The cell death regulator GRIM-19 is an inhibitor of signal transducer and activator of transcription 3

Abstract

GRIM-19 (gene associated with retinoid-IFN-induced mortality 19), isolated as a cell death activator in a genetic screen used to define mechanisms involved in IFN-beta- and retinoic acid-induced cell death, codes for a approximately 16-kDa protein that induces apoptosis in a number of cell lines. Antisense ablation of GRIM-19 caused resistance to cell death induced by IFN plus retinoic acid and conferred a growth advantage to cells. To understand the molecular bases for its cell death regulatory activity, we used a yeast two-hybrid screen and identified that the transcription factor STAT3 (signal transducer and activator of transcription 3) binds to GRIM-19. GRIM-19 inhibits transcription driven by activation of STAT3, but not STAT1. It neither inhibits the ligand-induced activation of STAT3 nor blocks its ability to bind to DNA. Mutational analysis indicates that the transactivation domain of STAT3, especially residue S727, is required for GRIM-19 binding. Because GRIM-19 does not bind significantly to other STATs, our studies identify a specific inhibitor of STAT3. Because constitutively active STAT3 up-regulates antiapoptotic genes to promote tumor survival, its inhibition by GRIM-19 also demonstrates an antioncogenic effect exerted by biological therapeutics.

Fig. 1.

Identification of STAT3 as a GRIM-19 binding protein. Yeast cells transformed with the indicated expression vectors were grown on media containing (A) or lacking (B) tryptophan, leucine, and histidine for 1 week. Plasmids transformed in each case were as follows: 1, pAct2; 2, pGBKT7; 3, STAT3; 4, GRIM-19; 5, GRIM-19+pACT2; 6, STAT3+GRIM-19. pGBKT7 and pAct2 express only the GAL4-DBD and GAL4-TAD, respectively.

Fig. 2.

Endogenous GRIM-19 binds to STAT3 specifically. (A–D) MCF-7 cells stimulated with IFN-β (500 units/ml) and RA (1 μM) were lysed, and IP or Western (WB) analysis was performed with specific antibodies. (E–G) Specific IP of STAT3 with GRIM-19. The STAT3 genotypes of cells are indicated. (H and I) GRIM-19 does not bind to other STATs. Lanes 1 and 2, lysates were immunoprecipitated with GRIM-19 antibodies. Lanes 3 and 4, lysates without IP. Western transfers were probed with the indicated antibodies. (J–L) STAT1 does not bind to GRIM-19. Cells were stimulated with murine IFN-γ (200 units/ml) for 30 min; lysates were processed and analyzed as indicated.

Fig. 3.

GRIM-19 inhibits STAT3-dependent gene expression. (A) MCF-7 cells were transfected with S3-Luc and treated with the indicated agents for 16 h. Luciferase activity in the lysates was measured. Each bar represents mean relative light units (RLU) ± SE of triplicate transfectants in the same experiment. IL-6 (10 ng/ml), IFN-β (500 units/ml), and RA (1 μM) were used in this experiment. IR, IFN-β/RA combination. (B) Transfection was performed as in A, in the presence of the empty vector pCXN2 or the GRIM-19 (G-19) expression plasmid. (C) Increasing amounts of STAT3 plasmid overcome GRIM-19-induced inhibition. Luciferase activity was determined as in A. Numbers across the STAT3 row indicate the fold excess of STAT3 used, relative to GRIM-19, in the cotransfection assays. GRIM-19 and the reporter were used at 100 ng per transfection in the STAT3^–/– cells. Cells were treated with soluble IL-6 receptor (100 ng/ml) and IL-6 (10 ng/ml). (D) Antisense GRIM-19 promotes STAT3-dependent gene expression. Cells stably expressing empty pTKO1 vector (empty bars) and antisense GRIM-19 (filled bars) were used for transfection analysis as in A. (E and F) The same cell lysates were monitored for GRIM-19 and STAT3 expression by Western analysis. (G and H) The specific inhibitory effect of GRIM-19 on STAT3. STAT1^–/– cells were transfected with S3-Luc or pIRE Luc along with GRIM-19, and cells were stimulated with the indicated agents. IFN-γ was used at 200 units/ml in H. STAT1 expression was reconstituted along with reporter where indicated. (I) Expression of STAT3-regulated genes in hTERT-HME cells is inhibited by GRIM-19. Stable cell lines expressing WT and Y705F mutant forms of STAT3 were transfected with mammalian expression vector pCXN2 (V) or the same vector expressing GRIM-19 (G). RNA was analyzed by the Northern method. (J and K) The same cells were transfected with luciferase reporters driven by the cyclin B1 and cdc2 promoters, and the influence of GRIM-19 on luciferase expression was measured. c, Control vector; W, WT mutant of STAT3; F, Y705F mutant of STAT3.

Fig. 4.

Effect of GRIM-19 on STAT3 activation and DNA binding. (A–C) MCF-7 cells stably transfected with empty vector (V) or vector expressing myc-tagged GRIM-19 (G) were stimulated with IL-6 (10 ng/ml). Cell lysates were analyzed by the Western method (WB) with antibodies specific for native or phospho-STAT3. (D) Nuclear extracts (5 μg) from IL-6-stimulated cells were used for EMSA with a STAT3 binding element. N, no extract. (E–G) Total nuclear protein (70 μg) was used in Western analyses. (H) The effect of recombinant GRIM-19 on DNA binding of STAT3. Reactions were incubated with buffer (B) or 4 μg of recombinant GRIM-19 (G). Lanes 1 and 2, no nuclear extract; lanes 3 and 4, nuclear extract from IL-6-treated cells.

Fig. 5.

The TAD of STAT3 is required for GRIM-19 binding. (A) Diagram of the modular structure of STAT3. Amino acid positions at which stop codons are introduced into the mutants and the coordinates of various domains are indicated. CCD, coiled-coil domain; LK, linker domain; NTD, N-terminal domain; SH2, Src homology 2 domain. (B and D) The N-terminally flag-tagged mutants were transfected into STAT3^–/– cells, and 60μg of lysate from each sample was used for Western analysis with anti-flag antibodies. STATDB has been described (16). (C and E) Total lysate (250 μg) was used for IP with GRIM-19 specific antibodies, followed by Western analysis with anti-flag antibodies.

Fig. 6.

The S727 residue is important for GRIM-19 binding to STAT3. (A) STAT3^–/– cells were transfected with flag-tagged WT or mutant STAT3 constructs and an IP with GRIM-19-specific antibodies was performed as in Fig. 5. The same lysates were analyzed for the expression of STAT3 (B) and actin (C). WB, Western analysis.