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The RAS effector RIN1 directly competes with RAF and is regulated by 14-3-3 proteins
Ying Wang1, Richard T Waldron, Ajay Dhaka
1Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, California 90095, USA.
Abstract:
Activation of RAS proteins can lead to multiple outcomes by virtue of regulated signal traffic through alternate effector pathways. We demonstrate that the RAS effector protein RIN1 binds to activated RAS with an affinity (K(d), 22 nM) similar to that observed for RAF1. At concentrations close to their equilibrium dissociation constant values, RIN1 and RAF1 compete directly for RAS binding. RIN1 was also observed to inhibit cellular transformation by activated mutant RAS. This distinguishes RIN1 from other RAS effectors, which are transformation enhancing. Blockade of transformation was mediated by the RAS binding domain but required membrane localization. RIN1 recognizes endogenous RAS following transient activation by epidermal growth factor, and a portion of RIN1 fractionates to the cell membrane in a manner consistent with a reversible interaction. RIN1 also binds to 14-3-3 proteins through a sequence including serine 351. Mutation of this residue abolished the 14-3-3 binding capacity of RIN1 and led to more efficient blockade of RAS-mediated transformation. The mutant protein, RIN1(S351A), showed a shift in localization to the plasma membrane. Serine 351 is a substrate for protein kinase D (PKD [also known as PKCmu]) in vitro and in vivo. These data suggest that the normal localization and function of RIN1, as well as its ability to compete with RAF, are regulated in part by 14-3-3 binding, which in turn is controlled by PKD phosphorylation.
Insights
RIN1, a RAS effector protein, competes with RAF1 for RAS binding and inhibits cellular transformation. Its interaction with 14-3-3 proteins, regulated by PKD phosphorylation, controls RIN1 localization and function.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncology
Background:
- RAS proteins are key regulators of cell signaling pathways.
- Dysregulation of RAS signaling is implicated in various cancers.
- Understanding RAS effector interactions is crucial for targeted therapies.
Purpose of the Study:
- To investigate the interaction of RIN1 with activated RAS proteins.
- To determine RIN1's role in RAS-mediated cellular transformation.
- To elucidate the regulatory mechanisms controlling RIN1 function.
Main Methods:
- Co-immunoprecipitation assays to assess protein-protein interactions.
- Cellular transformation assays using activated RAS mutants.
- Site-directed mutagenesis to investigate specific residue functions.
- Confocal microscopy to analyze protein localization.
Main Results:
- RIN1 binds activated RAS with high affinity, competing with RAF1.
- RIN1 inhibits RAS-mediated cellular transformation.
- Mutation of serine 351 in RIN1 enhances its transformation inhibitory activity and alters its localization.
- RIN1 binding to 14-3-3 proteins is regulated by PKD phosphorylation.
Conclusions:
- RIN1 acts as a negative regulator of RAS-mediated transformation.
- 14-3-3 binding, controlled by PKD phosphorylation, modulates RIN1's localization and function.
- RIN1 represents a potential therapeutic target for RAS-driven cancers.