The results described in this study indicate that GILZ binds Ras, inhibits downstream Ras-dependent signals, functions as a physiological brake on cell proliferation, and is required for the antiproliferative activity of GCs.
We have previously shown that GILZ binds to Raf and downmodulates ERK activation (39
). Here, we show that GILZ binds not only Raf but also Ras and that this binding depends on different molecular domains. In fact, while the first 60 aa of GILZ are responsible for binding to Raf at the N-terminal domain, binding to Ras is mediated via the TSC, which by itself is necessary and sufficient for binding. In addition, we found that Ras, Raf, and GILZ interact to form a ternary complex, the formation of which depends on the activation state of Ras. This suggests that GILZ is particularly important in activated cells. However, the formation of the trimeric complex does not preclude the formation of dimeric GILZ-Ras and GILZ-Raf complexes from occurring. As Ras activation levels regulate Ras-GILZ and Ras-Raf binding, it is reasonable to propose that a trimeric conformation may coexist with dimeric Ras-GILZ and Raf-GILZ conformations. When Ras is not activated, GILZ mainly binds Raf. However, the activation of Ras leads to both a greater affinity of GILZ for Ras and an increase in the affinity of Raf for Ras that is greater than the affinity of Raf for GILZ. In addition, more GILZ was found in the trimeric conformation when Ras was less activated, which suggests that, for trimeric conformation, Raf-GILZ binding is more important than Ras-GILZ binding. Importantly, GILZ interactions with Ras and Raf are physiological, as they are detectable in primary spleen T lymphocytes and thymocytes and their presence suggests that GILZ has a regulatory role on Ras activity.
As a consequence of binding to Ras and Raf, GILZ inhibits downstream signals such as ERK, AKT, and Rb phosphorylation as well as cyclin D1 expression, which are important mediators of cell proliferation. In fact, we found that stable GILZ transfection of NIH-3T3 cells induces a significant decrease in cell number and [3
H]-thymidine uptake as well as an enrichment of the G0
phase of the cell cycle, which correlates with inhibition of ERK, AKT, and Rb phosphorylation and cyclin D1 expression. In particular, GILZ binding to Ras is necessary for GILZ antiproliferative activity, as GILZΔTSC did not bind to Ras and did not inhibit Ras-triggered cell proliferation, while GILZ-TSC-LZ, which binds Ras, inhibited Ras-driven proliferation. This suggests that, by binding Ras, GILZ blocks the propagation of signaling to Raf and AKT, which are both involved in mediating Ras-triggered cell proliferation (12
). In fact, the inhibitory effect of GILZ can be overcome by either activated AKT or activated Raf.
Interestingly, GILZ inhibited Ras- and Raf-induced cell proliferation, thus suggesting that the antiproliferative effect may be mediated by trimeric Ras-Raf-GILZ or dimeric Ras-GILZ and Raf-GILZ conformations. Raf-ERK and PI3K-AKT are considered the 2 major pathways modulating cyclin D1 transcriptional activation (17
), wherein ERK, but not AKT, is considered important for Raf-regulated cell signaling and both ERK and AKT are considered important for Ras-regulated cell signaling (11
). Because GILZ inhibits AKT and ERK activation and affects Ras- and Raf-dependent cell proliferation, it is conceivable that GILZ may target Ras-dependent AKT and ERK activation as well as Raf-dependent ERK activation. Thus, GILZ can bind directly to Raf and inhibit ERK but not AKT, and this is sufficient to inhibit the cell proliferation. On the other hand, the binding of GILZ to Ras, by an allosteric effect that has a negative impact on the activation of ERK and AKT (generally thought to be indispensable for mediating the transforming potential of Ras [ref. 45
]), inhibits both pathways, which cooperate in inducing cell proliferation (11
A notable result here is that Ras-GILZ interaction affects cell transformation. The effect of GILZ on the Ras-induced transformation of NIH-3T3, together with the signaling regulation, supports previous reports that Ras transformation requires multiple downstream pathways including ERK (43
) and AKT (47
). The inhibitory effect of GILZ on Ras-mediated transformation suggests that GILZ could contribute to modulation of Ras oncogenic activity and warrants further investigation as to the possible structural and/or functional alteration of GILZ in human tumors.
The antiproliferative action of GILZ suggests that it plays a physiological role in the control of cell growth and that its upregulation may be a mechanism of pharmacological importance. Endogenous GCs, the end-effectors of the hypothalamus-pituitary-adrenal axis, play an important role in regulating the homeostasis of the immune system (48
). Moreover, GCs are used as pharmacological agents due to their immunosuppressive and antiinflammatory properties. Their activity is due to their complex action on innate and adaptive immunity reflecting GC capacity to inhibit T lymphocyte activation (49
). Previous studies demonstrate that GCs inhibit T cell proliferation (5
). In particular, GCs have been shown to block antigen and mitogen-induced T lymphocyte proliferation, and various molecular mechanisms have been suggested as being responsible for this effect (5
). Our results show that GILZ inhibition by siRNA results in increased cell proliferation consequent to ConA stimulation, which suggests that GILZ plays a role in regulating T cell activation and growth. Most notable, silencing GILZ in the 3DO T cell line completely inhibited the DEX-induced antiproliferative effect. These results indicate that GILZ has a physiological role in cell proliferation machinery and is required for the pharmacological action of GCs.