The Ste20 kinases SPAK and OSR1 travel between cells through exosomes.

Koumangoye R, Delpire E
Am J Physiol Cell Physiol. 2016 311 (1): C43-53

PMID: 27122160 · PMCID: PMC4967142 · DOI:10.1152/ajpcell.00080.2016

Proteomics studies have identified Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) in exosomes isolated from body fluids such as blood, saliva, and urine. Because proteomics studies likely overestimate the number of exosome proteins, we sought to confirm and extend this observation using traditional biochemical and cell biology methods. We utilized HEK293 cells in culture to verify the packaging of these Ste20 kinases in exosomes. Using a series of centrifugation and filtration steps of conditioned culture medium isolated from HEK293 cells, we isolated nanovesicles in the range of 40-100 nm. We show that these small vesicles express the tetraspanin protein CD63 and lack endoplasmic reticulum and Golgi markers, consistent with these being exosomes. We show by Western blot and immunogold analyses that these exosomes express SPAK, OSR1, and Na-K-Cl cotransporter 1 (NKCC1). We show that exosomes are not only secreted by cells, but also accumulated by adjacent cells. Indeed, exposing cultured cells to exosomes produced by other cells expressing a fluorescently labeled kinase resulted in the kinase finding its way into the cytoplasm of these cells, consistent with the idea of exosomes serving as cell-to-cell communication vessels. Similarly, coculturing cells expressing different fluorescently tagged proteins resulted in the exchange of proteins between cells. In addition, we show that both SPAK and OSR1 kinases entering cells through exosomes are preferentially expressed at the plasma membrane and that the kinases in exosomes are functional and maintain NKCC1 in a phosphorylated state.

Copyright © 2016 the American Physiological Society.

MeSH Terms (19)

Cell Communication Cell Membrane Coculture Techniques Culture Media, Conditioned Exosomes HEK293 Cells HeLa Cells Humans Luminescent Proteins Microscopy, Fluorescence Particle Size Phosphorylation Protein-Serine-Threonine Kinases Protein Transport Recombinant Proteins Solute Carrier Family 12, Member 2 Tetraspanin 30 Time Factors Transfection

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