Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

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.

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.

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).

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.

(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.

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.

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.