Regulation Of Rho Family GTPases Research Articles

Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI

Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code," where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42, etc.). This work attempts to understand the molecular mechanism behind this specific phosphorylation-induced reduction in binding affinity. Using several microseconds long atomistic molecular dynamics simulations of the wild-type and phosphorylated states of the RhoA-RhoGDI complex, we propose a molecular-interaction-based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N-terminal region of RhoGDI that contribute most to the binding affinity. Molecular mechanics Poisson-Boltzmann surface area binding energy calculations show a significant weakening of interaction on phosphorylation at the RhoA-specific site of RhoGDI. In contrast, phosphorylation at a Rac1-specific site does not affect the overall binding affinity significantly, which confirms the presence of a phosphorylation code. RhoA-specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N-terminal region of RhoGDI, which manifests a reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long-range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.

CAMP: A master regulator of cadherin-mediated binding in endothelium, epithelium and myocardium.

Regulation of cadherin-mediated cell adhesion is crucial not only for maintaining tissue integrity and barrier function in the endothelium and epithelium but also for electromechanical coupling within the myocardium. Therefore, loss of cadherin-mediated adhesion causes various disorders, including vascular inflammation and desmosome-related diseases such as the autoimmune blistering skin dermatosis pemphigus and arrhythmogenic cardiomyopathy. Mechanisms regulating cadherin-mediated binding contribute to the pathogenesis of diseases and may also be used as therapeutic targets. Over the last 30 years, cyclic adenosine 3',5'-monophosphate (cAMP) has emerged as one of the master regulators of cell adhesion in endothelium and, more recently, also in epithelial cells as well as in cardiomyocytes. A broad spectrum of experimental models from vascular physiology and cell biology applied by different generations of researchers provided evidence that not only cadherins of endothelial adherens junctions (AJ) but also desmosomal contacts in keratinocytes and the cardiomyocyte intercalated discs are central targets in this scenario. The molecular mechanisms involve protein kinase A- and exchange protein directly activated by cAMP-mediated regulation of Rho family GTPases and S665 phosphorylation of the AJ and desmosome adaptor protein plakoglobin. In line with this, phosphodiesterase 4 inhibitors such as apremilast have been proposed as a therapeutic strategy to stabilize cadherin-mediated adhesion in pemphigus and may also be effective to treat other disorders where cadherin-mediated binding is compromised.

SH3 domain regulation of RhoGAP activity: Crosstalk between p120RasGAP and DLC1 RhoGAP

RhoGAP proteins are key regulators of Rho family GTPases and influence a variety of cellular processes, including cell migration, adhesion, and cytokinesis. These GTPase activating proteins (GAPs) downregulate Rho signaling by binding and enhancing the intrinsic GTPase activity of Rho proteins. Deleted in liver cancer 1 (DLC1) is a tumor suppressor and ubiquitously expressed RhoGAP protein; its activity is regulated in part by binding p120RasGAP, a GAP protein for the Ras GTPases. In this study, we report the co-crystal structure of the p120RasGAP SH3 domain bound directly to DLC1 RhoGAP, at a site partially overlapping the RhoA binding site and impinging on the catalytic arginine finger. We demonstrate biochemically that mutation of this interface relieves inhibition of RhoGAP activity by the SH3 domain. These results reveal the mechanism for inhibition of DLC1 RhoGAP activity by p120RasGAP and demonstrate the molecular basis for direct SH3 domain modulation of GAP activity.

Phosphoproteomics identify arachidonic-acid-regulated signal transduction pathways modulating macrophage functions with implications for ovarian cancer.

Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions.Methods: AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca2+ accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release.Results: We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca2+-triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A2 group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options.Conclusion: Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression.

Roles of the cytoskeleton, cell adhesion and rho signalling in mechanosensing and mechanotransduction.

All cells sense and respond to various mechanical forces in and mechanical properties of their environment. To respond appropriately, cells must be able to sense the location, direction, strength and duration of these forces. Recent progress in mechanobiology has provided a better understanding of the mechanisms of mechanoresponses underlying many cellular and developmental processes. Various roles of mechanoresponses in development and tissue homeostasis have been elucidated, and many molecules involved in mechanotransduction have been identified. However, the whole picture of the functions and molecular mechanisms of mechanotransduction remains to be understood. Recently, novel mechanisms for sensing and transducing mechanical stresses via the cytoskeleton, cell-substrate and cell-cell adhesions and related proteins have been identified. In this review, we outline the roles of the cytoskeleton, cell-substrate and cell-cell adhesions, and related proteins in mechanosensing and mechanotransduction. We also describe the roles and regulation of Rho-family GTPases in mechanoresponses.

Cell-extracellular matrix and cell-cell adhesion are linked by syndecan-4

Cell-extracellular matrix (ECM) and cell-cell junctions that employ microfilaments are sites of tension. They are important for tissue repair, morphogenetic movements and can be emblematic of matrix contraction in fibrotic disease and the stroma of solid tumors. One cell surface receptor, syndecan-4, has been shown to regulate focal adhesions, junctions that form at the ends of microfilament bundles in response to matrix components such as fibronectin. Recently it has been shown that signaling emanating from this proteoglycan receptor includes regulation of Rho family GTPases and cytosolic calcium. While it is known that cell-ECM and cell-cell junctions may be linked, possible roles for syndecans in this process are not understood. Here we show that wild type primary fibroblasts and those lacking syndecan-4 utilize different cadherins in their adherens junctions and that tension is a major factor in this differential response. This corresponds to the reduced ability of fibroblasts lacking syndecan-4 to exert tension on the ECM and we now show that this may extend to reduced tension in cell-cell adhesion.

FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions.

Guanine-nucleotide dissociation inhibitors (GDI) are negative regulators of Rho family GTPases that sequester the GTPases away from the membrane. Here we ask how GDI-Cdc42 interaction regulates localized Cdc42 activation for cell motility. The sensitivity of cells to overexpression of Rho family pathway components led us to a new biosensor design (GDI.Cdc42 FLARE), in which Cdc42 was modified with a FRET ‘binding antenna’ that selectively reported Cdc42 binding to endogenous GDI. Similar antennae could also report GDI-Rac1 and GDI-RhoA interaction. Through computational multiplexing and simultaneous imaging, we determined the spatiotemporal dynamics of GDI-Cdc42 interaction and Cdc42 activation during cell protrusion and retraction. This revealed a remarkably tight coordination of GTPase release and activation on a time scale of 10 seconds, suggesting that GDI-Cdc42 interactions are a critical component in the spatiotemporal regulation of Cdc42 activity, and not merely a mechanism for global sequestration of an inactivated pool of signaling molecules.

MgcRacGAP interacts with cingulin and paracingulin to regulate Rac1 activation and development of the tight junction barrier during epithelial junction assembly.

The regulation of Rho-family GTPases is crucial to direct the formation of cell-cell junctions and tissue barriers. Cingulin (CGN) and paracingulin (CGNL1) control RhoA activation in epithelial cells by interacting with RhoA guanidine exchange factors. CGNL1 depletion also inhibits Rac1 activation during junction assembly. Here we show that, unexpectedly, Madin-Darby canine kidney epithelial cells depleted of both CGN and CGNL1 (double-KD cells) display normal Rac1 activation and tight junction (TJ) formation, despite decreased junctional recruitment of the Rac1 activator Tiam1. The expression of the Rac1 inhibitor MgcRacGAP is decreased in double-KD cells, and the barrier development and Rac1 activation phenotypes are rescued by exogenous expression of MgcRacGAP. MgcRacGAP colocalizes with CGN and CGNL1 at TJs and forms a complex and interacts directly in vitro with CGN and CGNL1. Depletion of either CGN or CGNL1 in epithelial cells results in decreased junctional localization of MgcRacGAP but not of ECT2, a centralspindlin-interacting Rho GEF. These results provide new insight into coordination of Rho-family GTPase activities at junctions, since apical accumulation of CGN and CGNL1 at TJs during junction maturation provides a mechanism to spatially restrict down-regulation of Rac1 activation through the recruitment of MgcRacGAP.

N‐cadherin, A Vascular Smooth Muscle Cell–Cell Adhesion Molecule: Function and Signaling for Vasomotor Control

Cell-cell adhesion complexes are increasingly recognized as an important cell-signaling site, similar to integrin-extracellular matrix FA. Furthermore, cell-cell adhesions are involved in the regulation of multi-cellular/tissue organization and organ, tissue, and cellular level functional behavior. Although N-cadherin is the major cell-cell adhesion molecule in VSM, only limited studies have been undertaken to understand its function in VSM. In contrast, N-cadherin signaling and functions have been extensively studied in neurons, fibroblasts, and myocytes, as well as in the context of epithelial-mesenchymal-transitions. Increasing evidence has indicated that N-cadherin-mediated cell-cell adhesions are important for tissue integrity and cell proliferation. Relevant to VSM, N-cadherin's role in actin cytoskeleton organization and contraction, as well as its role in regulation of Rho family GTPases are of particular interest. This article briefly reviews the fundamentals of N-cadherin biology that help shape our current understanding of its function and signaling mechanisms. In particular, attention is given to applications of this knowledge to VSM. The review points to the need for more research effort that is directed at understanding the role of N-cadherins in the regulation of vascular function.

Cytoskeletal function in the immune system

Cytoskeletal function in the immune system

Centrosomal localization of RhoGDIβ and its relevance to mitotic processes in cancer cells

Rho GDP-dissociation inhibitors (RhoGDIs) are regulators of Rho family GTPases. RhoGDIβ has been implicated in cancer progression, but its precise role remains unclear. We determined the subcellular localization of RhoGDIβ and examined the effects of its overexpression and RNAi knockdown in cancer cells. Immunofluorescence staining showed that RhoGDIβ localized to centrosomes in human cancer cells. In HeLa cells, exogenous GFP-tagged RhoGDIβ localized to centrosomes and its overexpression caused prolonged mitosis and aberrant cytokinesis in which the cell shape was distorted. RNAi knockdown of RhoGDIβ led to increased incidence of monopolar spindle mitosis resulting in polyploid cells. These results suggest that RhoGDIβ has mitotic functions, including regulation of cytokinesis and bipolar spindle formation. The dysregulated expression of RhoGDIβ may contribute to cancer progression by disrupting these processes.

Centralspindlin: at the heart of cytokinesis.

The final step in the cell cycle is the formation of two genetically identical daughter cells by cytokinesis. At the heart of cytokinesis in animal cells is the centralspindlin complex which is composed of two proteins, a kinesin-like protein, Mitotic kinesin-like protein 1, and a Rho GTPase activating protein (RhoGAP), CYK-4. Through its targeted localization to a narrow region of antiparallel microtubule overlap immediately following chromosome segregation, centralspindlin initiates central spindle assembly. Centralspindlin has several critical functions during cell division including positioning of the division plane, regulation of Rho family GTPases, as well as midbody assembly and abscission. In this review, we will examine the biochemistry of centralspindlin and its multiple functions during cell division. Remarkably, several of its critical functions are somewhat unexpected. Although endowed with motor domains, centralspindlin has an important role in generating stable, antiparallel microtubule bundles. Although it contains a Rho family GAP domain, it has a central role in the activation of RhoA during cytokinesis. Finally, centralspindlin functions as a motor protein complex, as a scaffold protein for key regulators of abscission and as a conventional RhoGAP. Because of these diverse functions, centralspindlin lies at the heart of the cytokinetic mechanism.

CdGAP regulates cell migration and adhesion dynamics in two‐and three‐dimensional matrix environments

CdGAP is a Rac1/Cdc42 specific GTPase activating protein (GAP) that localizes to cell-matrix adhesions through an interaction with the adhesion scaffold α-parvin/actopaxin to regulate lamellipodia formation and cell spreading. Herein, we demonstrate, using a combination of siRNA-mediated silencing and overexpression, that cdGAP negatively regulates directed and random migration by controlling adhesion maturation and dynamics through the regulation of both adhesion assembly and disassembly. Interestingly, cdGAP was also localized to adhesions formed in three-dimensional (3D) matrix environments and cdGAP depletion promoted cancer cell migration and invasion through 3D matrices. These findings highlight the importance of GAP proteins in the regulation of Rho family GTPases and the coordination of the cell migration machinery..

MKK6 increases the melanocyte dendricity through the regulation of Rho family GTPases

Melanocyte dendrites serve as the principal conduit for melanosome transfer. The dendrite formation requires actin polymerization mediated by Rho family GTPases including RhoA, Rac1 and Cdc42. The aim of this study is to investigate and explore the involvement of p38 MAPK in melanocyte dendrite formation. We transduced melanoma cells with adenovirus harboring the expression cassette for constitutive active form of MKK6, an upstream MAPKK for p38 MAPK. We investigated the effect of melanogenic inducers on melanocyte dendricity, using SK-mel-24 melanoma cells because that this cell line is refractory to several melanogenic inducers in terms of melanogenesis. TPA-induced the phosphorylation of p38 MAPK and the elongation of dendrite length, suggesting that MKK6 may be involved in this process. Overexpression of the constitutive active form of MKK6 resulted in significant elongation of dendrites in the melanoma cell line SK-mel-24. Moreover, overexpression of MKK6 ultimately led to the upregulation of Cdc42 and Rac1, suggesting that MKK6 acts as a crucial upstream signaling molecule for Rho family GTPases. When overexpressed in normal human epidermal melanocytes, MKK6 led also the increase of dendrite length. These results suggest that MKK6 is an authentic regulator for melanocytes dendricity, through the modulation of Rho family GTPases.

The 19-Amino Acid Insertion in the Tumor-associated Splice Isoform Rac1b Confers Specific Binding to p120 Catenin

The Rac1b splice isoform contains a 19-amino acid insertion not found in Rac1; this insertion leads to decreased GTPase activity and reduced affinity for GDP, resulting in the intracellular predominance of GTP-bound Rac1b. Here, using co-precipitation and proteomic methods, we find that Rac1b does not bind to many common regulators of Rho family GTPases but that it does display enhanced binding to SmgGDS, RACK1, and p120 catenin (p120(ctn)), proteins involved in cell-cell adhesion, motility, and transcriptional regulation. We use molecular modeling and structure analysis approaches to determine that the interaction between Rac1b and p120(ctn) is dependent upon protein regions that are predicted to be unstructured in the absence of molecular complex formation, suggesting that the interaction between these two proteins involves coupled folding and binding. We also find that directed cell movement initiated by Rac1b is dependent upon p120. These results define a distinct binding functionality of Rac1b and provide insight into how the distinct phenotypic program activated by this protein may be implemented through molecular recognition of effectors distinct from those of Rac1.

Wnt/Fz signaling and the cytoskeleton: potential roles in tumorigenesis

Wnt/beta-catenin regulates cellular functions related to tumor initiation and progression, cell proliferation, differentiation, survival, and adhesion. Beta-catenin-independent Wnt pathways have been proposed to regulate cell polarity and migration, including metastasis. In this review, we discuss the possible roles of both beta-catenin-dependent and -independent signaling pathways in tumor progression, with an emphasis on their regulation of Rho-family GTPases, cytoskeletal remodeling, and relationships with cell-cell adhesion and cilia/ciliogenesis.

The BAR Domain Superfamily: Membrane-Molding Macromolecules

Membrane-shaping proteins of the BAR domain superfamily are determinants of organelle biogenesis, membrane trafficking, cell division, and cell migration. An upsurge of research now reveals new principles of BAR domain-mediated membrane remodeling, enhancing our understanding of membrane curvature-mediated information processing.

New Exchanges in Eph-Dependent Growth Cone Dynamics

The Eph receptor tyrosine kinases and their ephrin ligands play a pivotal role during axon pathfinding and neural circuitry formation. A prominent way in which Eph receptors sculpt cellular morphology is by remodeling the actin cytoskeleton and the surrounding plasma membrane through the regulation of Rho family GTPases. Two articles in this issue of Neuron (Sahin et al. and Cowan et al.) shed light on how Eph receptors recruit guanine nucleotide exchange factors for Rho family GTPases to modulate growth cone dynamics.

Translocation t(X;11)(q13;q23) in B‐cell chronic lymphocytic leukemia disrupts two novel genes

Deletion of chromosome region 11q22-q23 defines a subgroup of patients with B-cell chronic lymphocytic leukemia (B-CLL) characterized by poor survival. Although the tumor-suppressor gene ATM in the consensus deletion region was found to be biallelically inactivated in about one third of B-CLL cases, in the majority of those who have this deletion, inactivation of the remaining ATM allele was not observed. To identify a second disease-associated gene, we investigated two B-CLL cases with translocation breakpoints in the critical 11q23 deletion region. In one case, a t(X;11)(q13;q23) was cloned and two novel genes were isolated. The breakpoint on 11q23 affected the ARHGAP20 gene, which encodes a protein predicted to be involved in the regulation of Rho family GTPases. The breakpoint on Xq13 occurred in BRWD3, which codes for a putative novel transcription factor. The rearrangement of ARHGAP20 and BRWD3 did not result in fusion transcripts, but it disrupted both genes. Mutation analysis of 28 B-CLL samples with monoallelic deletions and two B-CLL samples with 11q23 translocations detected no deleterious mutation in the remaining copy of ARHGAP20. Quantitative expression analysis in 22 B-CLLs revealed significant up-regulation of ARHGAP20 in CLL B cells, whereas BRWD3 was slightly down-regulated. Thus, deregulation of ARHGAP20 by altered gene expression or by gene disruption (but not point mutation) might be a general molecular mechanism of B-CLL leukemogenesis.

Control of axonal branching and synapse formation by focal adhesion kinase.

The formation of neuronal networks in the central nervous system (CNS) requires precise control of axonal branch development and stabilization. Here we show that cell-specific ablation of the murine gene Ptk2 (more commonly known as fak), encoding focal adhesion kinase (FAK), increases the number of axonal terminals and synapses formed by neurons in vivo. Consistent with this, fak mutant neurons also form greater numbers of axonal branches in culture because they have increased branch formation and reduced branch retraction. Expression of wild-type FAK, but not that of several FAK variants that prevent interactions with regulators of Rho family GTPases including the p190 Rho guanine nuclear exchange factor (p190RhoGEF), rescues the axonal arborization phenotype observed in fak mutant neurons. In addition, expression of a mutant p190RhoGEF that cannot associate with FAK results in a phenotype very similar to that of neurons lacking FAK. Thus, FAK functions as a negative regulator of axonal branching and synapse formation, and it seems to exert its actions, in part, through Rho family GTPases.