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Published on: January 16, 2020
Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells
Hagit Niv1, Orit Gutman, Yoel Kloog
1Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, 69978, Israel.
Abstract:
Ras-membrane interactions play important roles in signaling and oncogenesis. H-Ras and K-Ras have nonidentical membrane anchoring moieties that can direct them to different membrane compartments. Ras-lipid raft interactions were reported, but recent studies suggest that activated K-Ras and H-Ras are not raft resident. However, specific interactions of activated Ras proteins with nonraft sites, which may underlie functional differences and phenotypic variation between different Ras isoforms, are unexplored. Here we used lateral mobility studies by FRAP to investigate the membrane interactions of green fluorescent protein-tagged H- and K-Ras in live cells. All Ras isoforms displayed stable membrane association, moving by lateral diffusion and not by exchange with a cytoplasmic pool. The lateral diffusion rates of constitutively active K- and H-Ras increased with their expression levels in a saturable manner, suggesting dynamic association with saturable sites or domains. These sites are distinct from lipid rafts, as the activated Ras mutants are not raft resident. Moreover, they appear to be different for H- and K-Ras. However, wild-type H-Ras, the only isoform preferentially localized in rafts, displayed cholesterol-sensitive interactions with rafts that were independent of its expression level. Our findings provide a mechanism for selective signaling by different Ras isoforms.
Insights
Ras proteins interact dynamically with specific membrane sites, not lipid rafts, influencing their distinct signaling roles. These interactions vary between H-Ras and K-Ras, explaining isoform-specific functions in cells.
Area of Science:
- Cellular biology
- Molecular signaling
- Oncogenesis
Background:
- Ras-membrane interactions are crucial for cellular signaling and cancer development.
- H-Ras and K-Ras isoforms possess distinct membrane-binding features, potentially leading to varied cellular localization and function.
- While Ras-lipid raft interactions were previously studied, the localization of activated Ras proteins in non-raft domains remains largely unexplored.
Purpose of the Study:
- To investigate the membrane interactions and lateral mobility of H-Ras and K-Ras isoforms in live cells.
- To determine if activated Ras proteins associate with lipid rafts or distinct non-raft membrane domains.
- To elucidate the mechanisms underlying isoform-specific Ras signaling.
Main Methods:
- Fluorescence Recovery After Photobleaching (FRAP) was employed to study the lateral mobility of green fluorescent protein-tagged H-Ras and K-Ras.
- Live-cell imaging techniques were used to observe Ras protein dynamics.
- Expression levels and localization of Ras isoforms were analyzed.
Main Results:
- All Ras isoforms exhibited stable membrane association, with lateral diffusion rather than exchange with a cytoplasmic pool.
- The lateral diffusion rates of activated H-Ras and K-Ras saturated with increasing expression levels, indicating association with saturable membrane sites.
- These sites are distinct from lipid rafts, as activated Ras mutants were not raft-resident. Furthermore, these sites appear to differ between H-Ras and K-Ras.
- Wild-type H-Ras, unlike its activated mutants, showed cholesterol-sensitive raft interactions independent of expression level.
Conclusions:
- Activated H-Ras and K-Ras associate with distinct, saturable non-raft membrane domains, providing a mechanism for their isoform-specific signaling.
- These findings challenge the notion of Ras proteins being raft-resident and offer new insights into Ras isoform selectivity.
- The differential interactions of Ras isoforms with specific membrane compartments contribute to functional diversity and phenotypic variation.
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