Ras oncogenes are frequently associated with cancer. Of the three isoforms of Ras, K-Ras is most often mutated in human tumors. Ras proteins are prototypical GTPases that are biologically active when associated with cellular membranes. K-Ras differs from the other isoforms in utilizing a polybasic region in conjunction with a farnesylated CAAX motif for association with the plasma membrane (PM). We have discovered that serine 181 within this polybasic region is a site for phosphorylation by PKC and that this modification neutralizes the positive charge to a sufficient degree to cause K-Ras to dissociate from the PM and translocate to internal membranes. The C-terminus of K-Ras thus constitutes a farnesyl-electrostatic switch regulated by PKC, analogous to the myristoyl-electrostatic switch of the MARCKS protein. Among the membranes for which phosphorylated, internalized K-Ras has affinity is the outer mitochondrial membrane. Moreover, we have found that K-Ras with a phosphomimetic residue at position 181 is a potent stimulator of apoptosis. These surprising new discoveries suggest a new strategy for interfering with the biological effects of oncogenic K-Ras by promoting its phosphorylation by PKC. As proof of principle we have shown that xenograft tumors in nude mice driven by oncogenic K-Ras are sensitive to the PKC agonist bryostatin 1 but tumors driven by oncogenic K-Ras that is phosphorylation-deficient are insensitive. Moreover, oncogenic K-Ras that lacks C-terminal phosphorylation sites is hyperactive in transforming cells suggesting a physiologic role for this modification in dampening K-Ras signaling. To extend these exciting preliminary findings we propose three aims:
Specific Aim 1. Regulation of the farnesyl-electrostatic switch: we will develop quantitative assays for K-Ras translocation and study the regulation by PKC of the farnesyl-electrostatic switch.
Specific Aim 2. Mechanisms of phospho-K-Ras mediated apoptosis: we will determine the molecular mechanism(s) through which phosphorylated K-Ras stimulates apoptosis.
Specific Aim 3. The Role of C-terminal Phosphorylation of K-Ras in carcinogenesis: We test the hypothesis that the PKC agonist bryostatin 1 will have anti-neoplastic effects against K-Ras transformed human cells. We will also construct a knock-in mouse with a K-Ras allele that lacks C-terminal phosphorylation sites and test the hypothesis that such mice will have more aggressive tumors when models of carcinogen-induced, K-Ras dependent tumors are applied. These studies will not only elucidate a new and unexpected area of Ras biology but will also have immediate application for the quest to develop K-Ras specific therapies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA116034-02
Application #
7088821
Study Section
Cancer Molecular Pathobiology Study Section (CAMP)
Program Officer
Blair, Donald G
Project Start
2005-07-01
Project End
2010-04-30
Budget Start
2006-05-01
Budget End
2007-04-30
Support Year
2
Fiscal Year
2006
Total Cost
$325,932
Indirect Cost
Name
New York University
Department
Pathology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Ahearn, Ian; Zhou, Mo; Philips, Mark R (2018) Posttranslational Modifications of RAS Proteins. Cold Spring Harb Perspect Med 8:
Choi, Byeong Hyeok; Philips, Mark R; Chen, Yuan et al. (2018) K-Ras Lys-42 is crucial for its signaling, cell migration, and invasion. J Biol Chem 293:17574-17581
Choi, Byeong Hyeok; Chen, Changyan; Philips, Mark et al. (2018) RAS GTPases are modified by SUMOylation. Oncotarget 9:4440-4450
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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
Zhou, Mo; Philips, Mark R (2017) Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells. J Vis Exp :
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
Frémin, C; Guégan, J-P; Plutoni, C et al. (2016) ERK1/2-induced phosphorylation of R-Ras GTPases stimulates their oncogenic potential. Oncogene 35:5692-5698
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

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