An amphipathic helix targets serum and glucocorticoid-induced kinase 1 to the endoplasmic reticulum-associated ubiquitin-conjugation machinery

Abstract

Serum- and glucocorticoid-induced kinase 1 (Sgk1) regulates many ion channels and transporters in epithelial cells and promotes cell survival under stress conditions. In this study we demonstrate that Sgk1 is a short-lived protein regulated by the endoplasmic reticulum (ER)-associated degradation system and subcellular localization to the ER. We identified a hydrophobic motif (residues 18-30) as the signal for ER localization and rapid degradation by the ubiquitin (Ub)/proteasome pathway in both yeast and mammalian cells. Deletion or reduction of hydrophobicity of the motif redistributes Sgk1 to the cytosol and nucleus and markedly increases its half-life. We determined that the Ub-conjugating UBC6 and UBC7 and the Ub ligase HRD1 are the ER-associated Ub enzymes that mediate degradation of Sgk1; thus, Sgk1 has been identified as a cytosolic substrate for mammalian HRD1. Compartmentalization of Sgk1 controls the functional and spatial specificities of Sgk1-mediated signaling pathways, whereas rapid protein turnover provides a means to rapidly adjust Sgk1 abundance in response to different hormonal and external stimuli that increase Sgk1 gene transcription.

Fig. 1.

Stability of Sgk1 is determined by the N-terminal domain. t_1/2 and densitometric analysis determined by pulse–chase experiments of transfected CHO cells. (A and B) Mouse Sgk1, Sgk2, and Akt1. (C and D) Progressively N-terminal truncations of Sgk1. (E and F) GST and the fusion protein 60Sgk1-GST. Data points are the average of four experiments ± SD. Lines represent the fit of the data to a single exponential.

Fig. 2.

Effects of the Sgk1 degron on stability and localization of 60Sgk1-GST fusion protein. (A) Helical wheel representation of Sgk1 residues R18–Q30. (Left) The WT sequence. (Right) The change in hydrophobicity pattern produced by four Asp (D) mutations at positions 21, 22, 26, and 27 of the α-helix. Gray and white circles represent hydrophobic and hydrophilic residues. (B) Pulse–chase of CHO cells transfected with WT GST, 60Sgk1-GST, or 60Sgk14D-GST. (C) Immunofluorescence of CHO cells transfected with the constructs indicated in B and labeled with anti-GST antibody and Hoescht dye. (1 and 2) GST. (3 and 4) 60Sgk1-GST. (5 and 6) 60Sgk14D-GST. (Magnification: C, ×60.)

Fig. 3.

Immunolocalization of WT Sgk1 and Δ60Sgk1 in transfected CHO. Cofocal images of CHO cells cotransfected with Sgk1 and Calnexin-V5 (Upper) or Δ60Sgk1 and Calnexin-V5 (Lower). (Magnification: ×60.)

Fig. 4.

Sgk1 degradation by the Ub-dependent pathway. (A) Pulse–chase of Sgk1 in transfected CHO cells with or without lactacystin (10 μM) for 12 h. (B) Western blot of ubiquitinated Sgk1 in M1 cells and +/− transfected CHO cells with Sgk1 with or without treatment with lactacystin. IP, immunoprecipitation; IB, immunoblot.

Fig. 5.

Ub-conjugating enzymes and Ub ligases involved in Sgk1 degradation in Saccharomyces cerevisiae. (A) (Left) Cyclohexamide (CHX) chase of Sgk1 in transformed yeast strains: WT, Ubc4/5 and Ubc6/7 null mutants. (Right) Calculated t_1/2 of Sgk1 in the indicated yeast strains. (B) (Left) [³⁵S]Methionine pulse–chase experiments of Sgk1 in WT and Doa10/Hrd1 double mutant strains. (Right) Calculated t_1/2 of Sgk1 from the corresponding experiments. (C) Immunostaining of spheroplasts from doa10D mutant yeast transformed with Sgk1-HA and Δ60Sgk1-HA stained with monoclonal HA and DAPI. (Scale bars: 4 μm.)

Fig. 6.

Ub-conjugating enzymes and Ub ligases involved in Sgk1 degradation in mammalian cells. (A) Pulse–chase experiments of Sgk1 in CHO transfected with Sgk1 alone or with UBC6 or UBC7. Similar experiments in cells cotransfected with three different ER-anchored E3 ligases and corresponding inactive mutants. IP, immunoprecipitation. (B) gp78 or gp78R2M. (C) TEB4 and TEB4_C9S. (D) HRD1 or HRD1_C291S. (E–G) Graphical representation of the fraction of initial protein at three different time points. Each data point represents the average of two to three measurements. (H) Pulse–chase of Sgk1 cotransfected with scrambled siRNA, siRNA of TEB4, or HRD1 in HEK-293 cells. (I) RT-PCR of endogenous TEB4 and HRD1 in HEK cells transfected with siRNAs specific for the indicated E3 ligases. Controls are mock-transfected cells and scrambled siRNA. (J) Western blot of endogenous TEB4 in HEK cells transfected with siRNA scrambled and siRNA specific for human TEB4. IB, immunoblot.

Fig. 7.

Degradation and localization of endogenous Sgk1 in M1 cells. (A) Autoradiography of representative pulse–chase experiment of endogenous Sgk1 (≈1 month of exposure). (B) Graphic of t_1/2 of endogenous Sgk1. (C) Autoradiography of immunoprecipitated Sgk1 in M1 cells nontransfected and transfected with scrambled siRNA and three different oligos specific for mouse HRD1 (4-day exposure). (D) Average of protein abundance of former experiments (n = 3). (E) Confocal images of M1 cells showing colocalization of endogenous Sgk1 with transfected calnexin. Peptide panel indicates preincubation of anti-Sgk1 antibody with cognate peptide. (Magnification: E, ×60.)