Although oncogenic pathways frequently co-opt the translational machinery for their own benefit, they also paradoxically open tumor-suppressive mechanisms that can lead to their own demises. The present study shows that the RalA oncogenic pathway fits this mold, with a few novel twists. Like other oncogenic pathways, RalA activation hijacks the translational machinery. This translational co-option, however, is not the driving force behind the transformation process, but rather, by suppressing the translation of the extrinsic apoptotic pathway inhibitor FLIPS, is itself the key that opens a pathway to cell death.
The present work links the RalA pathway to control of the extrinsic apoptotic pathway activated by TRAIL. The mitogen-activated protein kinase pathway, but not the Ral or PI3K pathways, has been previously linked to the stabilization of myc, increased DR5 expression, increased caspase activation, and enhanced TRAIL-induced apoptosis in fibroblasts and HEK cells (29
). In the present study, there was no indication of RalA-induced DR5 up-regulation or myc stabilization (unpublished data), although RalA but not a Raf-selective T35S Ras effector mutant did increase caspase activation. These differences may be a consequence of different TRAIL exposure conditions used or alternatively may reflect tissue-specific differences in the control of the extrinsic apoptotic pathway and subtle differences in the balance between the multiple factors that set the apoptotic threshold. The finding that Ras, Raf, Ral, and myc all suppress TRAIL-induced caspase activation in multiple cell lines under multiple conditions (29
), however, suggests that the regulation of DISC function, including that by FLIP up-regulation, may be a common means by which oncogenic pathways regulate the extrinsic apoptotic pathway. While FLIPS
regulation can be accomplished transcriptionally, as has been previously reported for myc (37
), the ability of the Ral pathway to do so translationally represents a novel means of controlling the extrinsic cell death pathway.
The means by which the RalA pathway is linked to the translational machinery is novel, as is the manner in which this oncogenic pathway impacts translation. The present work shows that RalA is linked to the control of FLIPS
translation not via mTOR, the most commonly identified and extensively studied regulator of translation, but instead via a second GTPase, cdc42, and by the ability of cdc42 to regulate the activity of the mTOR target S6K1. Our observation that cdc42 activates S6K1 is consistent with previous reports that suggest that cdc42 coimmunoprecipitates with and activates S6K1 (7
). How a GTPase such as cdc42 activates S6K1, however, remains unclear, although previous studies using isoprenylation-deficient cdc42 mutants suggest that membrane targeting of cdc42 is critical for S6K1 activation (7
). Membrane localization of the cdc42-S6K complex may allow other membrane-associated proteins such as PDK1 to activate S6K1, although it is clear that at least part of the ability of cdc42 to activate S6K1 is distinct from that of PDK1 (26
). The observation that both RalBP1 (this study) and cdc42 (32
) function in a membrane-associated manner is consistent with the idea that RalBP1, cdc42, and S6K may be brought into intimate contact by RalA activation in the cell membrane, after which S6K1 activation can occur (15
The Ral-mediated control of translation noted in the present study is also unusual in that input from the RalA oncogenic pathway negatively impacts translation, at least with regard to FLIPS
mRNA. While the full range of Ral-mediated translational regulation remains to be determined, it seems unlikely that Ral pathway activation negatively regulates the translation of all mRNAs, particularly in light of recent studies showing that even complete disruption of PDK, an activator of Akt-mTOR signaling, suppresses the translation of only a subset of mRNAs and enhances the polysomal association of others (44
). Rather, the Ral-mediated suppression of S6K1 activity may suppress the translation of only a subset of structurally related mRNAs. If this is the case, the examination of the differential sensitivity of the closely related FLIPS
mRNAs to Ral-mediated translational regulation may prove informative. Alternatively, RalA may be linked to the translational apparatus by both an mTOR-independent pathway that suppresses the translation of FLIPS
and an mTOR-dependent pathway that does not regulate FLIPS
mRNA translation but does regulate the translation of other mRNAs critical for Ral-mediated transformation. It's worth noting that RalA appears to control not only FLIPS
translation but also FLIPS
stability, as RalA pathway activation significantly decreases the half-life of the FLIPS
protein in a manner that is reversible by the overexpression of S6K1 (unpublished observations). RalA may therefore have multiple inputs into translational and posttranslational regulation and multiple means of controlling the levels of proteins critical for the translation process.
It is finally worth noting that the present studies also suggest that mTOR contributes in a unique manner to the control of the extrinsic apoptotic pathway by enhancing FLIPS
localization to the DISC. Although mTOR has not been reported to be involved in protein trafficking or to interact with FLIPS
is extensively posttranslationally modified in ways that could potentially be altered by mTOR (49
). Furthermore, because mTOR plays a critical role in Akt-mediated enhancement of FLIPS
), the ability of mTOR to facilitate proper FLIPS
localization may ensure proper shutdown of the extrinsic apoptotic pathway in tumors with activated Akt pathways. The ability of the Ral pathway to bypass this shutdown and to reopen the extrinsic cell death pathway independently of mTOR and even in the face of high Akt levels (unpublished data) suggests that therapies designed to block mTOR and the oncogenic stimulation of translation should be compatible and perhaps synergistic with approaches designed to activate the extrinsic pathway opened by RalA. It may also be possible that part of the effect of mTOR inhibition itself is the unmasking of apoptotic pathways stimulated by RalA, particularly if the Ral pathway suppresses the expression/translation of a broad set of antiapoptotic proteins.
The present studies define several unique properties of the Ral oncogenic pathway. RalA activation is linked to the translational machinery in a novel, mTOR-independent, negative manner. Furthermore, unlike other oncogenic pathways, RalA uses this link not to drive transformation but to sensitize cells to cell death. While the exact means by which RalA can cooperate with other Ras effectors to bring about transformation remain to be fully defined, the present studies provide a basis for understanding the linkage between Ral pathway activation and translation and a mechanistic understanding of how the extrinsic apoptotic pathway could be manipulated for therapeutic benefit in the multiple malignancies in which Ras and RalA activation play key roles.