The Ral GTPases are mainly known for their positive contribution to Ras-driven oncogenesis. In particular, it has been reported that RalA supports anchorage independent growth and cell transformation downstream of oncogenic Ras. However, one report describes RalA also as suppressor of early stages of Ras-induced carcinoma progression, proposing that RalA might also have tumor suppressive activities in specific settings, such as stress signaling. In tumorigenesis, regulation of Stress-Activated map Kinase (SAPK) pathways may determine cell survival or death in response to tumor environmental cues. While RalA regulates TNF-α signaling by contributing to SAPK activation, the role of Ral GTPases in response to other tumor environment driven stresses is largely unknown. Here, we describe the serine/threonine protein kinase NDR1 as a new partner of RalA signaling in control of SAPK. We report that under osmotic and oxidative stresses the Ste20-like MAP4K4 kinase, an effector of RalA via the Exocyst complex, directly phosphorylates NDR1 on Thr444, a key regulatory residue for NDR1 activation. Moreover, we found that apoptosis induction triggered by TNF-α cytokine treatment or RASSF1A over expression signals through the RalA-MAP4K4-NDR1 pathway. This novel and unexpected pro-apoptotic role of RalA suggests that the RalA GTPase can positively signal in tumor suppressor pathways, in addition to its proto-oncogenic role downstream of Ras.
The Ral GTPases are key actors in Ras-dependent oncogenesis . The mammalian RalA and RalB GTPases play roles on different levels of tumorigenesis ranging from basic hallmarks of cancer, such as anchorage independent growth, to invasion and metastasis formation . Ral GTPases belong to the Ras superfamily of small GTPases . They are activated by Guanosine Exchange Factors (Ral GEFs), and are direct effectors of Ras in human tumorigenesis [4-7]. RalA functions in human cell transformation [4,8,9], while RalB promotes tumor cell survival and regulates cell motility [10,11]. More specifically, RalA loss-of-function inhibits anchorage independent growth of transformed human cells, and RalB knockdown causes tumor cell apoptosis and blocks cancer cell motility . The specificity of the distinct biological functions of RalA and RalB has remained elusive, although different post-translational modifications of Ral which regulate Ral subcellular localization and activity are likely involved to separate functions [8,12-15]. However, this functional specificity of RalA and RalB is lost in mice, since knockout of both Ral GTPases is necessary to inhibit Ras-induced tumorigenesis , suggesting that RalA and RalB have redundant functions in this model system. Moreover, a recent report describes RalA as a suppressor of early stages of Ras-induced carcinoma progression , indicating that RalA might have tumor suppressive functions besides its reported oncogenic role downstream of Ras.
Downstream effectors of Ral GTPases include components of the Exocyst complex, Sec5 and Exo84, which support Ral functions in tumorigenesis . It has been reported that the RalB-Sec5 complex activates TBK1 to promote tumor cell survival , and the RalB-Exo84 complex functions in autophagy . However, the RalA-Exocyst signaling pathway remains poorly understood in cancer cells . In this context of RalA-Exocyst signaling, the relationship of RalA to MAP4K4 is also yet to be explored in much more detail. MAP4K4 is a serine/threonine protein kinase of the GCK-IV family [21,22], and has been reported to support cellular transformation, tumor cell adhesion and invasion [23-25]. MAP4K4 is characterized by a unique C-terminal Citron Homology (CNH) domain , which is important for homodimerization and protein-protein interaction with Sec5, MEKK1 and beta1-integrin [10,26-28]. MAP4K4 has been defined as a Stress-Activated MAP Kinase (SAPK), driving the activation of MAPK-JNK cascades [22,29,30]. In Drosophila Msn, the fly counterpart of MAP4K4 , is part of a Ral-Sec5-Msn-JNK cascade regulating developmental-induced apoptosis . However, the role of MAP4K4 as signaling partner of RalA and/or RalB has not been defined yet in human cells.
RalA mediates TNF-α-induced p38 MAPK activation and JNK-dependent activation of FOXO [26,31]. Moreover, the SAPK cascade can influence the fate of cancer cells in response to environmental and cellular stresses . Together these findings suggested that RalA might regulate SAPK activation and stress responses. Here, we addressed these possible connections between RalA and MAP4K4 experimentally. More specifically, our study expands our understanding of how RalA signaling pathways mediate SAPK activation and determines the role of RalA in apoptosis induced responses to different types of environmental stresses. We identified MAP4K4 as a new upstream kinase of NDR1 (also known as STK38), an AGC serine/threonine protein kinase [33,34] that can be activated by Fas, TNF-α, osmotic and oxidative stresses [35-37]. Moreover, we demonstrate that RalA regulates NDR1 activation, via Exocyst and MAP4K4 signaling, to trigger apoptosis in response of extracellular stresses. Apoptosis triggered by overexpression of the tumor suppressor protein RASSF1A was also regulated by the RalA-Exocyst-MAP4K4-NDR1 pathway.
Deconvolution of the means and pathways used by Ras oncogenes has largely been a result of the disentangling of the Ras interactome into three main branches: the Raf-MEK-ERK kinase cascade, the PI3K-AKT kinase pathway, and equally important the Ral-dependent branch . Within the Ral-dependent branch human alA and RalB are essential and in some context instructive for oncogenic transformation . In regard of the oncogenic aspect, RalA and RalB play different biochemical and biological roles, although both, RalA and RalB, utilize the same signal transduction platform, namely the Exocyst complex . Our understanding of RalB signaling and functions has steadily progressed by discoveries showing that RalB plays a role in the capacity of cancer cells to dodge apoptosis , and by elucidating RalB’s permissive and instructive role in autophagy . In contrast, RalA beyond the Exocyst complex has found less solid grounds to justify its apparently contradictory functions in cellular transformation . On the one hand, current evidence suggests that RalA is needed for tumorigenesis by playing a positive role in anchorage independent growth of cancer cells [4,8,9] and by also regulating the turnover of cadherins at their physiological plasma membrane localization . On the other hand, RalA paradoxically can display tumor suppressive properties in squamous cell carcinoma progression .
iming at establishing a comprehensive RalA interactome, we undertook a systematic Y2H approach through which we uncovered and validated a novel RalA-Exocyst-MAP4K4-NDR1 signal transduction pathway that is necessary for efficient stress and apoptosis signaling in our settings (Figure 7). This discovery will not only fuel future studies of RalA-related cell biology, but also reveals an overlap between the RalA pathway and NDR-Hippo signaling, in which NDR1 functions as central player . Our study also sheds light on a new signaling wiring upstream of the NDR1 kinase (Figure 7). More specifically, in addition to being regulated by the MST1/2 Hippo kinases [36,40] and MST3 [52,59], we show here that the NDR1 kinase can also be regulated by other members of the Ste20-like kinase family, namely the MAP4K4 kinase in our settings. This observation revealed that a Ste20-like kinase outside of the GCKII and GCKIII subfamilies of Ste20-like kinases is required for the activation of NDR1 by hydrophobic motif phosphorylation. This suggests an additional level of redundancy for NDR1 activation to ensure a proper regulation of the tumor suppressive NDR1 kinase . Perhaps, given that the regulation of NDR1 by MST1 is dependent on the co-activator MOB1 [33,61], the regulation of NDR1 by MAP4K4 represents an hMOB1-independent level. Therefore, future research into the regulation of the RalA-Exocyst-MAP4K4-NDR1 pathway by the tumor suppressor MOB1 is warranted.
Figure 7: Model of RalA-Exocyst-MAP4K4-NDR1 stress/apoptotic signaling.
Based on our findings we are tempted to propose a model illustrating how RalA and NDR1 can function together in stress/apoptotic signaling. Upon activation of RalA by stress (e.g. osmotic or oxidative stress) and/or apoptotic stimuli, RalA supports the activation of MAP4K4 facilitated by the Exocyst complex. Once activated MAP4K4 phosphorylates and thereby activates NDR1 kinase, which subsequently modulates p38 signaling and the response to stress and/or apoptotic stimuli. Notably, our model does not exclude the possibility that each component can also be regulated by additional factors at each individual signaling level.
Our work shows further that the RalA-Exocyst-MAP4K4-NDR1 cascade can be activated by different stress stimuli, such as TNF-? which triggers apoptotic signaling or sorbitol which mimics high osmotic stress. Therefore, the RalA-Exocyst-MAP4K4-NDR1 pathway is likely to play a role in a broader stress signaling spectrum. Particularly, the pro-apoptotic role of NDR1 downstream of RalA-MAP4K4 signaling could be of specific interest in selected cancer settings. In this context, the pro-apoptotic role of NDR1 downstream of RalA-MAP4K4 signaling is in full agreement with the previously reported pro-apoptotic function of NDR1 in the prevention of T-cell lymphoma formation . Therefore, future clinical and animal studies addressing the pro-apoptotic role of RalA-MAP4K4-NDR1 signaling in complex multicellular contexts are warranted.
Given that we also observed that the RalA-MAP4K4-NDR1 axis is required for apoptotic signaling upon RASSF1A overexpression, this pro-apoptotic role of RalA highlights a potential paradoxical role of RalA in tumor suppression vs. tumor promotion. In this regard, it is worth mentioning that it has been already documented that at early stages of cancer progression RalA suppression is required for Ras-dependent invasive properties of squamous carcinoma cells associated with E-cadherin loss . Therefore, current evidence supports paradoxically opposite roles for RalA in tumor progression and suppression, which appear to be contradictory. On the one hand, RalA supports cellular transformation downstream of Ras [4,8,9]. On the other hand, our work reported herein suggests that RalA might also function in tumor suppressor signaling by promoting cancer cell apoptosis. Our findings further propose that the RalA GTPase could control tumor environment-induced cell death, while RalB plays a role in the cell autonomous survival of cancer cells [10,11]. Therefore, future research addressing this paradox is warranted to elucidate the mechanistic switch dictating whether RalA should support tumor promotion or tumor suppression. Nevertheless, our work provides the first molecular lead into how RalA could execute a role in tumor suppression. Based on our findings it is tempting to speculate that at first RalA would support stress-related signaling pathways, which in case of excessive stress would interfere with tumor growth. Thus, our work opens new perspectives into the contribution of RalA to tumor environment stress sensing.
We thank G. Scita (IEO, Milano, Italy) for providing reagents and all Hybrigenics staff for Y2H analysis. This work was supported by a GenHomme Network Grant (02490-6088) to Hybrigenics and Institut Curie, and by Fondation ARC pour la Recherche sur le Cancer , by Ligue National contre le Cancer to MCP (RS14/75-54), and association Christelle Bouillot RS was supported by ARC; AB and CJ were supported by Ligue Nationale contre le cancer, and CJ also by INCA, respectively. AH is a Wellcome Trust Research Career Development fellow (090090/Z/09/Z) at the UCL Cancer Institute. The work was also supported by the grant ANR-08-NLAN-0290-01 to J.C.
Citation: Mycielska ME, Wachsmuth CJ, Dettmer K, Wagner C, Schlitt HJ, et al. (2014) Hsp90 Inhibition Affects Cell Metabolism by Disrupting Mitochondrial Protein Insertion. J Cell Biol Cell Metab 1: 001.
Copyright: © 2014 Rasim Selimoglu, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.