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Methods Enzymol. 2007;428:297-312.

Hyperosmotic induction of mitogen-activated protein kinase scaffolding.

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Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.


Eukaryotic cells respond to hyperosmotic conditions by expunging water from the cell, leading to cell shrinkage. This is counteracted by adaptive responses that restore cell volume and strengthen the cytoskeletal architecture. In the budding yeast Saccharomyces cerevisiae, this response is mediated primarily by the mitogen-activated protein kinase (MAPK) cascade CDC42-STE50-STE11-Pbs2-Hog1. In mammalian cells, MAPK scaffold proteins facilitate the efficiency of signaling within the cascade by placing a kinase near its substrate and also regulate the subcellular localization of the signaling. Our laboratory has discovered a scaffold that coordinates the analogous Hog1 signal in mammalian cells, termed OSM (osmosensing scaffold for MEKK3). OSM organizes a complex consisting of the small GTPase Rac, MEKK3, and MKK3 for the activation of p38 MAPK. Interactions among OSM, Rac, and MEKK3 are augmented in response to sorbitol and are also localized to membrane ruffles, sites of rapid actin turnover. Suppression of the expression of OSM or MEKK3 by RNA interference strongly inhibits the sorbitol-dependent activation of p38. Furthermore, mutations in OSM were concurrently found to cause cerebral cavernous malformations (CCM), a disease of the central nervous system characterized by thin-walled, leaky blood vessels that become hemorrhagic. Our laboratory has also demonstrated that Krit1, another gene harboring mutations that lead to CCM, binds OSM and its interaction is enhanced in response to sorbitol in a similar manner as the MEKK3-OSM interaction. This chapter describes the cell biological and biochemical methods used for assaying protein-protein interactions in live cells using fluorescence resonance energy transfer, in vitro kinase assays for MEKK3-MKK3-p38 pathway members, and gene suppression by RNA interference to study hyperosmotic stress-dependent signaling.

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