Mechanism of multi-site phosphorylation from a ROCK-I:RhoE complex structure

The ROCK-I serine/threonine protein kinase mediates the effects of RhoA to promote the formation of actin stress fibres and integrin-based focal adhesions. ROCK-I phosphorylates the unconventional G-protein RhoE on multiple N- and C-terminal sites. These phosphorylation events stabilise RhoE, which functions to antagonise RhoA-induced stress fibre assembly. Here, we provide a molecular explanation for multi-site phosphorylation of RhoE from the crystal structure of RhoE in complex with the ROCK-I kinase domain. RhoE interacts with the C-lobe αG helix of ROCK-I by means of a novel binding site remote from its effector region, positioning its N and C termini proximal to the ROCK-I catalytic site. Disruption of the ROCK-I:RhoE interface abolishes RhoE phosphorylation, but has no effect on the ability of RhoE to disassemble stress fibres. In contrast, mutation of the RhoE effector region attenuates RhoE-mediated disruption of the actin cytoskeleton, indicating that RhoE exerts its inhibitory effects on ROCK-I through protein(s) binding to its effector region. We propose that ROCK-I phosphorylation of RhoE forms part of a feedback loop to regulate RhoA signalling.     

The Rho Family Member RhoE Interacts with Skp2 and Is Degraded at the Proteasome during Cell Cycle Progression

RhoE/Rnd3 is an atypical member of the Rho family of small GTPases. In addition to regulating actin cytoskeleton dynamics, RhoE is involved in the regulation of cell proliferation, survival, and metastasis. We examined RhoE expression levels during cell cycle and investigated mechanisms controlling them. We show that RhoE accumulates during G₁, in contact-inhibited cells, and when the Akt pathway is inhibited. Conversely, RhoE levels rapidly decrease at the G₁/S transition and remain low for most of the cell cycle. We also show that the half-life of RhoE is shorter than that of other Rho proteins and that its expression levels are regulated by proteasomal degradation. The expression patterns of RhoE overlap with that of the cell cycle inhibitor p27. Consistently with an involvement of RhoE in cell cycle regulation, RhoE and p27 levels decrease after overexpression of the F-box protein Skp2. We have identified a region between amino acids 231 and 240 of RhoE as the Skp2-interacting domain and Lys²³⁵ as the substrate for ubiquitylation. Based on our results, we propose a mechanism according to which proteasomal degradation of RhoE by Skp2 regulates its protein levels to control cellular proliferation.     

Neuronal polarization is impaired in mice lacking RhoE expression

Proper development of neuronal networks relies on the polarization of the neurons, thus the establishment of two compartments, axons and dendrites, whose formation depends on cytoskeletal rearrangements. Rnd proteins are regulators of actin organization and they are important players in several aspects of brain development as neurite formation, axon guidance and neuron migration. We have recently demonstrated that mice lacking RhoE/Rnd3 expression die shortly after birth and have neuromotor impairment and neuromuscular alterations, indicating an abnormal development of the nervous system. In this study, we have further investigated the specific role played by RhoE in several aspects of neuronal development by using hippocampal neuron cultures. Our findings show that neurons from a mice lacking RhoE expression exhibit a decrease in the number and the total length of the neurites. We also show that RhoE-deficient neurons display a reduction in axon outgrowth and a delay in the process of neuronal polarization. In addition, our results suggest an involvement of the RHOA/ROCK/LIMK/COFILIN signaling pathway in the neuronal alterations induced by the lack of RhoE. These findings support our previous report revealing the important role of RhoE in the normal development of the nervous system and may provide novel therapeutic targets in neurodegenerative disorders.     

RhoE participates in the stimulation of the inflammatory response induced by ethanol in astrocytes

Astroglial cells are involved in the neuropathogenesis of several inflammatory diseases of the brain, where the activation of inflammatory mediators and cytokines plays an important role. We have previously demonstrated that ethanol up-regulates inflammatory mediators in both brain and astroglial cells. Since Rho GTPases are involved in inflammatory responses of astrocytes where loss of stress fibers takes place and RhoE/Rnd3 disorganizes the actin cytoskeleton, the aim of the present study was to investigate the implication of this protein in the stimulation of inflammatory signaling induced by ethanol. Our findings show that RhoE expression induces a decrease in both RhoA and Rac. In addition, RhoE not only induces actin cytoskeleton disorganization but it also stimulates both the IRAK/ERK/NF-kappaB pathway and the COX-2 expression associated with the inflammatory response in these cells. Our results also show that ethanol exposure induces RhoE signaling in astrocytes. Preincubation of astrocytes with GF109203X, an inhibitor of PKCs, reduces the RhoE levels and abolishes the ethanol-induced activation of IRAK, NF-kappaB and the COX-2 expression. Furthermore, RhoE overexpression restores ethanol responses in astrocytes treated with the PKCs inhibitor. Altogether, our findings suggest that this small GTPase is involved in the stimulation of the inflammatory signaling induced by ethanol in astrocytes. These findings provide new insights into the molecular mechanism involved in the inflammatory responses in astrocytes.     

RhoE interferes with Rb inactivation and regulates the proliferation and survival of the U87 human glioblastoma cell line

Rho GTPases are important regulators of actin cytoskeleton, but they are also involved in cell proliferation, transformation and oncogenesis. One of this proteins, RhoE, inhibits cell proliferation, however the mechanism that regulates this effect remains poorly understood. Therefore, we undertook the present study to determine the role of RhoE in the regulation of cell proliferation. For this purpose we generated an adenovirus system to overexpress RhoE in U87 glioblastoma cells. Our results show that RhoE disrupts actin cytoskeleton organization and inhibits U87 glioblastoma cell proliferation. Importantly, RhoE expressing cells show a reduction in Rb phosphorylation and in cyclin D1 expression. Furthermore, RhoE inhibits ERK activation following serum stimulation of quiescent cells. Based in these findings, we propose that RhoE inhibits ERK activation, thereby decreasing cyclin D1 expression and leading to a reduction in Rb inactivation, and that this mechanism is involved in the RhoE-induced cell growth inhibition. Moreover, we also demonstrate that RhoE induces apoptosis in U87 cells and also in colon carcinoma and melanoma cells. These results indicate that RhoE plays an important role in the regulation of cell proliferation and survival, and suggest that this protein may be considered as an oncosupressor since it is capable to induce apoptosis in several tumor cell lines.