The Ste20 kinases SPAK and OSR1 travel between cells through exosomes.
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.
Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, Tennessee.
Rainelli
R
Koumangoye
Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, Tennessee eric.delpire@vanderbilt.edu.
Eric
E
Delpire
D002450:Cell Communication; D002462:Cell Membrane / Q000201:enzymology*; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000378:metabolism; D055354:Exosomes / Q000201:enzymology*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010316:Particle Size; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2 / Q000378:metabolism; D060149:Tetraspanin 30 / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
C554586:CD63 protein, human; D017077:Culture Media, Conditioned; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C578877:SLC12A2 protein, human; D064506:Solute Carrier Family 12, Member 2; D060149:Tetraspanin 30; C118135:OXSR1 protein, human; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human
HEK293 cells; HeLa cells; NKCC1; Na-K-2Cl cotransport; extracellular vesicles