Regulation of a third conserved phosphorylation site in SGK1

Abstract

SGK1 (serum- and glucocorticoid-induced kinase 1) is a member of the AGC branch of the protein kinase family. Among well described functions of SGK1 is the regulation of epithelial transport through phosphorylation of the ubiquitin protein ligase Nedd4-2 (neuronal precursor cell expressed developmentally down-regulated 4-2). The activation of SGK1 has been widely accepted to be dependent on the phosphorylation of Thr256 in the activation loop and Ser422 in the hydrophobic motif near the C terminus. Here, we report the identification of two additional phosphorylation sites, Ser397 and Ser401. Both are required for maximum SGK1 activity induced by extracellular agents or by coexpression with other protein kinases, with the largest loss of activity from mutation of Ser397. Coexpression with active Akt1 increased the phosphorylation of Ser397 and thereby SGK1 kinase activity. SGK1 activation was further augmented by coexpression with the protein kinase WNK1 (with no lysine kinase 1). These findings reveal further complexity underlying the regulation of SGK1 activity.

FIGURE 1.

SGK1 is activated by active Akt1. Wild-type (WT) or kinase-dead (KD) ΔSGK1 was coexpressed with Myc-PDK1, HA-Akt1, or HA-Myr-Akt1 in HeLa cells. Protein expression was probed with anti-Myc, anti-FLAG, and anti-HA antibodies (upper two panels). Immunoprecipitated ΔSGK1 was assayed with GST-Nedd4-2 as substrate; shown are an autoradiogram and quantitation of three experiments (lower panel, plotted comparing wild-type SGK1 and kinase-dead ΔSGK1).

FIGURE 2.

Active Akt enhances phosphorylation of SGK1 Ser³⁹⁷. A, FLAG-SGK1 was coexpressed with vector or HA-Myr-Akt1 in HeLa cells. Immunoprecipitated SGK1 was subjected to mass spectrometry. Mass spectroscopic data of the phosphopeptide phospho-Ser⁴⁰¹ in the control sample and phospho-Ser⁴⁰¹ and phospho-Ser³⁹⁷ in samples from cells also expressing Myr-Akt1 are shown. B, shown is an amino acid sequence alignment of the tail region of selected AGC kinases. Conserved phosphorylation sites aligning with Ser³⁹⁷ are in boldface, and phosphorylation sites aligning with Ser⁴⁰¹ are in italics. PKAc, protein kinase A. C, FLAG-ΔSGK1 or mutant ΔSGK1(S397A) or ΔSGK1(S401A) was coexpressed with Myc-PDK1, HA-Akt1, or HA-Myr-Akt1 in HeLa cells. Protein expression was probed with anti-Myc, anti-FLAG, and anti-HA antibodies. Immunoprecipitated ΔSGK1 proteins were assayed with GST-Nedd4-2 as substrate (n = 4). WT, wild-type.

FIGURE 3.

Effects of mutating phosphorylatable residues to Ala or Asp. FLAG-tagged ΔSGK1 (A) and SGK1 (B) and the indicated mutants of each were expressed in HeLa cells. Protein expression was probed with anti-FLAG, anti-SGK1 phospho-Thr²⁵⁶, or anti-SGK1 phospho-Ser⁴²² antibodies. Immunoprecipitated SGK1 proteins were assayed with GST-Nedd4-2 as substrate (n = 3 in A and B). WT, wild-type.

FIGURE 4.

Mutation of S397A blocks SGK1 activation by H₂O₂ and IGF1. A, FLAG-ΔSGK1 or the indicated mutants were expressed in HeLa cells. Cells were untreated (–) or treated (+) with 2 mm H₂O₂ for 25 min or with 50 ng/ml IGF1 for 30 min before harvest. Protein expression was probed with the anti-FLAG antibody. Immunoprecipitated ΔSGK1 or mutants were assayed with GST-Nedd4-2 as substrate (n = 4). B, full-length FLAG-SGK1 or the indicated mutants were expressed in HeLa cells. Cells were untreated (–) or treated (+) with 2 mm H₂O₂ for 40 min. Protein expression was probed with the anti-FLAG antibody. Immunoprecipitated SGK1 or mutants were assayed with GST-Nedd4-2 as substrate (n = 3). WT, wild-type; Ctrl, control.

FIGURE 5.

WNK1 and Akt1 are involved in SGK1 activation by H₂O₂ and IGF1. HeLa cells were cotransfected with plasmid encoding FLAG-ΔSGK1 and WNK1 (Wi), Akt1 (Ai), or scrambled (Ci) small interfering RNA (siRNA) oligonucleotides. Cells were untreated (–) or treated (+) with 2 mm H₂O₂ for 25 min or with 50 ng/ml IGF1 for 30 min before harvest. Lysates were probed with anti-FLAG, anti-WNK1, anti-Akt1, anti-phospho-ERK1/2, and either anti-OSR1 (A) or anti-ERK1/2 (B) antibodies. Immunoprecipitated ΔSGK1 was assayed with GST-Nedd4-2 as substrate. A, WNK1 knockdown; B, Akt1 knockdown (n = 3 in A and B). Ctrl, control.

FIGURE 6.

WNK1 and Akt1 cooperate to activate SGK1. A, FLAG-ΔSGK1 was coexpressed with Myc-WNK1-(1–491), Myc-WNK1-(1–491)(T58A), HA-Akt1, HA-Myr-Akt1, or HA-Myr-Akt1KM (kinase-dead) in different combinations in HeLa cells. Protein expression was probed with anti-Myc, anti-FLAG, and anti-HA antibodies. Immunoprecipitated ΔSGK1 was assayed with GST-Nedd4-2 as substrate (n = 3). B, FLAG-SGK1 (full-length) or the indicated mutants were coexpressed with Myc-WNK1-(1–491) or Myc-WNK1-(1–491)(T58A) in HeLa cells. Protein expression was probed with anti-Myc and anti-FLAG antibodies. Immunoprecipitated SGK1 proteins were assayed with GST-Nedd4-2 as substrate (n = 4). WT, wild-type.

FIGURE 7.

Overexpressed WNK1-(1–491) co-immunoprecipitates with overexpressed Akt1. HA-Akt1, Myc-WNK1-(1–491), Myc-WNK1-(1–491)(T58A), Myc-WNK1-(1–220), or Myc-WNK1-(1–220)(T58A) was coexpressed in HeLa cells in the indicated combinations. Cells were harvested in detergent-free lysis buffer and lysed by aspiration through a 22-gauge needle 30 times. Anti-HA (A) or anti-Myc (B) immunoprecipitates (IP) were washed with lysis buffer, resolved on gels, and immunoblotted (IB) with the anti-Myc or anti-HA antibody.

FIGURE 8.

The figure shows the superposition of the SGK1 model (dark blue) with the SGK1 crystal structure (light blue; Protein Data Bank code 2r5t). The SGK1 model is based on the protein kinase A structure (Protein Data Bank code 1atp) using the homology modeling tool SWISS-MODEL (35). The activation loop, ATP-binding loop, and hydrophobic motif in the SGK1 model are colored yellow, orange, and green, respectively. Ser³⁹⁷ and Thr²⁵⁶ are shown as sticks. Arrows indicate the positions of Gly³⁷⁸, α-helix C (α_C), and β-strand 5 (β₅).