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Molecular and Cellular Biology, July 2008, p. 4377-4385, Vol. 28, No. 13
0270-7306/08/$08.00+0     doi:10.1128/MCB.00050-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Electrostatic Interactions Positively Regulate K-Ras Nanocluster Formation and Function

Institute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane 4072, Australia,¹ Centre for Microscopy and Microanalysis, University of Queensland, St. Lucia, Brisbane 4072, Australia²

Received 10 January 2008/ Returned for modification 18 February 2008/ Accepted 18 April 2008

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 epidermal growth factor 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.

Published ahead of print on 5 May 2008.