Rho GTPase expression in tumourigenesis: evidence for a significant link

Rho proteins belong to the small GTPases superfamily. They function as molecular switches that, in response to diverse stimuli, control key signaling and structural aspects of the cell. Although early studies proposed a role for Rho GTPases in cellular transformation, this effect was underestimated due to the fact that no genetic mutations affecting Rho-encoding genes were found in tumors. Recently, it has become evident that Rho GTPases participate in the carcinogenic process by either overexpression of some of the members of the family with oncogenic activity, downmodulation of other members with suggested tumor suppressor activity, or by alteration of upstream modulators or downstream effectors. Thus, alteration of the levels of expression of different members of the family of Rho GTPases has been detected in many types of human tumors leading to a great interest in the cellular effects elicited by these oncoproteins. This essay reviews the current evidence of dysregulation of Rho signaling by overexpression in human tumors.     

I’m coming to GEF you: Regulation of RhoGEFs during cell migration

Cell migration is a highly regulated multistep process that requires the coordinated regulation of cell adhesion, protrusion, and contraction. These processes require numerous protein–protein interactions and the activation of specific signaling pathways. The Rho family of GTPases plays a key role in virtually every aspect of the cell migration cycle. The activation of Rho GTPases is mediated by a large and diverse family of proteins; the guanine nucleotide exchange factors (RhoGEFs). GEFs work immediately upstream of Rho proteins to provide a direct link between Rho activation and cell–surface receptors for various cytokines, growth factors, adhesion molecules, and G protein-coupled receptors. The regulated targeting and activation of RhoGEFs is essential to coordinate the migratory process. In this review, we summarize the recent advances in our understanding of the role of RhoGEFs in the regulation of cell migration.     

Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets

Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that – beside the GAP domain – the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation.     

Rho-GTPase signaling drives melanoma cell plasticity

To investigate mechanisms that underlie different modes of tumor cell movement we have studied how regulation of the activity of the Rho family GTPases determines the mode of tumor cell movement. Guanine nucleotide exchange factors (GEFs) and GTPase accelerating proteins (GAPs) are key regulators of the activity of small GTPases with GEFs promoting activation to the GTP bound state and GAPs promoting inactivation by stimulating GTP hydrolysis. We identified two important signaling pathways regulating amoeboid and mesenchymal types of motility in melanoma. Here, we discuss our findings in the context of how specificity of Rho signaling is achieved by GEFs and GAPs.     

IAPs on the move: role of inhibitors of apoptosis proteins in cell migration

Inhibitors of Apoptosis Proteins (IAPs) are a class of highly conserved proteins predominantly known for the regulation of caspases and immune signaling. However, recent evidence suggests a crucial role for these molecules in the regulation of tumor cell shape and migration by controlling MAPK, NF-κB and Rho GTPases. IAPs directly control Rho GTPases, thus regulating cell shape and migration. For instance, XIAP and cIAP1 function as the direct E3 ubiquitin ligases of Rac1 and target it for proteasomal degradation. IAPs are differentially expressed in tumor cells and have been targeted by several cancer therapeutic drugs that are currently in clinical trials. Here, we summarize the current knowledge on the role of IAPs in the regulation of cell migration and discuss the possible implications of these observations in regulating tumor cell metastases.     

Rac and Rho mediate opposing hormonal regulation of the ether-a-go-go-related potassium channel.

BACKGROUND: Previous studies of ion channel regulation by G proteins have focused on the larger, heterotrimeric GTPases, which are activated by heptahelical membrane receptors. In contrast, studies of the Rho family of smaller, monomeric, Ras-related GTPases, which are activated by cytoplasmic guanine nucleotide exchange factors, have focused on their role in cytoskeletal regulation. RESULTS: Here we demonstrate novel functions for the Rho family GTPases Rac and Rho in the opposing hormonal regulation of voltage-activated, ether-a-go-go-related potassium channels (ERG) in a rat pituitary cell line, GH(4)C(1). The hypothalamic neuropeptide, thyrotropin-releasing hormone (TRH) inhibits ERG channel activity through a PKC-independent process that is blocked by RhoA(19N) and the Clostridium botulinum C3 toxin, which inhibit Rho signaling. The constitutively active, GTPase-deficient mutant of RhoA(63L) rapidly inhibits the channels when the protein is dialysed directly into the cell through the patch pipette, and inhibition persists when the protein is overexpressed. In contrast, GTPase-deficient Rac1(61L) stimulates ERG channel activity. The thyroid hormone triiodothyronine (T3), which antagonizes TRH action in the pituitary, also stimulates ERG channel activity through a rapid process that is blocked by Rac1(17N) and wortmannin but not by RhoA(19N). CONCLUSIONS: Rho stimulation by G(13)-coupled receptors and Rac stimulation by nuclear hormones through PI3-kinase may be general mechanisms for regulating ion channel activity in many cell types. Disruption of these novel signaling cascades is predicted to contribute to several specific human neurological diseases, including epilepsy and deafness.     

Nadrin GAP activity is isoform- and target-specific regulated by tyrosine phosphorylation

Cytoskeletal reorganization is crucial for platelet adhesion and thrombus formation to avoid excessive bleeding. Major regulators of cytoskeletal dynamics are small GTPases of the Rho family. Rho GTPases become activated by G-protein coupled receptor activation, downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors and by outside-in signaling of integrins. They act as molecular switches and cycle between active and inactive states. GTPase activating proteins (GAPs) stimulate the hydrolysis of GTP to GDP to terminate Rho signaling. Nadrin is a RhoGAP that was recently identified in platelets. Five Nadrin isoforms are known consisting of a unique GAP and an N-terminal BAR domain responsible for the selective regulation of RhoA, Cdc42 and Rac1. Besides BAR domain mediated regulation of Nadrin GAP activity nothing is known about the regulation of Nadrin and the impact on cytoskeletal reorganization. Here we show that Nadrin becomes tyrosine phosphorylated upon platelet activation. We found Src family proteins (Src, Lyn, Fyn) to be responsible to control Nadrin GAP activity by phosphorylation. Interestingly, phosphorylation of Nadrin leads to tightly regulated Rho activation that was found to be Nadrin isoform- and (Rho) target-specific. Src-phosphorylation of Nadrin5 mediated inactivation of Cdc42 while RhoA and Rac1 became activated upon Src-mediated phosphorylation of Nadrin2. Our results suggest a critical role for spatial and temporal regulation of Nadrin and thus for the control of Rho GTPases in platelets.     

ARAP3 is a PI3K- and rap-regulated GAP for RhoA

Rho and Arf family small GTPases are well-known regulators of cellular actin dynamics. We recently identified ARAP3, a member of the ARAP family of dual GTPase activating proteins (GAPs) for Arf and Rho family GTPases, in a screen for PtdIns(3,4,5)P(3) binding proteins. PtdIns(3,4,5)P(3) is the lipid product of class I phosphoinositide 3OH-kinases (PI3Ks) and is a signaling molecule used by growth factor receptors and integrins in the regulation of cell dynamics. We report here that as a Rho GAP, ARAP3 prefers RhoA as a substrate and that it can be activated in vitro by the direct binding of Rap proteins to a neighbouring Ras binding domain (RBD). This activation by Rap is GTP dependent and specific for Rap versus other Ras family members. We found no evidence for direct regulation of ARAP3's Rho GAP activity by PtdIns(3,4,5)P(3) in vitro, but PI3K activity was required for activation by Rap in a cellular context, suggesting that PtdIns(3,4,5)P(3)-dependent translocation of ARAP3 to the plasma membrane may be required for further activation by Rap. Our results indicate that ARAP3 is a Rap-effector that plays an important role in mediating PI3K-dependent crosstalk between Ras, Rho, and Arf family small GTPases.     

Direct modifications of Rho proteins: deconstructing GTPase regulation

Small GTPases of the Rho protein family are master regulators of the actin cytoskeleton and are targeted by potent virulence factors of several pathogenic bacteria. Their dysfunctional regulation can lead to severe human pathologies. Both host and bacterial factors can activate or inactivate Rho proteins by direct post‐translational modifications: such as deamidation and transglutamination for activation, or ADP‐ribosylation, glucosylation, adenylylation and phosphorylation for inactivation. We review and compare these unconventional ways in which both host cells and bacterial pathogens regulate Rho proteins.     

PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes

Signaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch-induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho-guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12-myristate 13-acetate, indicating that phorbol ester-sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral-mediated gene transfer of wild-type (WT) and dominant-negative (DN) mutants of PKCalpha and delta, we found that stretch-induced activation of Rho GTPases and phosphorylation of Rho-GDI were mainly regulated by PKCalpha. PKCdelta was involved in regulation of the activation of Rac1. Stretch-induced increases in [(3)H]-leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCalpha and delta, suggesting that PKCalpha and delta are both required in stretch-induced hypertrophy, through Rho GTPases-mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen-activated protein (MAP) kinases. Stretch-stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCalpha and delta, and that of MKK3/p38 inhibited by DN PKCdelta. The phosphorylation of ERK and JNK induced by overexpression of WT PKCalpha, and the phosphorylation of p38 induced by WT PKCdelta, were regulated by Rho GTPases. This study represents the first evidence that PKCalpha and delta are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch-induced hypertrophic process.     

RHO GTPase Signaling for Axon Extension: Is Prenylation Important?

Many lines of evidence indicate the importance of the Rho family guanine nucleotide triphosphatases (GTPases) in directing axon extension and guidance. The signaling networks that involve these proteins regulate actin cytoskeletal dynamics in navigating neuronal growth cones. However, the intricate patterns that regulate Rho GTPase activation and signaling are not yet fully defined. Activity and subcellular localization of the Rho GTPases are regulated by post-translational modification. The addition of a geranylgeranyl group to the carboxy (C-) terminus targets Rho GTPases to the plasma membrane and promotes their activation by facilitating interaction with guanine nucleotide exchange factors and allowing sequestering by association with guanine dissociation inhibitors. However, it is unclear how these modifications affect neurite extension or how subcellular localization alters signaling from the classical Rho GTPases (RhoA, Rac1, and Cdc42). Here, we review recent data addressing this issue and propose that Rho GTPase geranylgeranylation regulates outgrowth.     

The polybasic region of Ras and Rho family small GTPases: a regulator of protein interactions and membrane association and a site of nuclear localization signal sequences

Many small GTPases in the Ras and Rho families have a C-terminal polybasic region (PBR) comprised of multiple lysines or arginines. The PBR controls diverse functions of these small GTPases, including their ability to associate with membranes, interact with specific proteins, and localize in subcellular compartments. Different signaling pathways mediated by Ras and Rho family members may converge when the small GTPases are directed by their PBRs to shared binding sites in specific proteins or at cell membranes. The PBR promotes the interactions of small GTPases with SmgGDS, which is a nucleocytoplasmic shuttling protein that stimulates guanine nucleotide exchange by small GTPases. The PBR of Rac1 was recently found to have a functional nuclear localization signal (NLS) sequence, which enhances the nuclear accumulation of protein complexes containing SmgGDS and Rac1. Sequence analysis demonstrates that canonical NLS sequences (K-K/R-x-K/R) are present in the PBRs of additional Ras and Rho family members, and are evolutionarily conserved across several phyla. These findings suggest that the PBR regulates the nucleocytoplasmic shuttling of some Ras and Rho family members when they are in protein complexes that are too large to diffuse through nuclear pores. These diverse functions of the PBR indicate its critical role in signaling by Ras and Rho family GTPases.