The protective role of a small GTPase RhoE against UVB-induced DNA damage in keratinocytes

RhoE, a p53 target gene, was identified as a critical factor for the survival of human keratinocytes in response to UVB. The Rho family of GTPases regulates many aspects of cellular behavior through alterations to the actin cytoskeleton, acting as molecular switches cycling between the active, GTP-bound and the inactive, GDP-bound conformations. Unlike typical Rho family proteins, RhoE (also known as Rnd3) is GTPase-deficient and thus expected to be constitutively active. In this study, we investigated the response of cultured human keratinocyte cells to UVB irradiation. RhoE protein levels increase upon exposure to UVB, and ablation of RhoE induction through small interfering RNA resulted in a significant increase in apoptosis and a reduction in the levels of the pro-survival targets p21, Cox-2, and cyclin D1, as well as an increase of reactive oxygen species levels when compared with control cells. These data indicate that RhoE is a pro-survival factor acting upstream of p38, JNK, p21, and cyclin D1. HaCat cells expressing small interfering RNA to p53 indicate that RhoE functions independently of its known associates, p53 and Rho-associated kinase I (ROCK I). Targeted expression of RhoE in epidermis using skin-specific transgenic mouse model resulted in a significant reduction in the number of apoptotic cells following UVB irradiation. Thus, RhoE induction counteracts UVB-induced apoptosis and may serve as a novel target for the prevention of UVB-induced photodamage regardless of p53 status.     

Rnd proteins: Multifunctional regulators of the cytoskeleton and cell cycle progression

Rnd3/RhoE has two distinct functions, regulating the actin cytoskeleton and cell proliferation. This might explain why its expression is often altered in cancer and by multiple stimuli during development and disease. Rnd3 together with its relatives Rnd1 and Rnd2 are atypical members of the Rho GTPase family in that they do not hydrolyse GTP. Rnd3 and Rnd1 both antagonise RhoA/ROCK‐mediated actomyosin contractility, thereby regulating cell migration, smooth muscle contractility and neurite extension. In addition, Rnd3 has been shown to have a separate role in inhibiting cell cycle progression by reducing translation of cell cycle regulators, including cyclin D1 and Myc. We propose that Rnd3 could act as a tumour suppressor to limit proliferation, but when mutations bypass this activity of Rnd3, it can promote cancer invasion through its effects in the actin cytoskeleton.     

A New Member of the Rho Family, Rnd1, Promotes Disassembly of Actin Filament Structures and Loss of Cell Adhesion

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell–substrate adhesion leading to cell rounding (hence Rnd for “round”). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.     

RhoE function is regulated by ROCK I-mediated phosphorylation

The Rho GTPase family member RhoE regulates actin filaments partly by binding to and inhibiting ROCK I, a serine/threonine kinase that induces actomyosin contractility. Here, we show that ROCK I can phosphorylate multiple residues on RhoE in vitro. In cells, ROCK I-phosphorylated RhoE localizes in the cytosol, whereas unphosphorylated RhoE is primarily associated with membranes. Phosphorylation has no effect on RhoE binding to ROCK I, but instead increases RhoE protein stability. Using phospho-specific antibodies, we show that ROCK phosphorylates endogenous RhoE at serine 11 upon cell stimulation with platelet-derived growth factor, and that this phosphorylation requires an active protein kinase C signalling pathway. In addition, we demonstrate that phosphorylation of RhoE correlates with its activity in inducing stress fibre disruption and inhibiting Ras-induced transformation. This is the first demonstration of an endogenous Rho family member being phosphorylated in vivo and indicates that phosphorylation is an important mechanism to control the stability and function of this GTPase-deficient Rho protein.     

RhoE binds to ROCK I and inhibits downstream signaling

RhoE belongs to the Rho GTPase family, the members of which control actin cytoskeletal dynamics. RhoE induces stress fiber disassembly in a variety of cell types, whereas RhoA stimulates stress fiber assembly. The similarity of RhoE and RhoA sequences suggested that RhoE might compete with RhoA for interaction with its targets. Here, we show that RhoE binds ROCK I but none of the other RhoA targets tested. The interaction of RhoE with ROCK I was confirmed by coimmunoprecipitation of the endogenous proteins, and the two proteins colocalized on the trans-Golgi network in COS-7 cells. Although RhoE and RhoA were not able to bind ROCK I simultaneously, RhoE bound to the amino-terminal region of ROCK I encompassing the kinase domain, at a site distant from the carboxy-terminal RhoA-binding site. Overexpression of RhoE inhibited ROCK I-induced stress fiber formation and phosphorylation of the ROCK I target myosin light chain phosphatase. These data suggest that RhoE induces stress fiber disassembly by directly binding ROCK I and inhibiting it from phosphorylating downstream targets.     

Socius is a novel Rnd GTPase-interacting protein involved in disassembly of actin stress fibers

Rho family small GTPases are key regulators of the actin cytoskeleton in various cell types. The Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been recently identified as new members of the Rho family of GTPases, and expression of Rnd1 or Rnd3 in fibroblasts causes the disassembly of actin stress fibers and the retraction of the cell body to produce extensively branching cellular processes. Here we have performed a yeast two-hybrid screening by using Rnd1 as bait and identified a novel protein that specifically binds to Rnd GTPases. We named this protein Socius. Socius directly binds to Rnd GTPases through its COOH-terminal region. When transfected into COS-7 cells, Socius is translocated to the cell periphery in response to Rnd1 and Rnd3 and colocalized with the GTPases. While expression of wild-type Socius in Swiss 3T3 fibroblasts has little effect on the actin cytoskeleton, the expression of a membrane-targeted form of Socius, containing a COOH-terminal farnesylation motif (Socius-CAAX), induces a dramatic loss of stress fibers. The inhibitory effect of Socius-CAAX on stress fiber formation is enhanced by truncation of its NH(2) terminus. On the other hand, the expression of Socius-CAAX or its NH(2) terminus-truncated form suppresses the Rnd-induced retraction of the cell body and the production of extensively branching cellular processes, although the disassembly of stress fibers is observed. We propose that Socius participates in the Rnd GTPase-induced signal transduction pathways, leading to reorganization of the actin cytoskeleton.     

Rnd3/RhoE induces tight junction formation in mammary epithelial tumor cells

Glucocorticoid hormones stimulate adherens and tight junction formation in Con8 mammary epithelial tumor cells through a multistep process in which the membrane organization of structural apical junction proteins and tight junction sealing is controlled by specific signal transduction components. We have previously shown that dexamethasone stimulation of apical junction formation requires down-regulation of the small GTPase RhoA. Here we identified Rnd3/RhoE, a GTPase-deficient Rho family member and RhoA antagonist, as a key regulator of apical junction dynamics. Exogenously expressed Rnd3/RhoE co-localized with actin at the cell periphery and induced the localization of the adherens junction protein beta-catenin and the tight junction protein ZO-1 to sites of cell-cell contact, and led to the formation of highly sealed tight junctions. Treatment with glucocorticoids was not required to achieve complete apical junction remodeling. Consistent with Rnd3/RhoE acting as an antagonist of RhoA, expression of Rnd3/RhoE rescued the disruptive effects of constitutively active RhoA on apical junction organization. Our results demonstrate a new role for the Rho family member Rnd3/RhoE in regulating the assembly of the apical junction complex and tight junction sealing.     

B-RAF regulation of Rnd3 participates in actin cytoskeletal and focal adhesion organization

The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.     

Rnd3 Regulates Lung Cancer Cell Proliferation through Notch Signaling

Rnd3/RhoE is a small Rho GTPase involved in the regulation of different cell behaviors. Dysregulation of Rnd3 has been linked to tumorigenesis and metastasis. Lung cancers are the leading cause of cancer-related death in the West and around the world. The expression of Rnd3 and its ectopic role in non-small cell lung cancer (NSCLC) remain to be explored. Here, we reported that Rnd3 was down-regulated in three NSCLC cell lines: H358, H520 and A549. The down-regulation of Rnd3 led to hyper-activation of Rho Kinase and Notch signaling. The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells. Mechanistically, Notch intracellular domain (NICD) protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation. Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase.     

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.     

RhoE deficiency produces postnatal lethality, profound motor deficits and neurodevelopmental delay in mice

Rnd proteins are a subfamily of Rho GTPases involved in the control of actin cytoskeleton dynamics and other cell functions such as motility, proliferation and survival. Unlike other members of the Rho family, Rnd proteins lack GTPase activity and therefore remain constitutively active. We have recently described that RhoE/Rnd3 is expressed in the Central Nervous System and that it has a role in promoting neurite formation. Despite their possible relevance during development, the role of Rnd proteins in vivo is not known. To get insight into the in vivo function of RhoE we have generated mice lacking RhoE expression by an exon trapping cassette. RhoE null mice (RhoE gt/gt) are smaller at birth, display growth retardation and early postnatal death since only half of RhoE gt/gt mice survive beyond postnatal day (PD) 15 and 100% are dead by PD 29. RhoE gt/gt mice show an abnormal body position with profound motor impairment and impaired performance in most neurobehavioral tests. Null mutant mice are hypoactive, show an immature locomotor pattern and display a significant delay in the appearance of the hindlimb mature responses. Moreover, they perform worse than the control littermates in the wire suspension, vertical climbing and clinging, righting reflex and negative geotaxis tests. Also, RhoE ablation results in a delay of neuromuscular maturation and in a reduction in the number of spinal motor neurons. Finally, RhoE gt/gt mice lack the common peroneal nerve and, consequently, show a complete atrophy of the target muscles. This is the first model to study the in vivo functions of a member of the Rnd subfamily of proteins, revealing the important role of Rnd3/RhoE in the normal development and suggesting the possible involvement of this protein in neurological disorders.     

Proapoptotic p53-interacting protein 53BP2 is induced by UV irradiation but suppressed by p53

p53 is an important mediator of the cellular stress response with roles in cell cycle control, DNA repair, and apoptosis. 53BP2, a p53-interacting protein, enhances p53 transactivation, impedes cell cycle progression, and promotes apoptosis through unknown mechanisms. We now demonstrate that endogenous 53BP2 levels increase following UV irradiation induced DNA damage in a p53-independent manner. In contrast, we found that the presence of a wild-type (but not mutant) p53 gene suppressed 53BP2 steady-state levels in cell lines with defined p53 genotypes. Likewise, expression of a tetracycline-regulated wild-type p53 cDNA in p53-null fibroblasts caused a reduction in 53BP2 protein levels. However, 53BP2 levels were not reduced if the tetracycline-regulated p53 cDNA was expressed after UV damage in these cells. This suggests that UV damage activates cellular factors that can relieve the p53-mediated suppression of 53BP2 protein. To address the physiologic significance of 53BP2 induction, we utilized stable cell lines with a ponasterone A-regulated 53BP2 cDNA. Conditional expression of 53BP2 cDNA lowered the apoptotic threshold and decreased clonogenic survival following UV irradiation. Conversely, attenuation of endogenous 53BP2 induction with an antisense oligonucleotide resulted in enhanced clonogenic survival following UV irradiation. These results demonstrate that 53BP2 is a DNA damage-inducible protein that promotes DNA damage-induced apoptosis. Furthermore, 53BP2 expression is highly regulated and involves both p53-dependent and p53-independent mechanisms. Our data provide new insight into 53BP2 function and open new avenues for investigation into the cellular response to genotoxic stress.     

PGC-1α, a key modulator of p53, promotes cell survival upon metabolic stress

Metabolic stress results in p53 activation, which can trigger cell-cycle arrest, ROS clearance, or apoptosis. However, what determines the p53-mediated cell fate decision upon metabolic stress is not very well understood. We show here that PGC-1α binds to p53 and modulates its transactivation function, resulting in preferential transactivation of proarrest and metabolic target genes. Thus glucose starvation results in p53-dependent cell-cycle arrest and ROS clearance, but abrogation of PGC-1α expression results in extensive apoptosis. Additionally, prolonged starvation results in PGC-1α degradation concomitant with induction of apoptosis. We have also identified RNF2, a Polycomb group (PcG) protein, as the cognate E3 ubiquitin ligase. Starvation of mice where PGC-1α expression is abrogated results in loss of p53-mediated ROS clearance, enhanced p53-dependent apoptosis, and consequent severe liver atrophy. These findings provide key insights into the role of PGC-1α in regulating p53-mediated cell fate decisions in response to metabolic stress.     

Protein kinase C delta regulates Ser46 phosphorylation of p53 tumor suppressor in the apoptotic response to DNA damage

The p53 tumor suppressor is activated in the cellular response to genotoxic stress. Transactivation of p53 target genes dictates cell cycle arrest and DNA repair or induction of apoptosis; however, a molecular mechanism responsible for these distinct functions remains unclear. Recent studies revealed that phosphorylation of p53 on Ser(46) was associated with induction of p53AIP1 expression, resulting in the commitment of the cell fate into apoptotic cell death. Moreover, upon exposure to genotoxic stress, p53DINP1 was expressed and recruited a kinase(s) to p53 that specifically phosphorylated Ser(46). Here, we show that the pro-apoptotic kinase, protein kinase C delta (PKCdelta), is involved in phosphorylation of p53 on Ser(46). PKCdelta-mediated phosphorylation is required for the interaction of PKCdelta with p53. The results also demonstrate that p53DINP1 associates with PKCdelta upon exposure to genotoxic agents. Consistent with these results, PKCdelta potentiates p53-dependent apoptosis by Ser(46) phosphorylation in response to genotoxic stress. These findings indicate that PKCdelta regulates p53 to induce apoptotic cell death in the cellular response to DNA damage.     

A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3

Clostridium botulinum C3 is the prototype of the family of the C3-like transferases that ADP-ribosylate exclusively RhoA, -B and -C. The ADP-ribose at Asn-41 results in functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton. We report on a new C3-like transferase produced by a pathogenic Staphylococcus aureus strain. The transferase designated C3(Stau) was cloned from the genomic DNA. At the amino acid level, C3(Stau) revealed an identity of 35% to C3 from C. botulinum and Clostridium limosum exoenzyme, respectively, and of 78% to EDIN from S. aureus. In addition to RhoA, which is the target of the other C3-like transferases, C3(Stau) modified RhoE and Rnd3. RhoE was ADP-ribosylated at Asn-44, which is equivalent to Asn-41 of RhoA. RhoE and Rnd3 are members of the Rho subfamily, which are deficient in intrinsic GTPase activity and possess a RhoA antagonistic cell function. The protein substrate specificity found with recombinant Rho proteins was corroborated by expression of RhoE in Xenopus laevis oocytes showing that RhoE was also modified in vivo by C3(Stau) but not by C3 from C. botulinum. The poor cell accessibility of C3(Stau) was overcome by generation of a chimeric toxin recruiting the cell entry machinery of C. botulinum C2 toxin. The chimeric C3(Stau) caused the same morphological and cytoskeletal changes as the chimeric C. botulinum C3. C3(Stau) is a new member of the family of the C3-like transferases but is also the prototype of a subfamily of RhoE/Rnd modifying transferases.     

Serine/threonine kinase 31 promotes PDCD5-mediated apoptosis in p53-dependent human colon cancer cells

Although programed cell death 5 (PDCD5) is an important protein in p53-mediated proapoptotic signaling, very little is known about PDCD5-related cell death. In this study, we report that serine/threonine kinase 31 (STK31) interacts with PDCD5, which maintains the stability of PDCD5. STK31 overexpression significantly activated PDCD5 stabilization and p53-mediated apoptosis in response to etoposide (ET). However, STK31 knockdown did not enhance apoptosis by ET treatment. Moreover, when STK31 was depleted, PDCD5 inhibited the activation of the p53 signaling pathway with ET, indicating that the PDCD5–STK31 network has an essential role in p53 activation. Importantly, STK31 activated the p53 signaling pathway by genotoxic stress through positive regulation of PDCD5-mediated apoptosis. We thus demonstrated that overexpression of STK31 greatly inhibited tumorigenic growth and increased the chemosensitivity of HCT116 human colorectal carcinoma cells. Taken together, these findings demonstrate that the STK31–PDCD5 complex network regulates apoptosis of cancer cells, and STK31 is a positive apoptosis regulator that inhibits tumorigenesis of colon cancer cells by inducing PDCD5-mediated apoptosis in response to genotoxic stress.     

Rnd1 and Rnd3 targeting to lipid raft is required for p190 RhoGAP activation

The Rnd proteins Rnd1, Rnd2, and Rnd3/RhoE are well known as key regulators of the actin cytoskeleton in various cell types, but they comprise a distinct subgroup of the Rho family in that they are GTP bound and constitutively active. Functional differences of the Rnd proteins in RhoA inhibition signaling have been reported in various cell types. Rnd1 and Rnd3 antagonize RhoA signaling by activating p190 RhoGAP, whereas Rnd2 does not. However, all the members of the Rnd family have been reported to bind directly to p190 RhoGAP and equally induce activation of p190 RhoGAP in vitro, and there is no evidence that accounts for the functional difference of the Rnd proteins in RhoA inhibition signaling. Here we report the role of the N-terminal region in signaling. Rnd1 and Rnd3, but not Rnd2, have a KERRA (Lys-Glu-Arg-Arg-Ala) sequence of amino acids in their N-terminus, which functions as the lipid raft-targeting determinant. The sequence mediates the lipid raft targeting of p190 RhoGAP correlated with its activation. Overall, our results demonstrate a novel regulatory mechanism by which differential membrane targeting governs activities of Rnd proteins to function as RhoA antagonists.     

Induction of apoptosis and stress response in ovarian carcinoma cell lines treated with ST1926, an atypical retinoid

To understand the molecular mechanisms mediating apoptosis induction by a novel atypical retinoid, ST1926, the cellular response to drug treatment was investigated in IGROV-1 ovarian carcinoma cells carrying wild-type p53 and a cisplatin-resistant p53 mutant subline (IGROV-1/Pt1). Despite a similar extent of drug-induced DNA strand breaks, the level of apoptosis was substantially higher in p53 wild-type cells. p53 activation and early upregulation of p53-target genes were consistent with p53-dependent apoptosis in IGROV-1 cells. Stress-activated protein kinases were activated in both cell lines in response to ST1926. This event and activation of AP-1 were more pronounced in IGROV-1/Pt1 cells, in which the modulation of DNA repair-associated genes suggests an increased ability to repair DNA damage. Inhibition of JNK or p38 stimulated ST1926-induced apoptosis only in IGROV-1 cells, whereas inhibition of ERKs enhanced apoptosis in both the cell lines. Such a pattern of cellular response and modulation of genes implicated in DNA damage response supports that the genotoxic stress is a critical event mediating drug-induced apoptosis. The results are consistent with apoptosis induction through p53-dependent and -independent pathways, regulated by MAP kinases, which likely play a protective role.