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

Abstract

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 (Isc). Cell lines expressing SGK1 (28.8 ± 1.3 μA/cm2) and PX-PKB (34.9 ± 3.4 μA/cm2) had significantly higher Isc than parent (22.0 ± 2.2 μA/cm2) and PKB (21.5 ± 2.0 μA/cm2) expressing cell lines. The cell line expressing PH-SGK1 (19.1 ± 1.1 μA/cm2) did not have a significantly different Isc than the parent cells. These results indicate that the N-terminal region of SGK1 is critical for the regulation of Isc and may be important for recognition of substrates involved in its effect on Na+current. NIH DK41841 & DK07508