Hsp90 binds and regulates Gcn2, the ligand-inducible kinase of the alpha subunit of eukaryotic translation initiation factor 2 [corrected]

Abstract

The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

FIG. 1

Hsp90 mutants stimulate GCN4-lacZ expression under repressed conditions. (A) β-Galactosidase activity assayed in extracts prepared from the different yeast strains expressing Hsp90 mutants transformed with the GCN4-lacZ reporter plasmid p180 (schematically represented). Enzyme activities are given in units. (B) β-Galactosidase activity assayed for the panel A strains transformed with the control reporter plasmid p227 containing point mutations (×) in the ATGs of the four uORFs in the 5′-untranslated region of the GCN4 mRNA. The values are the means of three to four independent experiments. + a.a., with amino acids; − a.a., without amino acids; hHsp90, human Hsp90β; 10%, strain 10% Hsp82wt; G313N, HH1a-pHCA/Hsp82 G313N; T525I, HH1a-pHCA/Hsp82 T525I.

FIG. 2

Gcn2 is required for the increased expression of the GCN4-lacZ reporter. β-Galactosidase activities of WT and G313N strains with a deletion of the gcn2 gene (strains OD1 and OD2) transformed with plasmid p180 are shown. Cells were grown only under repressed conditions (with amino acids). The experiments were carried out twice with less than 20% difference.

FIG. 3

The G313N mutation is responsible for increased GCN4-lacZ expression under repressed conditions. β-Galactosidase activity from reporter plasmid p180 was assayed in extracts prepared from the different yeast strains (WT or Hsp82 G313N or Hsp82 G313N transformed with the plasmid pTCA/Hsp82 which codes for the WT allele of Hsp82). The experiments were carried out twice. + a.a., with amino acids; − a.a., without amino acids.

FIG. 4

Gcn2 binds Hsp90 in vivo. (A) Antibodies to human Hsp90β coprecipitate Gcn2. Equal amounts of yeast extracts (0.5 mg) from cells expressing human Hsp90β transformed with the plasmid p2U/GSTGcn2 were used with different antibodies as indicated below the panel. GSTGcn2 was revealed by immunoblotting with an anti-Gcn2 antiserum. α-Hsp90, antibody against human Hsp90; α-lacZ, antibody against β-galactosidase (Promega); α-flu, antibody against flu-tag (Aves Laboratory); α-Gcn2, polyclonal antibody against Gcn2 (77). IP, immunoprecipitation. (B) Antibodies to Gcn2 coprecipitate human Hsp90β. Equal amounts of yeast extracts (0.5 mg) from cells expressing the human Hsp90β transformed with the plasmid p2U/GSTGcn2 were used with different antibodies as indicated below the panel. Hsp90 was revealed by immunoblotting with the monoclonal antibody against Hsp90 (H90-10). (C) FLAG-tagged Hsp82 associates with GSTGcn2. Equal amounts of yeast extracts (0.5 mg) from isogenic strains expressing Hsp82wt or G313N with or without the FLAG tag were used with the FLAG antibody. All strains also expressed GSTGcn2 from plasmid p2U/GSTGcn2. Gcn2 was revealed by immunoblotting with an anti-Gcn2 antiserum. (D) Endogenous Gcn2 interacts with human Hsp90. Equal amounts of yeast extracts from cells expressing the human Hsp90β were used with the monoclonal anti-human Hsp90 antibody. Gcn2 was revealed as described for panel A. Lane 1, strain expressing human Hsp90; lane 2, strain expressing human Hsp90 with a gcn2 deletion; lane 3, strain expressing the yeast Hsp82. (E) Endogenous Gcn2 interacts with FLAG-tagged Hsp82. Equal amounts of yeast extracts from cells expressing the FLAG-tagged yeast Hsp82 were used with the FLAG antibody. Lane 1, strain with GCN2; lane 2, strain carrying a gcn2 deletion.

FIG. 5

Ansamycin benzoquinones block kinase activity of Gcn2 in vitro. Gcn2 was translated at 30°C in rabbit reticulocyte lysate. Macbecin I (Mc) and GA were added during synthesis, where indicated. After immunoprecipitation with an anti-Gcn2 antiserum, the samples were used for in vitro kinase assays and electrophoresed on an SDS-polyacrylamide gel electrophoresis gel. The two panels represent the same gel with differential detection of incorporated ³⁵S (upper panel) and ³²P (lower panel) by using two sheets of X-ray film. The numbers above the panels represent the concentrations of compounds used (in micromolar). Note that the slightly reduced levels of immunoprecipitated Gcn2 in the presence of Mc were specific to this particular experiment and are not generally observed. −, no drug added.

FIG. 6

GA increases the association of Hsp90 with Gcn2 in vitro. Gcn2 was translated at 30°C in rabbit reticulocyte lysate. GA was added during synthesis at a concentration of 20 μM. The top sections represent the input, and the bottom sections represent the immune pellets after coimmunoprecipitation (IP) with the anti-Hsp90 monoclonal antibody. (A) Plasmids pYes/GSTGcn2, pYes/Gcn2, and pYes/Gcn2ΔN were used for in vitro translation; (B) plasmids pYes/Gcn2 and pYes/Gcn2^K559V were used. The migration positions of the different Gcn2 variants are indicated with arrows.

FIG. 7

Macbecin I reduces Gcn2 levels in vivo. Strain HH1a-pHCA/Hsp82 was transformed with plasmids pYes/Gcn2 (A) and pYes/Gcn2ΔN (B). Gcn2 variants were induced for 24 h by 2% galactose in the presence or absence of macbecin I (50 μM) and immunoprecipitated with an anti-Gcn2 antiserum. (A) Gcn2 was revealed by immunoblotting with an antiserum against Gcn2. Lanes 1 and 2, induction in the presence of galactose (10-fold less material was loaded in lane 2 than in lane 1; lane 3, cells grown with 2% glucose; lane 4, induction with galactose in the presence of 50 μM macbecin I. (B) Gcn2ΔN was either visualized by immunoblot analysis with an antibody against Gcn2 or used for an in vitro kinase assay. Lanes 1 and 2, parent strain; lanes 3 and 4, strain transformed with the plasmid pYes/Gcn2ΔN. (C) The WT strain was transformed with the plasmid pLG/LUC and grown with glucose or galactose in the presence or absence of macbecin I for 24 h. The plotted luciferase activity represents the mean values from two independent experiments.

FIG. 8

Inactivation of Hsp90 abolishes Gcn2 accumulation and activity in vivo. Strain HH1a-G170D was transformed with plasmids pYes/Gcn2ΔN (A) and pLG/LUC (B). Cells grown to mid-logarithmic phase at 30°C in glucose were shifted to galactose and 37°C simultaneously for 6 h to induce the expression of Gcn2ΔN (A) or luciferase (B). (A) Cell extracts from the different treatments were immunoprecipitated with an anti-Gcn2 antiserum, and an immunoblotting experiment with the same antibody (top) and a kinase assay (bottom) were performed. Lanes 1 and 2, shift to galactose. Cells were grown at 30°C (lanes 1 and 3) or at 37°C (lane 2). (B) Cells were grown with galactose (lanes 1, 2, and 3) or glucose (lane 4) at 30°C (lanes 1, 3, and 4) or at 37°C (lane 2) for 6 h. Lane 3, cells treated also with macbecin I (50 μM) for 24 h. Following immunoprecipitation with an anti-luciferase antiserum, luciferase was revealed with the same antibody. IgH, immunoglobulin heavy chain.

FIG. 9

Model for the role of Hsp90 in the maturation and inhibition of Gcn2. GA may block the transition from step II to III.