Oral Presentation 2014 International Biophysics Congress

Dissecting oncogenic RAS signaling by NMR (#8)

Matthew J. Smith 1 , Mohammad Mazhab-Jafari 2 , Zhenhao Fang 2 , Christopher B. Marshall 1 , Mitsu Ikura 1 2
  1. University Health Network, Toronto, ON, Canada
  2. Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada
Rat Sarcoma viral oncogene hololog (RAS) proteins are frequently mutated in cancer (~25% of all human tumours) and have been known as major drivers of tumourgenesis for decades. However, the development of clinically effective, RAS-targeted cancer therapies has been unsuccessful, and RAS-driven cancers remain among the most refractory to available treatments. This is largely owing to the lack of understanding of how mutant RAS functions and alters related signaling pathways in tumours. To overcome this, we sought to develop better functional assays for, and mechanistic insights into oncogenic RAS-affected pathways. We employ NMR spectroscopy to study RAS enzymatic kinetics and the underlying mechanisms of catalysis, as well as interactions with downstream effector proteins. We have extensively used real-time NMR-based GTPase assays we developed previously (1-3) to characterize oncogenic Ras mutations and Noonan syndrome-derived mutations in the Ras guanine nucleotide exchange factor (GEF) Son-of-Sevenless (SOS) expressed in mammalian cells. Our parallel and quantitative NMR assays of RAS-effector interactions demonstrated that mutant RAS G12V alters properties of the integrated RAS network (4). RAS is prenylated and functions on the plasma membrane, but previous mechanistic studies were mainly carried out using a truncated, soluble form of RAS. We employed the nanodisc technology developed by Sligar and coworkers to study membrane-anchored RAS, which enabled us to characterize how the biological membrane affects the structure and function of wild-type and mutant RAS. Recent findings also suggest that the RAS pathway has a cross talk with calcium signaling pathways through its interaction with calmodulin and inositol 1,4,5-trisphosphate receptor (IP3R), which will be discussed in the talk together with our crystallographic and electron microscopic studies on IP3R. These structural and functional characterizations of RAS by NMR may help to design new therapeutic strategies for anti-cancer agents and diagnostic tools.  (Supported by CFI, CIHR, CCSRI, HSFO, and CRS)
  1. 1. Marshall CB, Ho J, Buerger C, Plevin MJ, Li GY, Li Z, Ikura M, Stambolic V. Sci Signal. 2009 Jan 27;2(55):ra3. 2. Marshall CB, Meiri D, Smith MJ, Mazhab-Jafari MT, Gasmi-Seabrook GM, Rottapel R, Stambolic V,Ikura M. Methods. 2012 Aug;57(4):473-85. 3. Smith MJ, Neel BG, Ikura M. Proc Natl Acad Sci U S A. 2013 Mar 19;110(12):4574-9. 4. Smith MJ, Ikura M. Nat Chem Biol. 2014 Mar;10(3):223-30.