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. 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.