Stress granule formation is a fundamental component of the organismal response to environmental stress (e.g., oxidative conditions, heat, UV irradiation, hypoxia). In response to these stresses, translation of selective mRNAs is halted by their retention into stress granules, to facilitate cell survival (Anderson and Kedersha, 2006
). We propose that RSK2 facilitates stress granule assembly to repress translation and to enhance cell survival (). This model is based on our observation that RSK2 regulates sequestration of TIA-1, which is critical for stress granule formation (Gilks et al., 2004
). In addition RSK2 expression is required for the organization of PABP1, a stress granule marker, into the granules. Dispersal of RSK2 throughout the cytoplasm by cycloheximide is also consistent with RSK2 association with stress granules. Moreover, RSK2 has been found to be associated with polyribosomes (Angenstein et al., 1998
), and components of polyribosomes and stress granules have been proposed to exist in equilibrium with each other (Kedersha et al., 2005
). RSK2 regulation of stress granule formation is physiologically important because silencing RSK2 decreased cell survival through the apoptotic pathway in response to stress. Therefore, RSK2 sequestration into stress granules is an integral component for cell survival and not simply a mechanism to control RSK2 activity.
The NTKD of RSK2 is responsible for direct interaction with the PRD of TIA-1. This conclusion is based on the observations that RSK2 directly interacts with the TIA-1 PRD in an in vitro binding assay using recombinant proteins, and that an inactivating point mutation in the RSK2 NTKD inhibits its interaction with TIA-1. Furthermore, the isolated RSK2 N-terminal domain binds to TIA-1 in an in vitro binding assay and is sequestered into stress granules.
RSK2 may serve as a scaffold for stress granule assembly, because TIA-1 association with granules is blocked more efficiently by silencing RSK2 than by just inhibiting its kinase activity. Thus, the amount of RSK2, as well as its kinase activity, is important in stress granule assembly. Consistent with a scaffolding function for RSK2 is the observation that RSK2 is stably sequestered in stress granules. Moreover, a kinase-dead RSK2 mutant does not interact with TIA-1 and does not go to stress granules, suggesting that sequestration of RSK2 is regulated by NTKD catalytic activity. However, it is unlikely that the interaction between RSK2 and TIA-1 is regulated by direct RSK2 phosphorylation of TIA-1, as it does not contain RSK2 consensus phosphorylation sites. It is more probable that the interaction with TIA-1 is controlled by conformational differences between the active and inactive forms of the NTKD.
In response to mitogen treatment RSK2 is slowly released from stress granules and upon release is able to shuttle rapidly in and out of the nucleus. RSK2 does not contain a classical polybasic NLS and the mechanism of RSK2 translocation has not been previously examined. We have discovered that the sequence responsible for RSK2 nuclear-cytoplasmic shuttling is within the C-terminal domain. This domain contains the CTKD and the ERK1/2 docking site. It is unlikely that ERK1/2 is involved in RSK2 nuclear translocation because activated ERK1/2 is released from RSK (Roux et al., 2003
). Curiously, RSK2 nuclear accumulation is dependent on TIA-1, but it is the N-terminal domain that interacts with TIA-1, and the isolated N-terminal domain is not able to shuttle into the nucleus. These data are consistent with the idea that the C-terminal domain associates with a nucleo-cytoplasmic shuttling protein but that TIA-1 enhances RSK2 nuclear accumulation, by interacting with a nuclear binding partner. Evidence for this hypothesis is provided by the fact that the kinase-dead RSK2 translocates in response to mitogen but neither accumulates in the nucleus nor binds TIA-1. TIA-1 does not contain a classical NLS (Zhang et al., 2005
) and, therefore, it is likely that both RSK2 and TIA-1 piggyback into the nucleus in association with an NLS-containing protein. However, TIA-1 import can also occur independently of RSK2 because altering the levels of RSK2 does not alter TIA-1 nuclear accumulation. We also propose that the NTKD regulates shuttling of the C-terminal domain by controlling its interaction with an NLS-containing protein. This suggestion is supported by the observations that the kinase-dead RSK2 mutant does not translocate in stressed cells even though it is dispersed in the cytoplasm; whereas, the isolated C-terminal domain is able to shuttle. In summary, we conclude that the RSK2 C-terminal domain contains a nuclear-cytoplasmic shuttling sequence but that the NTKD controls translocation by regulating the binding of an NLS-containing protein and also determines nuclear accumulation via its interaction with TIA-1.
In the nuclei of MCF7 cells, cyclin D1 is a critical RSK2 target for regulation of proliferation. This reasoning is based on the following data: (1) silencing or inhibition of RSK2 reduces cyclin D1 levels and proliferation; (2) ectopic expression of cyclin D1 prevents the inhibition of proliferation resulting from knockdown of RSK2; and (3) forced nuclear localization of RSK2 increases cyclin D1 levels in the absence of activating any other signaling pathway. The CCND1 (cyclin D1 gene) promoter is regulated by the transcription factor, CREB, and RSK was earlier proposed to activate CREB. However, CREB does not appear to be a physiological substrate for RSK (Sapkota et al., 2007
; Wiggin et al., 2002
). In agreement with the literature, inhibition of RSK did not alter basal or the mitogen-induced increase in CREB phosphorylation (Fig. S3D
). Furthermore, NLS-RSK2 did not increase phosphorylation of CREB (Fig. S3E
). The transcription factor, c-fos, a RSK substrate is known to regulate the CCND1 promoter. However, although RSK phosphorylation does contribute to the stabilization of the c-fos protein, ERK1/2 phosphorylation is required for further stabilization and for c-fos activation (Murphy et al., 2002
). Importantly, NLS-RSK2, in the absence of active ERK1/2, is able to stimulate cyclin D1 levels. Therefore, it is unlikely that the RSK2-induced increase in cyclin D1 is mediated by c-fos. Thus, our data are the first to establish a specific function for RSK2 in connection with cyclin D1 regulation. We conclude that RSK2 is a pivotal regulatory factor linking the stress response to survival and ultimately to proliferation through its association with TIA-1.
We have found that RSK2 is important for cell survival in response to stress. We hypothesize that RSK2 protects cells through its control of stress granule formation. This conclusion is supported by our observations that silencing RSK2 decreases cell survival and stress granule formation. Stress granules represent an ancient mechanism in eukaryotes for the post-translational regulation of mRNA. For example, stress granule-like mRNA granules have been shown to form in the unicellular eukaryote, trypanosome, when in the intestinal tract of the insect vector or in starvation conditions in culture (Cassola et al., 2007
). A number of human viral pathogens inhibit or induce stress granule formation (McInerney et al., 2005
; White et al., 2007
; Smith et al., 2006
; Emara and Brinton, 2007
). The difference between how these viruses alter the host’s stress response is related to their particular replication requirements. Stress granules have also been shown to form in vivo
in hypoxic areas within tumors and are thought to contribute to the radioresistance of the tumor vasculature (Moeller et al., 2004
). Additionally, the persistence of stress granule formation in the hippocampal cornu ammonis 1 neurons is thought to prevent their recovery in in vivo
stroke models (DeGracia et al., 2007
). Stress granules have also been found in muscle biopsies of patients with sporadic inclusion body myositis, an inflammatory muscle disease, and not in the controls; but the causal relationship of these stress granules to the disease is unknown (Nakano et al., 2005
). There is also in vivo
evidence demonstrating that inhibition of stress granule formation decreases organism survival in response to stress (McEwen et al., 2005
). Thus there is substantial evidence for the physiological significance of stress granules and we provide the first evidence that RSK2 may play a fundamental role in regulating the response of an organism to stress.