicole; Tojal da Silva, Israel; Ramirez, Craig et al. (2017) The G protein-coupled receptor GPR31 promotes membrane association of KRAS. J Cell Biol 216:2329-2338
Court, Helen; Ahearn, Ian M; Amoyel, Marc et al. (2017) Regulation of NOTCH signaling by RAB7 and RAB8 requires carboxyl methylation by ICMT. J Cell Biol 216:4165-4182
Zhou, Mo; Wiener, Heidi; Su, Wenjuan et al. (2016) VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking. J Cell Biol 214:445-58
Tsai, Frederick D; Lopes, Mathew S; Zhou, Mo et al. (2015) K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif. Proc Natl Acad Sci U S A 112:779-84
Cox, Adrienne D; Der, Channing J; Philips, Mark R (2015) Targeting RAS Membrane Association: Back to the Future for Anti-RAS Drug Discovery? Clin Cancer Res 21:1819-27
Tsai, Frederick D; Wynne, Joseph P; Ahearn, Ian M et al. (2014) Metabolic labeling of Ras with tritiated palmitate to monitor palmitoylation and depalmitoylation. Methods Mol Biol 1120:33-41
Sung, Pamela J; Tsai, Frederick D; Vais, Horia et al. (2013) Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors. Proc Natl Acad Sci U S A 110:20593-8
Court, Helen; Amoyel, Marc; Hackman, Michael et al. (2013) Isoprenylcysteine carboxylmethyltransferase deficiency exacerbates KRAS-driven pancreatic neoplasia via Notch suppression. J Clin Invest 123:4681-94
Ahearn, Ian M; Haigis, Kevin; Bar-Sagi, Dafna et al. (2012) Regulating the regulator: post-translational modification of RAS. Nat Rev Mol Cell Biol 13:39-51
Philips, Mark R (2012) Ras hitchhikes on PDE6?. Nat Cell Biol 14:128-9

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