Electrostatic interactions positively regulate K-Ras nanocluster formation and function.
The organization of Ras proteins into plasma membrane nanoclusters is essential for high-fidelity signal transmission, but whether the nanoscale environments 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.
Plowman
Sarah J
SJ
Institute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane 4072, Australia.
Ariotti
Nicholas
N
Goodall
Andrew
A
Parton
Robert G
RG
Hancock
John F
JF
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D016716:PC12 Cells; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D051381:Rats; D055672:Static Electricity; D014157:Transcription Factors / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism*
D020033:Protein Isoforms; D014157:Transcription Factors; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins