Elsevier

Cellular Signalling

Volume 22, Issue 4, April 2010, Pages 668-675
Cellular Signalling

RhoA/ROCK1 signaling regulates stress granule formation and apoptosis

https://doi.org/10.1016/j.cellsig.2009.12.001Get rights and content

Abstract

Cells form stress granules (SGs), in response to unfavorable environments, to avoid apoptosis, but it is unclear whether and how SG formation and cellular apoptosis are coordinately regulated. In this study we detected the small GTPase, Ras homolog gene family member A (RhoA), and its downstream kinase, Rho-associated, coiled-coil containing protein kinase 1 (ROCK1), in SG, and found that their stress-induced activities were important for SG formation and subsequent global translational repression. Importantly, only activated RhoA and ROCK1 were sequestered into SG. Sequestration of activated ROCK1 into SG prevented ROCK1 from interacting with JNK-interacting protein 3 (JIP-3) and its activation of c-Jun N-terminal kinase (JNK), a pathway triggering apoptosis, thereby protecting cells from apoptosis. This study identifies a specific signaling pathway, mediated by RhoA and ROCK1, which determines cell fate by promoting SG formation or initiating apoptosis during stress.

Introduction

Stress granules (SGs) are subcellular structures that are formed in cells responding to various environmental stresses, such as high temperature, heavy metal poisoning or nutrient starvation [1], [2], [3], [4], [5]. It has been suggested that SG functions to protect housekeeping mRNAs and repress their translation by recruiting these mRNAs and certain mRNA-associated proteins. Through this mechanism, cells could also conserve energy and increase the availability of translation machinery for stress-related gene expression. The heat shock protein 70 transcript, for example, is excluded from SG and is actively translated under stress [3]. Recently, studies have shown that SG can protect cells from apoptosis, because actively disrupting SG would result in the release of apoptosis-inducing components from SG to trigger cell death [6], [7]. However, it is still unclear whether and how the decision to form SG or induce apoptosis, under stress, can be regulated by any specific signaling pathway.

RhoA, along with Rac1 and Cdc42, is the most studied Rho GTPases that regulate a wide variety of cellular processes, such as vesicle trafficking, cell-cycle progression and cytoskeleton rearrangement [8], [9]. The regulation of RhoA activity is mediated by the GDP/GTP activation cycle. Upon activation, GTP-bound RhoA signal is transmitted to many downstream effectors. Among these signal effectors, Rho-associated coiled-coil forming kinase (ROCK) is the most widely examined. The ROCK family, including ROCK1 and ROCK2, comprises serine/threonine kinases that phosphorylate various downstream substrates. ROCK contains a Rho-binding domain (RBD) which, upon binding by RhoA, induces a conformational change at the carboxyl terminus and the activation of the kinase domain leading to actin filament stabilization and increased myosin ATPase activity [10], [11]. While it is generally believed that ROCK is important for cytoskeletal reorganization, it is unclear whether, under stress condition, ROCK plays a regulatory role in SG formation, which would require cytoskeleton and motor complex rearrangement [12], [13], [14], [15], [16]. In fact, ROCK1 can phosphorylate JIP-3 and activate JNK, which in turn would cause cytochrome C release and subsequent caspase-3 activation, thereby mediating UV irradiation-triggered apoptosis [14], [15], [17]. It is puzzling how ROCK1 can actively participate in events triggering stress response to determine cellular survival.

We have previously identified several critical SG components regulating SG dynamics, including a RNA-binding protein, growth factor receptor-bound protein 7 (Grb7) and dynein motor light chain (DLC2A) [16], [18]. In the current study, we found an additional regulatory signaling pathway, the RhoA/ROCK1 pathway that is important in cellular decision, to either form SG (survival) or trigger apoptosis (death). We found that under stress, RhoA and ROCK1 were activated, and both their activities were required for efficient SG formation. Further, both activated RhoA and ROCK1 were sequestered into SG. Forced disruption of SG formation resulted in the dissociation of RhoA and ROCK1 from other SG components. The released and activated ROCK1 would then interact with, and phosphorylate, JIP-3, thereby triggering apoptosis. This apoptotic pathway is inhibited in cells containing SG. This study provides the first example for a stress-initiated signaling pathway that determines cell fate by either promoting SG formation or triggering apoptosis.

Section snippets

Antibodies, reagents and plasmid constructions

The antibodies were from Santa Cruz Biotechnology (anti-Actin, anti-ROCK1, anti-ROCK2, anti-Cdc42, anti-Rac1, anti-RhoA, anti-JIP-3, anti-Dcp1a, anti-PABP, anti-HuR and anti-TIA-1), Upstate (anti-GST and anti-Phospho-Serine), Chemicon (anti-FMRP) and Cell Signaling (anti-JNK, anti-phospho-JNK). DAPI and AMP-PNP was from Roche. Annexin V apoptosis kit, EHNA, Y-27632, protease K, cycloheximide and puromycin are from Sigma Aldrich. C3 exotoxin was from BIOMOL International. RhoA activation kit and

RhoA and its downstream kinase, ROCK1, are both present in SG

The most widely studied small GTPases, RhoA, Rac1 and Cdc42, are known to modulate cytoskeleton rearrangement in cells under stress. To first determine if these signaling molecules were physically associated with SG, we used a heat shock-triggered stress model of mouse embryonal carcinoma P19 cells [16], [18], to examine their cellular localization. We found that in the stressed P19 cytoplasm, the endogenous RhoA, but neither Rac1 nor Cdc42, was mostly localized in distinct foci overlapping

Discussion

In this study, by using the heat shock-induced cellular stress model, we show that stress-activated RhoA and ROCK1 are important for SG formation in stressed cells. Active RhoA and ROCK1 are both sequestered into SG, which prevents their interaction with JIP-3, a critical component for JNK-mediated apoptosis. This is the first study identifying a signaling pathway that is able to trigger both SG formation and apoptosis (Fig. 5C). The fate for cells to survive or die is determined, at least

Acknowledgement

This work is supported in part by NIH Grants DA11190, DA11806, DK54733, DK60521 and K02-DA13926.

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