Glycogen synthase kinase 3 phosphorylates hypoxia-inducible factor 1alpha and mediates its destabilization in a VHL-independent manner

Abstract

Hypoxia-inducible transcription factor 1alpha (HIF-1alpha) is a key player in the response to hypoxia. Additionally, HIF-1alpha responds to growth factors and hormones which can act via protein kinase B (Akt). However, HIF-1alpha is not a direct substrate for this kinase. Therefore, we investigated whether the protein kinase B target glycogen synthase kinase 3 (GSK-3) may have an impact on HIF-1alpha. We found that the inhibition or depletion of GSK-3 induced HIF-1alpha whereas the overexpression of GSK-3beta reduced HIF-1alpha. These effects were mediated via three amino acid residues in the oxygen-dependent degradation domain of HIF-1alpha. In addition, mutation analyses and experiments with von Hippel-Lindau (VHL)-defective cells indicated that GSK-3 mediates HIF-1alpha degradation in a VHL-independent manner. In line with these observations, the inhibition of the proteasome reversed the GSK-3 effects, indicating that GSK-3 may target HIF-1alpha to the proteasome by phosphorylation. Thus, the direct regulation of HIF-1alpha stability by GSK-3 may influence physiological processes or pathophysiological situations such as metabolic diseases or tumors.

FIG. 1.

Regulation of HIF-1α and the HIF-1 target PAI-1 gene by the inhibition of GSK-3. (A) HepG2 cells were cultured under normoxia (16% O₂) for 48 h and then stimulated with either LiCl or insulin and further cultured for 4 h under normoxic or hypoxic (8% O₂) conditions. HIF-1α protein levels were measured by Western blot analysis. The HIF-1α protein levels under normoxic conditions (16% O₂) were set at 1. (B, D, and F) Representative Western blots. One hundred micrograms of total protein from HepG2 cells was analyzed with antibodies against HIF-1α, phospho-GSK-3α/β (p-GSK-3α/β), and GSK-3α/β. PAI-1 levels in 100 μg of protein from the culture medium were determined and analyzed with an antibody against human PAI-1. (C) HepG2 cells were transfected with siRNA against GSK-3β and cultured for 72 h under normoxia. Then cells were further cultured under normoxia (16% O₂) or hypoxia (8% O₂) for the next 24 h. The HIF-1α and PAI-1 protein levels were measured by Western blot analysis, and protein levels under normoxic conditions (16% O₂) were set at 1. Values are means ± SEM of results from at least three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results with 16% O₂ and those with 8% O₂; **, significant difference between results with 8% O₂ and those with 8% O₂ and a stimulus or siRNA; ***, significant difference between results with 16% O₂ and those with 16% O₂ and a stimulus or siRNA; P ≤ 0.05. (E) HepG2 cells were transfected with either the PAI-1 promoter construct (pGL3-hPAI-796 Luc; PAI-766) or the HRE mutant (pGL3-hPAI-796HREm Luc; PAI-766HREm) construct and an empty control vector or an expression vector carrying GSK-3β. Then cells were cultured under normoxia (16% O₂; control) or exposed to hypoxia (8% O₂) or to insulin for the next 24 h. In each experiment, the percentage of Luc activity relative to the activity in the pGL3PAI-766 and pGL3PAI-766M2 controls, which was set at 100%, was determined.

FIG. 2.

GSK-3 downregulates HIF-1α via the TAD-N. (A) HepG2 cells were cotransfected with expression vectors for GSK-3β, Gal4-HIF-1α-TAD-N-C, Gal4-HIF-1α-TAD-N, and Gal4-HIF-1α-TAD-C and the p5GE1B-Luc gene construct. After transfection, cells were cultured with fresh culture medium for the next 48 h under normoxia (16% O₂). In each experiment, the percentage of Luc activity relative to that in the Gal4-HIF-1α-TAD-N-C, Gal4-HIF-1α-TAD-N, or Gal4-HIF-1α-TAD-C control, which was set at 100%, was determined. The values represent means ± SEM of results from three independent experiments. Numbers below the upper left panel represent amino acid positions. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the control and those for GSK-3β-transfected cells (P ≤ 0.05); bHLH, basic helix-loop-helix; −, control. (B) Representative Western blot. One hundred micrograms of total protein from the transfected HepG2 cells was analyzed by Western blotting with antibodies against Flag M2 and the Golgi membrane (GM). (C) HepG2 cells were cotransfected with expression vectors for GSK-3 S9A and Gal4-HIF-1α-TAD-N and the p5GE1B-Luc gene construct and cultured as described for panel A. In each experiment, the percentage of Luc activity relative to that in the Gal4-HIF-1α-TAD-N control, which was set at 100%, was determined. Values represent means ± SEM of results from three independent experiments. *, significant difference between results for the control and those for myrPKB and/or GSK-3 S9A (P ≤ 0.05). (D) Representative Western blot analyzed as described for panel B.

FIG. 3.

Mutation of the GSK-3 target sites abolishes the destabilization of HIF-1α. (A) HepG2 cells were cotransfected with expression vectors for GSK-3β, wild-type Gal4-HIF-1α-TAD-N (TADN), or the Gal4-HIF-1α-TAD-N S551A, Gal4-HIF-1α-TAD-N T555V, Gal4-HIF-1α-TAD-N S589A, or Gal4-HIF-1α-TAD-N S551S/T555V/S589A (S/T/S) mutant and the p5GE1B-Luc gene construct. After transfection, cells were cultured with fresh culture medium for the next 48 h under normoxia (16% O₂). In each experiment, the percentage of Luc activity relative to that in the wild-type Gal4-HIF-1α-TAD-N control, which was set at 100%, was determined. The values represent means ± SEM of results from three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the control and those for GSK-3β-transfected cells; **, significant difference between results for wild-type Gal4-HIF-1α-TAD-N and those for Gal4-HIF-1α-TAD-N mutants; P ≤ 0.05. bHLH, basic helix-loop-helix. (B) Representative Western blot. One hundred micrograms of total protein from the transfected HepG2 cells was analyzed by Western blotting with antibodies against Flag M2 and GSK-3α/β. (C) HepG2 cells were cotransfected with expression vectors for GSK-3β, Myc- or V5-tagged full-length wild-type human HIF-1α (WT), or the human HIF-1α S551A, human HIF-1α T555V, human HIF-1α S589, human HIF-1α S551A/T555V (S/T), or human HIF-1α S551A/T555V/S589A (S/T/S) mutant. After transfection, cells were cultured with fresh culture medium for the next 24 h. The HIF-1α protein levels were measured by Western blotting, and the wild-type HIF-1α levels in the controls were set at 100%. Values are means ± SEM of results from at least three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the wild type in control and GSK-3β-transfected cells; **, significant difference between results for the mutants and those for the wild type in GSK-3β-transfected cells; ***, significant difference between results for the wild type and those for the mutants; P ≤ 0.05. −, control. (D) Representative Western blot. One hundred micrograms of total protein from the transfected HepG2 cells was analyzed by Western blotting with antibodies against the Myc, HA, and V5 tags and the Golgi membrane (GM).

FIG. 4.

The destabilization of HIF-1α by GSK-3 is independent of the hydroxylation site proline 564. (A) HepG2 cells were cotransfected with expression vectors for GSK-3β, wild-type Gal4-HIF-1α-TAD-N (TADN), or the Gal4-HIF-1α-TAD-N P564A (P564A), Gal4-HIF-1α-TAD-N P564A/S551A (P/S), or Gal4-HIF-1α-TAD-N P564A/T555V/S589A (P/T/S/) mutant and the p5GE1B-Luc gene construct. After transfection, cells were cultured with fresh culture medium for the next 48 h under normoxia (16% O₂). In each experiment, the percentage of Luc activity relative to that in the wild-type Gal4-HIF-1α-TAD-N control, which was set at 100%, was determined. The values represent means ± SEM of results from three independent experiments. Numbers below the upper left panel represent amino acid positions. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the respective control and those for GSK-3β-transfected cells; **, significant difference between results for the TAD-N wild type and those for TAD-N mutants; P ≤ 0.05. bHLH, basic helix-loop-helix; −, control. (B) Representative Western blot. One hundred micrograms of total protein from the transfected HepG2 cells was analyzed by Western blotting with antibodies against Flag M2 and the HA tag.

FIG. 5.

GSK-3-mediated destabilization of HIF-1α is independent of the hydroxylation sites within HIF-1α and of pVHL. (A) HepG2 cells were cotransfected with expression vectors for GSK-3β, full-length V5-tagged wild-type human HIF-1α (WT), or the human HIF-1α P402A/P564A/N803A (P/P/N), human HIF-1α P402A/P564A/N803A/S551A/T555V (P/P/N/S/T), or human HIF-1α P402A/P564A/N803A/S551A/T555V/S589A (P/P/N/S/T/S) mutant. After transfection, the cells were cultured with fresh culture medium for the next 24 h. The HIF-1α protein levels were measured by Western blotting, and the wild-type HIF-1α levels in the controls were set at 100%. Values are means ± SEM of results from at least three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the control and those for GSK-3β-transfected cells; **, significant difference between results for the mutants and those for the wild type in GSK-3β-transfected cells; ***, significant difference between results for the mutants and those for the wild type; P ≤ 0.05. bHLH, basic helix-loop-helix. (B and E) Representative Western blots. One hundred micrograms of total protein from the cells was analyzed by Western blotting with antibodies against HIF-1α, pVHL, actin, and the V5 and HA tags. +, present; −, absent. (C) Determination of HIF-1α protein half-life. HepG2 cells were cotransfected with vectors for HA epitope-tagged GSK-3β or either V5-tagged full-length wild-type HIF-1α or the respective HIF-1α mutant human HIF-1α P402A/P564A/N803A or human HIF-1α P402A/P564A/N803A/S551A/T555V/S589A. After the inhibition of protein synthesis with cycloheximide (CHX; 100 μg/ml), the HIF-1α protein levels were determined by Western analysis with an antibody against the V5 tag. (D) The VHL-defective RCC4 cells (−) and the RCC4 cells with reintroduced VHL (+) were transfected with GSK-3β expression vectors or an empty control vector. After transfection, the cells were cultured with fresh culture medium for the next 24 h. The HIF-1α levels were measured by Western blotting, and the wild-type HIF-1α levels in the RCC4 cells were set at 100%. Values are means ± SEM of results from at least three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results for the control and those for GSK-3β-transfected cells; **, significant difference between results for RCC4/VHL cells and those for RCC4 cells; ***, significant difference between results for RCC4/VHL cells and RCC4 cells transfected with GSK-3β; P ≤ 0.05. (F) Immunoblot (IB) analysis of anti-V5 and anti-HA immunoprecipitates (IP) and whole-cell extracts (WCE) of HepG2 cells treated with the proteasomal inhibitor MG132 after transfection with the indicated plasmids. GM, Golgi membrane.

FIG. 6.

GSK-3 mediates the destabilization of HIF-1α in a VHL-independent manner via the proteasome. HepG2 cells were cotransfected with expression vectors for GSK-3β, full-length V5-tagged wild-type human HIF-1α, or the human HIF-1α P402A/P564A/N803A or human HIF-1α P402A/P564A/N803A/S551A/T555V/S589A mutant. After transfection, cells were cultured for the next 24 h under normoxia (16% O₂) and then they were treated with MG132 and further cultured for 4 h under normoxia or hypoxia (8% O₂). (A) The HIF-1α protein levels were measured by Western blotting, and the levels of wild-type human HIF-1α and the human HIF-1α P402A/P564A/N803A and human HIF-1α P402A/P564A/N803A/S551A/T555V/S589A mutants under hypoxia were set at 100%. Values are means ± SEM of results from at least three independent experiments. Statistics were calculated by using the Student t test for paired values. *, significant difference between results with 16% O₂ and those with 8% O₂; **, significant difference between results for the respective control and those for GSK-3β-transfected cells; ***, significant difference between results for the respective control and those for MG132-treated cells; P ≤ 0.05. bHLH, basic helix-loop-helix. (B) Representative Western blot. One hundred micrograms of total protein from the transfected HepG2 cells was analyzed by Western blotting with antibodies against human HIF-1α, the V5 tag, the HA tag, and the Golgi membrane (GM). WT, wild type; P/P/N, PPN mutant; P/P/N + S/T/S, PPNSTS mutant. Numbers represent oxygen concentrations.

FIG. 7.

HIF-1α is phosphorylated by GSK-3. (A) GST-HIF-1α-TAD-N wild-type (WT) or mutant (listed by mutations) fusion proteins were prepared from E. coli, and 20 μg of these fusion proteins was incubated with 50 mU of active GSK-3β and 10 μCi of [γ-³²P]ATP for 30 min at 30°C. (B) Afterwards, the phosphorylated proteins were separated from unbound radioactivity by electrophoresis on a 10% SDS gel. Radioactive proteins were visualized by phosphorimaging.