DEFINITION
Ras homolog gene family, member A (RhoA) is a small GTPase protein known to regulate the actin cytoskeleton in the formation of stress fibers. In humans, it is encoded by the gene RhoA .
RhoA is part of a larger family of related proteins known as the Ras superfamily; proteins involved in the regulation and timing of cell division.
Other names are ARH12,ARHA,H12,RHO12,RHOH12 .
Molecular weight:21KDa.
RhoA protein is expressed in all tissues tested.
THE GENE RHOA
This gene can be found on Chromosome 3 at location: 49,371,585-49,424,530.The DNA sequence contains 5 exons and the transcript length is of 1919 bps translated to a 193 residues protein.
Entrez Gene cytogenetic band 3p21.3 Ensembl cytogenetic band 3p21.31 HGNC cytogenetic band: 3p21.3
CHEMICAL STRUCTURE AND IMAGES
Rho protein structures consist of a single domain with a six-stranded beta-sheet surrounded by alpha-helices .The b-sheet of RhoA contains two antiparallel (B2 and B3) and five parallel beta-strands (B3, B1, B4-B6) , five a-helices (A1, A3, A3′, A4, A5) , and three 310-helices (H1-H3) . When compared to Ras, Rho proteins contain three insertions and one deletion. The 13-residue insertion (Asp 124 to Gln 136 in RhoA) between strand B5 and helix A4 is the most distinctive feature that clearly differentiates Rho from other Ras-related GTPases. The amino acid sequence in this insert varies considerably among different Rho proteins, suggesting functional implications. Interestingly, the conformation of this helix does not depend on the type of nucleotide bound to the protein.
Conformational differences between RhoA*GDP and RhoA*GTPgS are restricted to the switch I and switch II regions (the nomenclature follows that used for Ras). Dramatic changes in switch I from the GDP- to the GTP-bound state are exemplified by displacements of 5.4 A and 6.4 A ( 1 A = 10-10m) at Pro36 and Phe39, respectively (Fig. 1b). The side chains of Pro36 Tyr34 become oriented toward the nucleotide, so that the ring of Tyr34 stacks on Pro36. Three hydrophobic residues, Val35, Val38, and Phe39 , become solvent exposed, suggesting their involvement in target binding following GTP-induced activation. An important difference between the GDP and GTPgS- 2+ 2+ bound RhoA relates to the mode of Mg ion binding. In the former, the Mg ion is coordinated by three water molecules, a g-phosphate oxygen atom, and two protein ligands, including the main-chain carbonyl of Thr37, whereas in the GTPgS structure the coordination is similar to p21Ras in its active form and involves the side chain of Thr37 .
a) The structure of RhoA in an inactive conformation with bound GDP and Mg2+ . The secondary structural elements are labeled, and the switch I and switch II regions are highlighted. Also indicated are the locations of the insert helix and residues Gly14, Thr37,and Asn41.
b) The conformational changes in switch I upon activation of RhoA . Alignment of the inactive and active structures of RhoA reveals dramatic changes in the switch I region. The GDP-bound conformation is shown as dark line from the same perspective as in A, and the aligned GTPgS-bound conformation is shown as the lighter line. PDB codes 1FTN and 1A2b.
The aminoacids more expressed in RhoA are Lysine and Valine .
The amminoacid more espressed in the same way in RhoA, RhoB and RhoC is Leucine ,while the less expressed in the same way in RhoA, RhoB and RhoC are Hystidine and Triptophan .
High content of lysine and arginine is due to increased synthesis of histones . Histones are positively charged basic proteins, as possessing a large number of amino acids with basic side chain, in particular lysine and arginine. Histones interact with DNA, which is negatively charged because of the abundance of phosphate groups to form structures called nucleosomes.
The methylation of histones is a reaction mediated by the class of enzymes histone methyl-transferase (HMTs; Histone Metiltransferases) and provides for the transfer of a methyl group to a lysine or arginine present N-terminal of histone H3 or H4. The donor of methyl groups is S-adenosyl-methionine (or SAM).
The effects may be different. An example is the methylation of lysine 9 of histone H3, which leads to the formation of a binding site for the main protein of heterochromatin (heterochromatin protein-1 or HP1), a protein able to inducesilencing and then packing . On the contrary a H3 lysine 4 methylation has the opposite effect and promotes the opening of chromatin leading to increased gene expression.
L-tryptophan is an essential amino acid necessary for protein synthesis in mammalian cells. Moreover, tryptophan is the precursor for the neurotransmitter serotonin , for the hormone melatonin , and contributes to the synthesis of the coenzymes NADH and NADPH . Degradation products of tryptophan have immunoregulatory functions. Mammalian cells cannot synthesize L-tryptophan, and thus depend on transport machineries for its uptake and protein turnover for its production. Because cell growth is strictly dependent on tryptophan serum levels, different cell types compete for this amino acid, for instance in the regulation of the immune response. Identified transporter proteins potentially involved in the uptake of tryptophan in human cells include the Na+-independent systems b0AT1 ; b0,+AT ; TAT1 ; y+LAT1 and y+LAT2 ; and LAT1, LAT2, LAT3, and LAT4. Of these, b0,+AT, LAT1, LAT2, y+LAT1, and y+LAT2 are amino acid exchangers; they countertransport cytosolic amino acids for extracellular ones. LAT1 is present in proliferative tissues and, as such, is up-regulated in many human primary tumor cells. Although regulated suppression of the immune system prevents autoimmunity and is important during pregnancy to protect the fetus or after organ transplant to block graft rejection, it can be harmful if co-opted by tumors to escape detection. T cells of the immune system normally recognize and destroy abnormal cells, including cancerous and grafted tissues. This process requires the amino acid tryptophan. Foreign grafts and cancer cells can dampen the immune response by starving T cells of tryptophan through a mechanism involving uptake and conversion to kynurenines using an enzyme called indoleamine-2,3-dioxygenase . Independent of tryptophan starvation, kynurenines induce T cell death when they are excreted through an unknown mechanism. Kynurenines are natural amino acids found in mammals; however, the transport machinery for their export across the cell membrane is not known. Nanosensor Detection of an Immunoregulatory Tryptophan Influx/Kynurenine Efflux Cycle
RhoA,RhoB and RhoC (collectively Rho) have the same aminoacid sequence in their
effector domains (approximately 32–41 amino acids- highly homologous 85% ), appear to be regulated in a similar manner, and seem to have similar functions.
RhoB FUNCTIONS
Regulator of protein signaling and trafficking:
Plays a pivotal role in the dynamic regulation of the actin cytoskeleton. Involved in intracellular protein trafficking of a number of proteins. Targets PRK1 to endosomes and is involved in trafficking of the EGF receptor from late endosomes to lysosomes. Also required for stability and nuclear trafficking of Akt which promotes endothelial cell survival during vascular development. Identified as a component of outside-in signaling pathways that coordinate Src activation with its translocation to transmembrane receptors.
Negative modifier of cancer progression:
Affects cell adhesion and growth factor signaling in transformed cells. Plays a negative role in tumorigenesis as RhoB deletion increases tumor formation initiated by Ras mutation. Limits the proliferation of transformed cells by facilitating turnover of oncogene c-Myc. Expression levels are dramatically decreased in lung, head and neck, and brain cancer, when tumors become more aggressive.
Modulator of cancer cell apoptosis:
Promotes proapoptotic signaling of regulators involved in cell cycle checkpoints, cell adhesion, vesicle trafficking, MAPK signaling, transcription, and immunity. Mediates apoptosis in neoplastically transformed cells after DNA damage. Is essential for apoptosis and antineoplastic activity of farnesyltransferase inhibitors in a mouse model. Is one of the targets of farnesyltransferase inhibitors which are currently under investigation as cancer therapeutics.
RhoC FUNCTIONS
Role of RhoC also been observed in limb development. The RhoC protein has been connected to cancer development. It is up-regulated in malignant pancreatic ductal carcinoma, inflammatory breast cancer tumors and highly metastatic melanoma. Ectopic over-expression of this gene increases the tumorigenic and metastasis properties of tumor progenitor cell. RhoC also induces the expression of angiogenic factors in human mammary epithelial cells, by facilitating the vascularization of tumors in which it is expressed.
SYNTESIS
Cellular signaling in rapid intestinal epithelial restitution
It has been recently reported that Ca2+-activated RhoA activity plays a critical role in regulation of cell migration after wounding in intestinal epithelial cells.Decreased [Ca2+]cyt concentration, either by reducing the Ca2+ driving force for Ca2+ influx via membrane depolarization by polyamine depletion or removal of [Ca2+]o from the culture medium inhibited RhoA expression and activity.Increasing [Ca2+]cyt through treatment with the Ca2+ ionophore ionomycin stimulated RhoA activity in intestinal epithelial cells.These results indicate that increasing [Ca2+]cyt activates RhoA activity and increases the formation of actomyosin stress fibers and stimulates intestinal epithelial migration during the early phase of mucosal restitution. (Cellular signaling in rapid intestinal epithelial restitution: implication of polyamines and K+ channels)
Rho signaling in vascular diseases
The upregulation of vascular RhoA,observed in hypertentsion,is hypothesized to enhance VSMC proliferation and inflammation in response to injury and atherogenesis. Inflammatory mediators including GPCR agonists,cytokines, and oxidatively modified LDL (oxLDL) signal through Rho to produce a variety of cellular responses involved in cell contraction, proliferation, and inflammation.These Rho-mediated responses contribute to the development of vascular diseases.Rho acting through Rho kinase has also been suggested to produce LIM kinase– and stress fiber–dependent activation of ERK,which in turn regulates the timing of cyclin D1 expression and cell cycle progression.It is likely that inflammatory mediators signal through Rho to activate NF-κB, increasing the expression of monocyte chemotactic peptide 1 (MCP-1),and adhesion molecules such as VCAM-1 and ICAM-1, leading to recruitment and adhesion of monocytes to the vascular wall.All of the signaling components mentioned above predispose to vascular disease. (RhoA-Rho kinase signaling mediates endothelium- and endoperoxide-dependent contractile activities characteristic of hypertensive vascular dysfunction TGFβ-induced RhoA activation and fibronectin production in mesangial cells require caveolae)
Regulation of smooth muscle contraction.
Various agonists (neurotransmitters as norepinephrine, hormones,Angiotensine II,endothelin etc.) bind to specific receptors to activate RhoA that induce the contraction in smooth muscle. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle
The activation of RhoA downstream of Ras is essenzial for proper induction of the cell cycle
Concerted activation of Ras through growth factors(EGF,PDGF etc) and integrins triggers the ERK pathway and also stimulates a series of events that result in the activation of RhoA. RhoA plays a crucial role in regulating the inhibitor p21 which prevents protein kinase activation of cyclinD/CDK4 or cyclinE/CDK2 which regulate DNA synthesis.
PATHWAY
To date, at least 11 proteins have been identified which directly interact with RhoA; Rho-kinase/ROK/ROCK1 e ROK2 (Rho-Associated Coiled-Coil-Containing Protein Kinase) (Role of RhoA/ROCK Signaling in Endothelial-Monocyte-Activating Polypeptide II Opening of the Blood-Tumor Barrier : Role of RhoA/ROCK Signaling in EMAP II Opening of the BTB,2011) , PKN (Protein Kinase-N) / PRK-1 , Rhotekin , Rhophilin , kinectin , Citron Kinase , MBS (Myosin-Binding Subunit) of Myosin PPtase (Myosin Phosphatase),p76RBE, PKC epsilon and DB1 transcription factor). Some of these have been shown to contribute to specific responses downstream of RhoA. Similarly to GEFs and GAPs, effectors bind to Rho both through the Switch 1 and 2 regions, but the amino acids involved in interaction with each target differ.
CELLULAR FUNCTIONS
CELLULAR LOCALIZATION
RhoA is localizated in the cytoplasm or in the cell membrane .
ENZYMATIC ACTIVITY
Rho proteins cycle between an active GTP -bound state and an inactive GDP -bound state. Their activation state is controlled by regulatory proteins such as GEFs , which catalyze the exchange of GDP for GTP thereby activating Rho, GDIs , which inhibit the release of GDP keeping Rho inactive, and GAPs , which increase the rate at which Rho hydrolyzes GTP and hence becomes inactivated. While the overall sequence of events leading to activation of Rho family proteins by extracellular signals is known, gaps remain in the molecular details of these pathways and are areas of intensive study.
BIOLOGICAL FUNCTION
Role in actin organization:
RhoA protein plays a central role in regulating cell shape, polarity and locomotion through their effects on actin polymerization , actomyosin contractility , cell adhesion , and microtubule dynamics . RhoA is believed to act primarily at the rear of migrating cells to promote detachment.
RhoA directly stimulates actin polymerization through activiation of diaphanous-related formins (DRFs, also known as Dia proteins). These stimulate addition of actin monomers to the fast-growing end of actin filaments. DRFs act together with ROCKs to mediate Rho-induced stress fiber formation. ROCK-mediated phosphorylation of LIMK and consequent inhibition of cofilin also contributes to the increase in actin filaments in response to Rho. In addition, ROCKs induce actomyosin-based contractility and phosphorylate several proteins involved in regulating myosins and other actin-binding proteins. Actomyosin contractility is important in migrating cells for detachment of the rear. Microtubules are essential for determining cell polarity as well as for vesicular locomotion and intracellular transport. The concerted action of ROCK and Dia is essential for the regulation of cell polarity and organization of microtubules. ROCK phosphorylates TAU and MAP2, proteins that regulate microtubule stability.
RhoA plays a key role in regulating the integrity of cell-extracellular matrix and cell-cell adhesions, the latter including both adherens junctions and tight junctions. Loss of cell-cell junctions is required form the migration of epithelial cells and may be regulated reciprocally by ROCKs and DRFs.
Role in cytokinesis:
Cytokinesis requires actomyosin-based contraction. Inhibition of ROCK or citron kinase causes defects in cytokinesis resulting in multinucleate cells. Diaphanous-related formins (DRFs) are also implicated in this process, the DRF mDia1 localizes to the cleavage furrow during cytokinesis. DRFs could contribute to cytokinesis by stimulating local actin polymerization and/or by coordinating microtubules with actin filaments at the site of the contractile ring.
Role in cell cycle regulation:
RhoA plays a pivotal role in G1 cell cycle progression, primarily through regulation of both cyclin D1 expression, and the levels of the cyclin-dependent kinase inhibitors p21 and p27(gene) . Multiple pathways seem to link Rho proteins to the control of cyclin D1 levels. Many of these involve the activation of protein kinases, leading to the subsequent modulation of transcription factor activity. RhoA suppresses p21 levels in multiple normal and transformed cell lines. This effect appears to occur through a transcriptional mechanism but is independent of p53, a major transcriptional regulator of p21. RhoA plays an important role in determining the levels of p27 through a pathway involving its effector, the Rho-associated kinases.
Role in development:
RhoA protein is required for processes involving cell migration in development including: neurite outgrowth, dorsal closure, bone formation, and myogenesis . Rho-loss of function is embryonically lethal in mouse development by E7. This is attributed to failure in gastrulation and an inability of cells to migrate.
Role in transcriptional control:
The relationship between many of the cellular functions mediated by RhoA with transcriptional regulation has been described.RhoA modulates the activity of SRF , NFkappaB, c/EBPb , Stat3 , Stat5 , FHL-2 , PAX6 , GATA-4 , E2F, Estrogen Receptor alpha , Estrogen Receptor beta , CREB , and transcription factors that depend on the JNK and p38 MAP kinasepathways. Substrates to these kinases include c-Jun, ELK, PEA3, ATF2, MEF2A, Max and CHOP/2GADD153.
DIAGNOSTIC USE
Several types of human cancers have been analyzed for RhoA mutations. Thus, breast, ovarian, lung and colon specimens were surveyed for RhoA gene mutations and performed chromosomal analysis on 3p21 .No mutations in RhoA were found, nor there a correlation between RhoA mRNA expression and the presence or absence of 3p21 deletions ( RHO–GTPases and cancer,2011 ).
a)Maintenance of normal epithelial polarity. RHOA , RAC1, CDC42 and PAR6 are required for cell polarity.
b)Benign tumours: loss of polarity and multilayering.
c)Locally invasive tumours: loss of tissue boundaries and increased motility.
d)Metastasis to a distant site: intravasation and extravasation. RHO and ROCK are required for tumour cells to cross endothelial cell layers.
BREAST CARCINOMA
RhoA protein levels were significantly increased in breast cancer compared with the corresponding normal tissue. Of particular note, protein levels of RhoA were barely detectable in normal mammary tissue, but were highly expressed in all breast tumors tested. Interestingly, RhoA protein levels correlated with increasing breast tumor grade.
OVARIAN CARCINOMA
RhoA mRNA is higher in ovarian carcinoma'ovaio , especially of serous histological type, than in benign tumors. The expression of the protein is further upregulated in tumors of stages III/IV when compared to those of stages I/II. Analysis of matched pairs of primary and metastatic lesions showed that expression of both RhoA mRNA was significantly higher in metastatic lesions of peritoneal dissemination than in the respective primary tumors.
LUNGS TUMORS
Of the two major forms of lung cancer, small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), the former has a greater metastatic potential. The expression and activation of RhoA is greater in SCLC than NSCLC cell lines. It has been observed that RhoA repress the expression of nitric oxide synthase-2 (NOS-2) in a lung cancer-derived cell line. Since NOS-2 activity is related to reduced proliferation, RhoA could be eliminating this antiproliferative signal in lung carcinogenesis. In addition, inhibition of RhoA by C3 exoenzyme or through ADP-ribosylation leads to an increase in cadherin-based adhesion and loss of motility of SCLC.
COLORECTAL CANCER
A high proportion of colon cancers overexpress RhoA and several aspects of colon tumor biology have been related to Rho GTPases. Leptin receptor and leptin-induced migration of colonic epithelial cancer cells is dependent on RhoA, since inhibition of the activity of the GTPase through introduction of dominant negative mutants completely abolishes the invasive capacity of the tumor cells.
Maria Giuseppa Volpe
Susan Arauco
