Mechanisms of ubiquitin -mediated degradation of serum - and glucocorticoid -induced protein kinase -1

Serum and glucocorticoid-induced protein kinase-1 (SGK-1) plays a critical role in the regulation of the epithelial sodium channel, ENaC. SGK-1 shares significant catalytic domain homology to AKT-1/PKB and is a downstream effector of anti-apoptotic PI-3 kinase signaling. Steady-state levels of SGK-1 are tightly regulated by rapid transcriptional activation and posttranslational modification including phosphorylation.;We show here that endogenous SGK-1 protein is polyubiquitinated and rapidly degraded by the 26S proteasome. In contrast to other rapidly degraded kinases, neither catalytic activity of SGK-1 nor activation site phosphorylation is required for its ubiquitin-modification and degradation. Instead, SGK-1 degradation required a lysine-less six amino acid (aa19-24) hydrophobic motif (GMVAIL) within the N-terminal domain. Deletion of aa19-24 significantly increased SGK1's half-life and prevented its ubiquitin modification. Interestingly, this minimal region is also required for association of SGK-1 with the endoplasmic reticulum (ER). Ubiquitin modification and degradation of SGK-1 is increasingly inhibited by the progressive mutation of six N-terminal lysine residues surrounding the GMVAIL motif. Mutation of all six lysines to arginine does not disrupt SGK-1's subcellular localization despite a significant decrease in ubiquitination implying that this modification per se was not required for targeting to the ER. These results suggest that constitutive ubiquitin-mediated degradation of SGK-1 is an important mechanism regulating its biological activity.;The research described in this thesis also identifies some putative interacting partners of SGK-1. Interestingly, many of these proteins are involved in metabolism and regulation of the protein synthesis. This implies a novel role for SGK-1 in regulation of metabolic pathways which in turn might explain the mechanisms by which SGK-1 acts as an anti-apoptotic kinase.