The data presented here indicate that GILZ, by binding to Raf-1, inhibits the MAPK pathway and suggest a new mechanism for GCH transcriptional control of this signaling circuitry.
T-cell activation is characterized by the release of a series of lymphokines that regulate apoptosis, T-cell proliferation, and clonal expansion (16
). GCH inhibition of T-cell activation and the consequent immunosuppression are achieved through a combination of genomic and nongenomic mechanisms and also through interference with the MAPK pathway (1
). In fact, interaction between GR and MAPK pathway components has been evoked to explain impaired IL-2 gene transcription and the consequent inhibition of cell activation and proliferation in different cells and physiologic settings. For example, recent evidence suggests that Raf-1, 14-3-3, and GR coimmunoprecipitate within the same protein complex in the cytoplasm of rat liver cells, and this provides a plausible explanation of the GCH effect on the Ras-Raf-1 signaling pathway (62
). Moreover, other studies with mast cells suggest that DEX inhibits activation of Raf-1, MEK, and ERK but does not affect Ras activation (53
). This effect is due to disruption of the Raf-1 multimeric complex without affecting proteins, such as 14-3-3, in this complex. According to this model, the loss of DEX-induced Hsp90 from the Raf-1 complex correlates with inhibition of antigen-stimulated Raf-1 activity and suggests that a loss of Hsp90 and the impairment of Raf activation are linked (8
). In addition, GCH increase the expression of MAPK phosphatase 1, and this effect is necessary for GCH-mediated inhibition of ERK-1/2 activation (33
). Finally, GR directly binds AP-1, thus inhibiting one of the final steps in the MAPK cascade (56
In the present study, we demonstrated that an indirect mechanism involving GILZ is responsible for GCH-mediated inhibition of the MAPK pathway, which could then contribute to inhibit T-cell activation. GILZ overexpression in 3DO clones suppressed the Raf pathway and, consequently, interfered with the c-Fos transcription. A direct protein-to-protein interaction, occurring both in vitro and in vivo (Fig. , 5, and 7), may be responsible for this inhibitory mechanism. In fact, GILZ fusion protein bound Raf, but not MEK and ERK, in cellular lysates from murine thymocytes and antibody anti-Raf coimmunoprecipitated GILZ, especially in DEX-treated mouse thymocytes (Fig. ).
Based on several protein motifs, it has been suggested that GILZ belongs to a TSC family (35
) of leucine zipper proteins comprising at least five other members (TSC-22, THG-1, KIAA0669, DIP, and shc), which can potentially homodimerize or heterodimerize by means of its leucine zipper pattern. All members of the TSC family share a high degree of homology in the dimerization domain (TSC-22 box and leucine zipper pattern) but have different N-terminal and C-terminal domains. Experimental evidence supports the hypothesis that TSC-22 and THG repressor activity resides in the unconserved N- and C-terminal domains (35
). Although GILZ shares a proline-rich region in the C-terminal domain with TSC-22 and THG-1, these domains may have totally different functions. Molecular modeling analysis (Fig. ) and experimental evidence suggested that GILZ interacts through its NH2
-terminal domain with the NH2
-terminal portion of Raf corresponding to the Ras-binding side. In fact, the GST-Raf-RBD fusion protein was able to bind GILZ in vitro (Fig. ) and in vivo in murine thymocytes (Fig. ). Deletion of the GILZ N-terminal region, but not of the proline-rich region, resulted in complete abrogation of binding to Raf-RBD and inhibition of AP-1 transcriptional activity (Fig. ). In contrast, the homodimerization state of GILZ was not required to bind Raf-1 (Fig. ).
Raf is known to bind directly to the GTP-bound form of Ras (13
). This Raf-Ras-GTP interaction does not, however, result directly in Raf activation. In fact, the Raf-Ras interaction is required for Raf recruitment and translocation from cytosol to plasma membrane (46
). Furthermore, Raf-1 bound in the cytosol to 14-3-3 protein, an arachidonate-selective acyltransferase and putative phospholipase A2. This protein associates with a number of key signaling proteins and cell cycle regulators; its role in MAPK activation is permissive for Raf-1 recruitment and activation, even though it is totally displaced when Ras recruits Raf to the plasma membrane (39
). Furthermore, Raf-1 kinase activity and function are regulated both positively and negatively by phosphorylation and dephosphorylation signals (7
). If Raf binds GILZ by using the same portion of the molecule that interacts with activated Ras, this raises the question of competition between GILZ and Ras for Raf binding. In fact, one hypothesis might be that GILZ binding Raf creates a steric obstacle for the Ras-Raf association and the consequent activation and recruitment of Raf in the cell membrane that could explain its diminished phosphorylation. Our data suggest that a steric interference of GILZ with Raf-Ras binding is possible (Fig. ). However, we cannot rule out the possibility that GILZ, rather than acting as a direct competitor for Raf-Ras binding, reduces the affinity of Raf for Ras and again induces an impaired Raf translocation to the cell membrane and consequently diminishes phosphorylation.
Whatever mechanism is triggered by the GILZ-Raf interaction, it is important to note that it inhibits Raf, MEK, and ERK activation and consequently impairs AP-1 activation.
Another mechanism has been proposed to account for the GILZ-induced defect in AP-1 activation. The in vitro interaction of recombinant GILZ with c-Fos and c-Jun inhibits AP-1 binding to its target DNA (42
). Although we obtained the same results (not shown), we demonstrate here that this was not the only mechanism of GILZ-induced AP-1 inhibition. The coexistence of multiple mechanisms is common to many biological systems where more than one molecular event can control any one pathway. In the case of GILZ, the redundancy may be due to the need to block the Raf-ERK cascade in order to inhibit not only c-Fos transcription but also the activity of other transcription factors controlled by this pathway. Were this the case, GILZ, as mediator of GCH immunosuppressive and anti-inflammatory activities, would have a larger spectrum of action.
The lack of IL-2 production, which was due, at least in part, to the block of MAPK driven by GILZ overexpression, resembles the state of functional unresponsiveness with impaired IL-2 production and Ras activation, a characteristic of anergic T cells (32
). Anergy, one of the responses of the immunity system to continuous antigen challenge, is also one of the T-cell responses to exogenous GCH (10
). Therefore, the observation that GILZ triggers the same molecular events caused by GCH and/or repeated exposure to the antigen once again confirms the functional cross talk between TCRs and GRs at various levels of signaling events (30
). Moreover, GILZ-mediated inhibition of the Raf-1 pathway suggests yet another mechanism accounting for the anti-inflammatory and immunosuppressive effects of GCH.