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The serine/threonine protein kinase SGK3 stimulates endosomal recycling of the potassium channel Kir2.2.
Serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the expression of various membrane proteins at the cell surface. In this study, the mechanism by which SGK3 increases the surface expression of the potassium channel Kir2.2 was investigated using two-electrode voltage clamp, luminometric...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2015
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| Zusammenfassung: | Serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the expression of various membrane proteins at the cell surface. In this study, the mechanism by which SGK3 increases the surface expression of the potassium channel Kir2.2 was investigated using two-electrode voltage clamp, luminometric surface expression measurements and fluorescence microscopy in a mammalian cell line (COS-7) and Xenopus laevis oocytes.
A number of different mechanisms by which SGK3 increases the membrane expression of various channel and transporter proteins has been proposed, including phosphorylation of the ubiquitin ligase Nedd4-2, phosphorylation of the transcription factor FOXO3a and phosphorylation of the phosphoinositide kinase PIKfyve. The results obtained in this study suggest that none of these mechanisms is responsible for the increased surface expression of Kir2.2 induced by SGK3. They furthermore indicate that SGK3 neither affects the amount of endocytosed Kir2.2 channels nor the number of newly synthesized channel molecules.
An antibody-based recycling assay showed that a substantial amount of Kir2.2 was internalized and recycled back to the plasma membrane during two 30 min time periods. It further indicated that coexpression of a constitutively active SGK3-mutant leads to an increased number of recycled channel proteins in comparison to coexpression of a dominant negative mutant.
Live-cell fluorescence imaging with two different colors revealed that Kir2.2 channels, SGK3 and the small G-protein Rab7 were extensively colocalized in a PI(3)P positive endosomal compartment. Furthermore, frequent interactions of Kir2.2-positive, SGK3-positive or Rab7-positive vesicles with the Rab11-positive recycling endosome were observed. I line with these results, a mutant of SGK3 that does not bind to PI(3)P had a much smaller effect on Kir2.2 surface expression than the wild-type kinase, suggesting that the intracellular localization of SGK3 to endosomal membranes plays a crucial role for its effect on Kir2.2.
Taken together, the results obtained in this study suggest that SGK3 promotes the recycling of Kir2.2 channels from a PI(3)P and Rab7-positive intracellular compartment that represents an intermediate stage of maturing early endosomes. The recycling of the channel back to the plasma membrane possibly occurs via Rab11 positive recycling endosomes. |
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| Umfang: | 129 Seiten |
| DOI: | 10.17192/z2015.0575 |