Overview
For more than two decades, the Philips Laboratory has focused on the interplay between subcellular trafficking and signaling of small GTPases, particularly as this relates to the products of RAS oncogenes. Early work on the role of carboxyl methylation of small GTPases in leukocyte activation (1) culminated in the cloning of isoprenylcysteine carboxyl methyltransferase (ICMT), the third of the three enzymes that sequentially modify the C-terminal CaaX motif of RAS and related proteins (2). Our discovery that ICMT is a polytopic membrane protein absolutely restricted to the endoplasmic reticulum (3) demonstrated that RAS proteins operate on membrane compartments other than the plasma membrane (PM). Our subsequent discovery that RAS proteins take distinct routes to the PM prompted a paradigm shift in RAS biology (4, 5). This led us to develop novel fluorescent probes that revealed RAS signaling on internal membranes (6, 7) and established the field of compartmentalized RAS signaling (8-11).
HeLa cell with green KRAS and red mitochondria
GRP1 signaling to NRAS on Golgi downstream of TCR
COS-1 cell with blue KRAS, red mitochondria and green Bcl-XL
COS-1 cells expressing GFP-ICMT
Space-filling atomic model of RAS
COS-1 cells expressing GFP-HRAS
RAS trafficking
MDCK cells expressing GFP-NRAS
These probes also allowed us to make significant contributions to inside-out-signaling to integrins through RAP1, a small GTPase closely related to RAS (12-16). We also analyzed localization and trafficking of RHO family small GTPases, which regulate the actin cytoskeleton (17, 18) as well as heterotrimeric G proteins, which include CaaX modified γ subunits (19). We were surprised to find that genetic ablation of ICMT exacerbated a genetically engineered mouse model (GEMM) of KRAS-driven pancreatic ductal adenocarcinoma (20) and subsequently found that the paradoxical effect was mediated through NOTCH signaling (21). We have focused on NRAS and KRAS which, when mutated, drive the majority of RAS-related malignancies. We showed that a large pool of farnesylated NRAS remains in the cytosol and discovered that VPS35, a component of the retromer, is a cytosolic chaperone for the GTPase (22). More recently we have found that NRAS membrane association is uniquely sensitive to inhibition of ICMT and we are exploring the efficacy of ICMT inhibitors in NRAS-driven melanoma (Ahearn et al. submitted). Turning to KRAS, we discovered that the association of KRAS4B with the PM is regulated by phosphorylation of S181 by PKC (23). We found that phosphorylation of activated KRAS4B limited cell survival (24), demonstrating that manipulation of KRAS4B subcellular localization alters its function. We are currently testing the relevance of this pathway in vivo with a GEMM. Recently we developed a quantitative assay for membrane association of KRAS4B, applied it to genome-wide RNAi and CRISPR screens, and identified several previously unappreciated genes involved in KRAS membrane targeting including several kinases, an orphan GPCR (25) and a nuclear factor that regulates the expression of prenyltransferases (Zhou et al., PNAS in revision). Because the products of the two KRAS splice variants, KRAS4A and KRAS4B, differ only in their membrane-targeting regions, we studied their distinct trafficking (26) and recently discovered that they differentially regulate tumor metabolism by virtue of differential affinity for the mitochondrial outer membrane that is regulated by palmitoylation, a stark example of compartmentalized signaling (27). This last discovery has caused us to explore other ways in which RAS proteins directly regulate metabolism and contribute to the metabolic rewiring observed in cancer cells.
The central, unifying theme of our work has been basic discovery in RAS trafficking and compartmentalized signaling. Because RAS proteins signal only when associated with cellular membranes, inhibition of membrane targeting of RAS has long been considered an attractive approach to anti-RAS drug discovery and affords our work significant translational potential.
References:
1. Philips, M.R. et al. Carboxyl methylation of ras-related proteins during signal transduction in neutrophils. Science 259, 977-980 (1993).
2. Dai, Q. et al. Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum. J Biol Chem 273, 15030-15034 (1998).
3. Wright, L.P. et al. Topology of mammalian isoprenylcysteine carboxyl methyltransferase determined in live cells with a fluorescent probe. Mol Cell Biol 29, 1826-1833 (2009).
4. Choy, E. et al. Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell 98, 69-80 (1999).
5. Ahearn, I.M. et al. FKBP12 binds to acylated H-ras and promotes depalmitoylation. Mol Cell 41, 173-185 (2011).
6. Chiu, V.K. et al. Ras signalling on the endoplasmic reticulum and the Golgi. Nat Cell Biol 4, 343-350. (2002).
7. Yeung, T. et al. Receptor activation alters inner surface potential during phagocytosis. Science 313, 347-351 (2006).
8. Bivona, T.G. et al. Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1. Nature 424, 694-698 (2003).
9. Mor, A. et al. The lymphocyte function-associated antigen-1 receptor costimulates plasma membrane Ras via phospholipase D2. Nat Cell Biol 9, 713-719 (2007).
10. Onken, B., Wiener, H., Philips, M. & Chang, E.C. Compartmentalized signaling of Ras in fission yeast. Proc Natl Acad Sci U S A 103, 9045-9050 (2006).
11. Sung, P.J. et al. Cytosolic Ras supports eye development in Drosophila. Mol Cell Biol 30, 5649-5657 (2010).
12. Bivona, T.G. et al. Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion. J Cell Biol 164, 461-470 (2004).
13. Mor, A. et al. Phospholipase D1 Regulates Lymphocyte Adhesion via Upregulation of Rap1 at the Plasma Membrane. Mol Cell Biol 29, 3297-3306 (2009).
14. Wynne, J.P. et al. Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector. J Cell Biol (2012).
15. Su, W. et al. Rap1 and its effector RIAM are required for lymphocyte trafficking. Blood 126, 2695-2703 (2015).
16. Chang, Y.C. et al. Molecular basis for autoinhibition of RIAM regulated by FAK in integrin activation. Proc Natl Acad Sci U S A 116, 3524-3529 (2019).
17. Michaelson, D. et al. Differential Localization of Rho GTPases in Live Cells. Regulation by hypervariable regions and rhogdi binding. J Cell Biol 152, 111-126. (2001).
18. Michaelson, D. et al. Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division. J Cell Biol 181, 485-496 (2008).
19. Michaelson, D., Ahearn, I., Bergo, M., Young, S. & Philips, M. Membrane Trafficking of Heterotrimeric G Proteins via the Endoplasmic Reticulum and Golgi. Mol Biol Cell 13, 3294-3302 (2002).
20. Court, H. et al. Isoprenylcysteine carboxylmethyltransferase deficiency exacerbates KRAS-driven pancreatic neoplasia via Notch suppression. J Clin Invest 123, 4681-4694 (2013).
21. Court, H., Ahearn, I.M., Amoyel, M., Bach, E.A. & Philips, M.R. Regulation of NOTCH signaling by RAB7 and RAB8 requires carboxyl methylation by ICMT. J Cell Biol 216, 4165-4182 (2017).
22. Zhou, M. et al. VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking. J Cell Biol 214, 445-458 (2016).
23. Bivona, T.G. et al. PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis. Mol Cell 21, 481-493 (2006).
24. Sung, P.J. et al. Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors. Proc Natl Acad Sci U S A 110, 20593-20598 (2013).
25. Fehrenbacher, N. et al. The G protein-coupled receptor GPR31 promotes membrane association of KRAS. J Cell Biol 216, 2329-2338 (2017).
26. Tsai, F.D. et al. K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif. Proc Natl Acad Sci U S A 112, 779-784 (2015).
27. Amendola, C.R. et al. KRAS4A directly regulates hexokinase 1. Nature 576, 482-486 (2019).