Electrostatic interactions positively regulate K-Ras nanocluster formation and function

Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, 4072, Australia; Centre for Microscopy and Microanalysis, University of Queensland, St Lucia, Brisbane, 4072, Australia

^* To whom correspondence should be addressed. Email: j.hancock{at}imb.uq.edu.au.

	Abstract

The organization of Ras proteins into plasma membrane nanoclusters is essential for high fidelity signal transmission, but whether the nanoscale enviroments of different Ras nanoclusters regulate effector interactions is unknown. We show using high-resolution spatial mapping that Raf-1 is recruited to and retained in K-Ras.GTP nanoclusters. In contrast, Raf-1 recruited to the plasma membrane by H-Ras is not retained in H-Ras.GTP nanoclusters. Similarly, upon EGF receptor activation, Raf-1 is preferentially recruited to K-Ras.GTP and not H-Ras.GTP nanoclusters. The formation of K-Ras.GTP nanoclusters is inhibited by phosphorylation of S181 in the C-terminal polybasic domain or enhanced by blocking S181 phosphorylation, with a concomitant reduction or increase in Raf-1 plasma membrane recruitment, respectively. Phosphorylation of S181 does not however regulate in vivo interactions with the nanocluster scaffold galectin-3 (Gal3), indicating separate roles for the polybasic domain and Gal3 in driving K-Ras nanocluster formation. Together these data illustrate that Ras nanocluster composition regulates effector recruitment and highlight the importance of lipid/protein nanoscale environments to the activation of signaling cascades.