Subcellular location and function studies of the serum and glucocorticoid induced kinase‐1 (SGK1)

SGK1 is involved in aldosterone‐induced Na + reabsorption by increasing epithelial Na + channel (ENaC) activity in cortical collecting duct (CCD) cells. Though it has similar substrate specificity to SGK1, the related protein kinase B (PKB) does not enhance ENaC current in A6 cells. We found SGK1‐GFP fusion protein is enriched in mitochondria, as is a truncated SGK1 consisting of the first 60 amino acids that includes a putative Phox homology (PX) domain and mitochondrial localization signal, suggesting this region is critical for the subcellular localization of SGK1. To further characterize the role of the N‐terminal region in subcellular localization, we transiently transfected CCD cells with chimeric constructs where the PX domain of SGK1 was swapped with the N‐terminal pleckstrin homology (PH) domain of PKB. We determined the location of PH‐SGK1, PX‐PKB, SGK1, and PKB fused to GFP on the C‐terminal. In contrast to the mitochondrial location of SGK1, both PKB and PH‐SGK1 were found in the cytoplasm and nucleus. Conversely, PX‐PKB co‐localized with SGK1 to mitochondria. To determine the roles of these domains in Na + transport we examined the effect of stably expressing SGK1, PKB, PH‐SGK1 or PX‐PKB in M1 cells on Na + current (I sc ). Cell lines expressing SGK1 (28.8 ± 1.3 μA/cm 2 ) and PX‐PKB (34.9 ± 3.4 μA/cm 2 ) had significantly higher I sc than parent (22.0 ± 2.2 μA/cm 2 ) and PKB (21.5 ± 2.0 μA/cm 2 ) expressing cell lines. The cell line expressing PH‐SGK1 (19.1 ± 1.1 μA/cm 2 ) did not have a significantly different I sc than the parent cells. These results indicate that the N‐terminal region of SGK1 is critical for the regulation of I sc and may be important for recognition of substrates involved in its effect on Na + current. NIH DK41841 & DK07508