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. 2002 Nov;22(22):7929-41.
doi: 10.1128/MCB.22.22.7929-7941.2002.

Glucocorticoid-induced leucine zipper inhibits the Raf-extracellular signal-regulated kinase pathway by binding to Raf-1

Emira Ayroldi  1 , Ornella Zollo, Antonio Macchiarulo, Barbara Di Marco, Cristina Marchetti, Carlo Riccardi
Affiliations

Glucocorticoid-induced leucine zipper inhibits the Raf-extracellular signal-regulated kinase pathway by binding to Raf-1

Emira Ayroldi et al. Mol Cell Biol. 2002 Nov.

Abstract

Glucocorticoid-induced leucine zipper (GILZ) is a leucine zipper protein, whose expression is augmented by dexamethasone (DEX) treatment and downregulated by T-cell receptor (TCR) triggering. Stable expression of GILZ in T cells mimics some of the effects of glucocorticoid hormones (GCH) in GCH-mediated immunosuppressive and anti-inflammatory activity. In fact, GILZ overexpression inhibits TCR-activated NF-kappaB nuclear translocation, interleukin-2 production, FasL upregulation, and the consequent activation-induced apoptosis. We have investigated the molecular mechanism underlying GILZ-mediated regulation of T-cell activation by analyzing the effects of GILZ on the activity of mitogen-activated protein kinase (MAPK) family members, including Raf, MAPK/extracellular signal-regulated kinase (ERK) 1/2 (MEK-1/2), ERK-1/2, and c-Jun NH(2)-terminal protein kinase (JNK). Our results indicate that GILZ inhibited Raf-1 phosphorylation, which resulted in the suppression of both MEK/ERK-1/2 phosphorylation and AP-1-dependent transcription. We demonstrate that GILZ interacts in vitro and in vivo with endogenous Raf-1 and that Raf-1 coimmunoprecipitated with GILZ in murine thymocytes treated with DEX. Mapping of the binding domains and experiments with GILZ mutants showed that GILZ binds the region of Raf interacting with Ras through the NH(2)-terminal region. These data suggest that GILZ contributes, through protein-to-protein interaction with Raf-1 and the consequent inhibition of Raf-MEK-ERK activation, to regulating the MAPK pathway and to providing a further mechanism underlying GCH immunosuppression.

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Figures

FIG. 1.
FIG. 1.
GILZ overexpression inhibits the binding of AP-1 to its DNA motif. (A) EMSA was performed with nuclear extract from untreated and anti-CD3-treated (1 μg/ml, 1 h) 3DO cells (lanes 2 and 6) or pcDNA3-transfected PV6 (lanes 3 and 7), pcDNA3-GILZ-transfected ST7 (lanes 4 and 8), or GIRL-19 (lanes 5 and 9) clones. Lane 1, probe alone. (B) Nuclear extract from untreated or anti-CD3-treated 3DO cells, alone (lane 5) or added with competitor cold probe (lane 3), with anti-c-Jun (lane 4), or with anti-c-Fos antibody (lane 6). Lane 1, labeled probe alone. C, untreated cells. (C) EMSA performed with NF-AT as the probe. Nuclear extract from untreated and anti-CD3-treated (1 μg/ml, 1 h) 3DO cells (lanes 2 and 6) or PV6 (lanes 3 and 7), ST7 (lanes 4 and 8), or GIRL-19 (lanes 5 and 9) clones. Lane 1, probe alone. The results are representative of three experiments. (D) 3DO cells were transfected with the AP-1 luciferase reporter geneand pcDNA3 (solid bar) or with the reporter gene and pcDNA3-GILZ (open bar) and stimulated for 4 and 18 h with plastic-bound anti-CD3 MAb. The values are expressed as the increase (n-fold) in luciferase activity. Each transfection was performed in triplicate. The standard errors were <10%.
FIG. 2.
FIG. 2.
GILZ overexpression inhibits c-Fos but not c-Jun trascription. Western blot analysis of c-Fos (A) and c-Jun (B) expression was performed. Nuclear cell lysates (10 μg) from PV6 and GIRL-19, stimulated for 1 h with immobilized anti-CD3 MAb (1 μg/ml), were probed with anti-c-Fos or anti-c-Jun antibodies (1 μg/ml). C, untreated cells. (C) The expression level of endogenous and exogenous GILZ was evaluated by Western blotting with anti-GILZ antiserum.
FIG. 3.
FIG. 3.
GILZ overexpression inhibits ERK-1/2, MEK-1/2, and Raf-1 but not JNK phosphorylation. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 5 or 60 min with plastic-bound anti-CD3 MAb. Whole-cell lysates were probed with an antibody specific for phosphorylated ERK-1/2 (pERK-1/2) (A), MEK-1/2 (pMEK) (B), or Raf-1 (pRaf-1) (C). Western blots were also performed with anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibodies to verify that no modulation of protein expression occurred or with β-tubulin to verify that an equivalent amount of proteins was loaded in each lane. PV6 or GIRL-19 was stimulated for the times indicated with plastic-bound anti-CD3 MAb. (D) Whole-cell lysates were probed with an antibody recognizing both phosphorylated forms of JNK: p54 and p46 (pSAPK/JNK). C, untreated cells.
FIG. 4.
FIG. 4.
GILZ interacts with endogenous Raf-1 in mouse thymocytes. Mouse thymocytes were treated for 6 h with DEX (100 nM), and cell lysates were incubated with GST or GST-GILZ beads. Binding of Raf-1 (A), MEK-1/2 (B), and ERK-1/2 (C) was visualized by Western blotting. Whole-cell lysates from thymocytes left untreated or treated with DEX were immunoprecipitated with an anti-Raf-1 or control isotype antibody (4 μg/500 μg of protein). (D and E) Nitrocellulose membrane was probed with an anti-GILZ antiserum (D) and then stripped and reprobed with anti-Raf-1 antibody (E).
FIG. 5.
FIG. 5.
GILZ interacts with Raf-1 in COS-7-transfected cells. COS-7 cells were cotransfected with pUSEamp-Raf-1 (2 μg) and myc-GILZ (2 μg) vectors. Immunoprecipitation was performed with anti-myc antibody (3 μg/500 μg of protein), and immunoreactive proteins were visualized with anti-Raf-1 (A) or anti-myc (B) antibodies. Whole-cell lysates were loaded to control GILZ and Raf-1 expression. Serum-starved COS-7 cells, either untransfected or transfected with myc-GILZ, were treated for 15 min with PMA (10 ng/ml). Raf-1 immunoprecipitates were analyzed for kinase activity in the presence of [γ-32P]ATP by using GST-MEK (C) or GST-MEK, GST-ERK, and MBP (D) as substrates. P.C., positive control performed with 10 U of purified Raf-1 kinase; N.C., negative control performed with Raf-1 immunocomplex from PMA-stimulated cells in the absence of MEK substrate. (E) MEK phosphorylation was also assayed by Western blot with an antibody specific for phosphorylated MEK (pMEK). myc-GILZ-Raf-cotransfected COS-7 cells were immunostained with anti-myc and anti-Raf antibodies. Single staining and superimposed images are shown. (F) Nuclei were visualized by DAPI staining.
FIG. 6.
FIG. 6.
(A) GILZ sequence alignment with a set of TSC family proteins (upper part of diagram) and alignment of NH2-terminal sequence of GILZ to 1vig as proposed by threading server (3DPSSM) (lower part of diagram). PSS indicates the predicted secondary structure for the N-terminal domain. SS indicates the known secondary structure of the library template 1vig. (B) 3D model of human GILZ. α-Helices are represented as light blue cylinders, β-sheets are represented as red ribbons, and proline residues are indicated in red CPK. (C) Molecular modeling of Raf-1-GILZ interaction. Distribution of solutions (i.e., the 100, 50, and 10 top solutions found) of docking experiments of Raf-1 and GILZ; the red balls indicate the center of mass of Raf-1, for each solution, interacting with GILZ.
FIG. 7.
FIG. 7.
The GST-Raf-RBD interacts with the GILZ amino-terminal region. (A) GST pulldown was performed with GST-Raf-RBD fusion protein corresponding to the human RBD (residues 1 to 149) of Raf or GST alone, attached to glutathione-Sepharose beads as bait and whole-cell lysates from untreated and DEX-treated thymocytes. The membrane was probed with anti-GILZ antiserum. Total lysates from DEX-treated and untreated thymocytes were loaded to control GILZ expression. (B) GST-Raf-RBD fusion protein was incubated for 18 h with the 35S-labeled in vitro-transcribed and translated proteins GILZ (lane 3), mutant 6 (lane 6), and mutant 13 (lane 9). Lane 1, GILZ; lane 2, GST plus GILZ; lane 4, mutant 6; lane 5, GST plus mutant 6; lane 7, mutant 13; lane 8, GST plus mutant 13. (C) GST-Raf-RBD fusion protein was incubated for 18 h with the 35S-labeled in vitro-transcribed and -translated protein GILZ (lane 3) or mutant 2 (lane 6) or mutant 117 (line 9). Lane 1, GILZ; lane 2, GST plus GILZ; lane 4, mutant 2; lane 5, GST plus mutant 2; lane 7, mutant 117; lane 8, GST plus mutant 117. (D) 3DO cells were transfected with the AP-1 luciferase reporter gene, along with GILZ, mutant 2, mutant 6, mutant 117, mutant 13, or GILZ plus activated Raf-1, and then stimulated for 18 h with plastic-bound anti-CD3 MAb. The values are expressed as the increase (n-fold) of luciferase activity compared to that in unstimulated cells. The values of transfected control groups are comparable; only one of them is shown in the figure. Each transfection was performed in triplicate. The standard errors were < 10%. (E) 3DO cells, transfected with the indicated vectors, were stimulated for 1 h with anti-CD3 MAb (1 μg/ml). Phosphorylation of ERK and MEK was visualized by Western blotting.
FIG. 8.
FIG. 8.
GILZ interferes with Ras-Raf-1 complex. (A) Activated Ras was expressed in COS-7 cells, and 100 μg of cellular extracts was incubated for 1 h with 2.5 μg of GST-Raf-RBD. The complex was purified by adsorption to glutathione-Sepharose beads, washed, and resuspended in PBS. Purified GILZ was added at the concentrations indicated. After 1 h at 4°C, the GST-Raf-RBD beads were washed and examined for associated proteins by Western blotting with anti-Ras, anti-GILZ, and anti-GST antibodies. N.T., nontransfected cells. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 1 h with plastic-bound anti-CD3 MAb. A total of 200 μg of cellular extracts was incubated for 1 h with 2.5 μg of GST-Raf-RBD. (B) Western blotting was performed with anti-Ras antibody. (C) The nitrocellulose membrane was then stripped and reprobed with anti-GILZ antibody.
FIG. 9.
FIG. 9.
DEX inhibits Raf-1, MEK, and ERK-1/2 phosphorylation. Mouse thymocytes left untreated or pretreated for 6 h with DEX (100 nM) were stimulated for 1 h with plastic-bound anti-CD3 MAb. Western blotting was performed with an anti-pERK-1/2 (A), anti-pMEK-1/2 (B), anti-pRaf-1 (C), or anti-GILZ (D) antibody. Western blotting was also performed with an anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibody to verify that no modulation of protein expression occurred or with β-tubulin to verify that equivalent amounts of proteins were loaded in all lanes.
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