BIOLOGICAL OVERVIEW

Members of the Rho family of small GTPases are required for many of the morphogenetic processes required to shape the animal body. The activity of this family is regulated in part by a class of proteins known as RhoGTPase Activating Proteins (RhoGAPs) that catalyse the conversion of RhoGTPases to their inactive state. In a search for genes that regulate Drosophila morphogenesis, several lethal alleles of crossveinless-c (cv-c) have been isolated. Molecular characterisation reveals that cv-c encodes the RhoGAP protein RhoGAP88C. During embryonic development, cv-c is expressed in tissues undergoing morphogenetic movements; phenotypic analysis of the mutants reveals defects in the morphogenesis of these tissues. Genetic interactions between cv-c and RhoGTPase mutants indicate that Rho1, Rac1 and Rac2 are substrates for Cv-c, and suggest that the substrate specificity might be regulated in a tissue-dependent manner. In the absence of cv-c activity, tubulogenesis in the renal or Malpighian tubules fails and they collapse into a cyst-like sack. Further analysis of the role of cv-c in the Malpighian tubules demonstrates that its activity is required to regulate the reorganisation of the actin cytoskeleton during the process of convergent extension. In addition, overexpression of cv-c in the developing tubules gives rise to actin-associated membrane extensions. Thus, Cv-c function is required in tissues actively undergoing morphogenesis, and it is proposed that its role is to regulate RhoGTPase activity to promote the coordinated organisation of the actin cytoskeleton, possibly by stabilising plasma membrane/actin cytoskeleton interactions (Denholm, 2005).

Morphogenesis describes a complex set of cell behaviours by which a tissue is formed; these include changes in cell shape, cell rearrangement and coordinated cell movement. Experimental evidence gathered over the past 15 years shows that the Rho family of small GTPases, which include the Rho, Rac and Cdc42 proteins, are central to the regulation of these processes. Although this protein family has pleiotropic roles in diverse cellular and developmental contexts, a commonality in their function is in the regulation of the cytoskeleton, particularly the actin cytoskeleton (Denholm, 2005).

The Rho GTPase family of proteins act as 'molecular switches' that cycle between two conformational states: a GTP-bound state in which they are active, and a GDP-bound inactive state. The balance between these states is controlled principally by two classes of regulatory proteins: the guanine nucleotide exchange factors (GEFs) that promote the active state by facilitating the release of GDP and subsequent rebinding of GTP, and the GTPase activating proteins (GAPs) that promote the inactive state by catalysing the weak intrinsic GTP hydrolysing capacity of the GTPase, thereby converting it to the inactive form. In many cases, it is crucial that the correct balance between these two states is properly regulated. This is evident in cases in which they are not -- deregulated GTPases can have catastrophic developmental consequences and can lead to cellular pathologies such as cancer (Denholm, 2005).

In all animal genomes sequenced to date, there are considerably more genes encoding for GAPs and GEFs than the GTPases they regulate. For example, the human genome encodes ~20 Rho-family GTPases, but in excess of 50 different GAPs and GEFs; similarly, the Drosophila genome encodes seven Rho-family GTPases but 21 GAPs and 20 GEFs. The preponderance of GAPs and GEFs probably reflects the importance of controlling the activity of RhoGTPase family members. The 21 GAP proteins in Drosophila show considerable diversity in their domain architecture and this may provide context specificity for the multitude of functions and outcomes of GTPase signalling. Although much is known about the catalytic function of the GAPs and GEFs, relatively little is known about how they function in a cellular or developmental context, e.g., their spatial and temporal regulation and the factors that control their specificity. Of the 21 Drosophila RhoGAPs, five have been studied to date. In most cases, these studies have used either gain-of-function analyses or dominant-negative approaches; therefore, the phenotypic defects in mutants have not been analysed (Denholm, 2005).

Evidence is presented that viable alleles in the crossveinless-c (cv-c) gene are hypomorphic alleles of the RhoGAP88C gene. Three cv-c alleles have been described previously. These alleles are characterised by partial or complete loss of the posterior crossvein (PCV) and variable detachment of the anterior crossvein (ACV) in the wing (Diaz-Benjumea, 1990; Edmondson, 1952; Stern, 1934). Despite having been first described over 70 years ago by Curt Stern and used as a marker for recombination studies, the molecular nature of the locus has until now remained a mystery (Denholm, 2005).

Lethal alleles of cv-c have been generated, and phenotypic analyses of these show that cv-c is required in the embryo in multiple tissues undergoing morphogenesis including the Malpighian tubules, midgut, head, posterior spiracles and the epidermis during dorsal closure. The Malpighian tubules have been chosen to examine the loss- and gain-of-function cv-c phenotypes in more detail and it has been found that Cv-c is autonomously required to regulate actin cytoskeleton dynamics during morphogenesis, and possibly in stabilising links between the cytoskeleton and the plasma membrane. In addition, genetic interactions between cv-c and RhoGTPase mutants show that Cv-c has specificity towards Rho, Rac1 and Rac2, and that it might regulate these GTPases in a tissue-specific manner. These data therefore suggest that cv-c acts to ensure coordinated assembly of the actin cytoskeleton in many tissues undergoing morphogenesis by regulating the activity of specific RhoGTPases (Denholm, 2005).

In three independent screens to detect mutations affecting tissue morphogenesis, novel alleles of cv-c were isolated. Phenotypic analyses show that Cv-c function is required in embryonic tissues for specific morphogenetic events: in the gut for normal midgut morphogenesis and for Malpighian tubule elongation; and in the epidermis for the invagination of the posterior spiracle, head involution and ordered dorsal closure. cv-c encodes RhoGAP88C, a regulator of Rho-family GTPases; cv-c interacts genetically with Rho and Rac GTPases, possibly in a tissue-specific manner. Defects in Malpighian tubule morphogenesis in cv-c mutants correlate with defects in the organisation of the subcortical actin cytoskeleton, and overexpression of cv-c leads to stable actin-filled membrane extensions (Denholm, 2005).

The common feature linking the morphogenetic processes in which cv-c activity is required is the coordinated reorganisation of large groups of cells during tissue remodelling. Such processes involve choreographed cell movements as well as alterations in cell shape. Attention was foucused on the role of Cv-c in the morphogenesis of the Malpighian tubules since they undergo convergent extension movements. In contrast to cell polarity, which is relatively normal in cv-c mutant embryos, it was found that the organisation of the actin cytoskeleton is aberrant. The normal strong subcortical accumulation of F-actin is lost in cv-c mutant Malpighian tubules. In particular, the striking concentration of F-actin on the luminal membrane at the onset of tubule elongation fails (Denholm, 2005).

How do these defects in the actin cytoskeleton relate to the Malpighian tubule morphogenetic phenotype? By a process of convergent extension, starting at stage 13 and continuing until stage 16, the Malpighian tubule cells remodel from short fat tubes, with 10-12 cells surrounding the lumen, to long thin tubes, with only two cells encircling the lumen. The convergent extension movements in the Malpighian tubules are likely to occur in a manner similar to those described in the epidermis during germband extension, whereby a coordinated reorganisation of cell partners drives a change in tissue dimension. Central to this reorganisation is the remodelling of zonula adherens junctions (ZAs), which are progressively disassembled as ZAs between new cell partners are formed. Remodelling of the actin cytoskeleton is thought to drive these changes, both to facilitate alterations in cell shape and finally to stabilise the new junctional configuration. It is suggested that the defects in the actin cytoskeleton observed in cv-c tubules perturbs the cell rearrangements that underlie convergent extension movements required during tubule elongation. As the Malpighian tubule phenotype becomes progressively more severe during the period when cell rearrangement normally occurs, it is suggested that it is the coordination of cell reorganisation rather than cell intercalation per se that is defective. In addition, morphogenesis of the Malpighian tubules requires the maintenance of a tubular structure and this also fails in cv-c mutants. It is likely that the accumulation of actin at the luminal membrane is required both to remodel cell-cell associations and to retain the integrity of the lumen. It is therefore proposed that Cv-c plays an important regulatory role to ensure that the actin cytoskeleton is properly remodelled during tubule morphogenesis. Furthermore, since cv-c is required in multiple tissues, it is predicted that cv-c is reiteratively used during development in the regulation of actin cytoskeletal remodelling during morphogenesis (Denholm, 2005).

It seems likely that Cv-c acts to orchestrate the actin cytoskeleton via the direct regulation of RhoGTPase activity; the finding that cv-c⁷ is defective in a specific crucial residue required for GAP enzymatic activity provides strong support for this hypothesis. Although the genetic interaction experiments highlight potential substrates and furthermore suggest the possibility that substrates might be regulated in a tissue-specific manner, additional biochemical analyses will be necessary to confirm this (Denholm, 2005).

Cv-c function appears to be evolutionarily conserved. The rat and human cv-c homologues, p122 and DLC2 respectively, have been shown to inhibit Rho-mediated assembly of actin stress fibres when overexpressed in cell culture (Ching, 2003; Nagaraja, 2004; Sekimata, 1999). Furthermore, the C. elegans cv-c homologue, GEI-1, is thought to provide a molecular link with the actin assembly machinery. GEI-1, was isolated as a binding partner of GEX-2, one of a group of interacting proteins that localise to the plasma membrane and are required for the process of ventral enclosure, a morphogenetic event similar to dorsal closure in the fly (Soto, 2002). The recent molecular characterisation of GEX-1 as a WAVE-family protein (M. Soto, personal communication to Denholm, 2005) sheds light on the function of these proteins in morphogenesis. Proteins of the WAVE-family are known to relay signals from Rho-family GTPases to the actin cytoskeleton, possibly by permitting the assembly of multi-protein complexes, including components that act to nucleate actin (such as Arp2/3-binding and actin-binding proteins, GTPase proteins, GAPs and GEFs). Thus, a molecular link exists between the Gex proteins, GTPases, their regulators and the actin cytoskeleton. Cv-c and its homologues may provide GAP activity within this large multi-protein complex, contributing to dynamic regulation of the actin cytoskeleton. For these reasons, it will be interesting to determine the relationship between Cv-c and the Gex homologues in the fly, Sra1/Gex2 and Kette/Gex3, and the fly SCAR/WAVE (Denholm, 2005).

The demonstration that overexpression of cv-c is sufficient to induce stable membrane extensions supported by F-actin suggests that Cv-c acts to coordinate or stabilise interactions that occur between the plasma membrane and the actin cytoskeleton. The Cv-c protein contains two domains implicated in lipid membrane binding, the START and SAM domains, suggesting that Cv-c localises to a membrane domain. This would place Cv-c in an ideal position to regulate an interaction between the plasma membrane and the actin cytoskeleton. There is compelling evidence that moesin is required to organise the cortical actin network which it does, at least in part, by linking the actin cytoskeleton to the plasma membrane. Given that moesin has been shown to antagonise Rho1 by altering its activation state, it will also be interesting to examine the requirement of Cv-c for moesin function (Denholm, 2005).

GENE STRUCTURE

cDNA clone length - 4486

Bases in 5' UTR - 324

Exons - 8

Bases in 3' UTR - 1108

PROTEIN STRUCTURE

Amino Acids - 1017

Structural Domains

The Drosophila RhoGAP88C gene spans a region of 14 kb and contains eight exons. The BDGP database contains a single RhoGAP88C cDNA (RE02250) of 4.4 kb in length. Conceptual translation of this cDNA reveals an open reading frame of 1017 amino acids. In addition to the GAP domain, RhoGAP88C contains two other previously described domains: a sterile alpha motif (SAM), originally defined as a protein-protein interaction domain, but more recently implicated in RNA and lipid membrane-binding; and a lipid transfer START domain so called because it was initially isolated in the steroidogenic acute regulatory (StAR) protein (Denholm, 2005).

Database searches identify several closely related RhoGAP proteins containing identical domain architecture to Drosophila RhoGAP88C. The closest related vertebrate proteins are the human deleted in liver cancer 1 and 2 [DLC1 and DLC2 (STARD13 -- Human Gene Nomenclature Database)], the mouse serologically defined colon cancer 13 antigen, and rat p122-RhoGAP. A C. elegans protein, gut on exterior-interacting protein (GEI) also shares a high level of identity with Drosophila RhoGAP88C in the GAP and START domains, but lacks the SAM motif (Denholm, 2005).

During body morphogenesis precisely coordinated cell movements and cell shape changes organize the newly differentiated cells of an embryo into functional tissues. Two genes, gex-2 and gex-3, are necessary for initial steps of body morphogenesis in Caenorhabditis elegans. In the absence of gex-2 and gex-3 activities, cells differentiate properly but fail to become organized. The external hypodermal cells fail to spread over and enclose the embryo and instead cluster on the dorsal side. Postembryonically gex-3 activity is required for egg laying and for proper morphogenesis of the gonad. GEX-2 and GEX-3 proteins colocalize to cell boundaries and appear to directly interact. GEX-2 and GEX-3 are highly conserved, with vertebrate homologs implicated in binding the small GTPase Rac and a GEX-3 Drosophila homolog, HEM2/NAP1/KETTE, that interacts genetically with Rac pathway mutants. These findings suggest that GEX-2 and GEX-3 may function at cell boundaries to regulate cell migrations and cell shape changes required for proper morphogenesis and development (Sato, 2002).

A signaling molecule, p122, shows a GTPase-activating activity specific for Rho and exhibits the ability to enhance the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C delta1 in vitro. The in vivo function of p122 has been analyzed. Microinjection of the GTPase-activating domain of p122 suppresses the formation of stress fibers and focal adhesions induced by lysophosphatidic acid, suggesting a GTPase-activating activity for Rho. Transfection of p122 induces the disassembly of stress fibers and the morphological rounding of various adherent cells. Analyses using deletion and point mutants demonstrates that the GTPase-activating domain of p122 is responsible for the morphological changes and detachment and that arginine residues at positions 668 and 710 and a lysine residue at position 706 in the GTPase-activating domain are essential. Using Fluo-3-based Ca2+ microscopy, it was found that p122 evokes a rapid elevation of intracellular Ca2+ levels, suggesting that p122 stimulates the phosphatidylinositol 4, 5-bisphosphate-hydrolyzing activity of phospholipase C delta1. These results demonstrate that p122 synergistically functions as a GTPase-activating protein specific for Rho and an activator of phospholipase C delta1 in vivo and induces morphological changes and detachment through cytoskeletal reorganization (Sekimata, 1999).

Hepatocellular carcinoma (HCC) is a major malignancy in many parts of the world, especially in Asia and Africa. Loss of heterozygosity (LOH) on the long arm of chromosome 13 has been reported in HCC. In search of tumor suppressor genes in this region, DLC2 (for deleted in liver cancer 2) has been identified at 13q12.3; DLC2 encodes a novel Rho family GTPase-activating protein (GAP). DLC2 mRNA is ubiquitously expressed in normal tissues but is significantly underexpressed in 18% (8/45) of human HCCs. DLC2 is homologous to DLC1, a previously identified tumor suppressor gene at 8p22-p21.3 frequently deleted in HCC. DLC2 encodes a novel protein with a RhoGAP domain, a SAM (sterile alpha motif) domain related to p73/p63, and a lipid-binding StAR-related lipid transfer (START) domain. Biochemical analysis indicates that DLC2 protein has GAP activity specific for small GTPases RhoA and Cdc42. Expression of the GAP domain of DLC2 sufficiently inhibits the Rho-mediated formation of actin stress fibers. Introduction of human DLC2 into mouse fibroblasts suppresses Ras signaling and Ras-induced cellular transformation in a GAP-dependent manner. Taken together, these findings suggest a role for DLC2 in growth suppression and hepatocarcinogenesis (Ching, 2003).

The cDNA for a Rho GTPase activating protein (GAP) mapping to chromosome 13q12 has been characterized. The cDNA is characterized by determining the complete sequence of a 4.8 kb cDNA clone that represents the 5' untranslated region (UTR), the translated region, and the 3' UTR. The protein has a sterile alpha-motif (SAM), a distinct GAP domain, and a conserved START (StAR related lipid transfer) domain. The cDNA has 5 instability motifs (ATTTA) in the 3' UTR and one motif in the translated region between GAP and START domains. The RhoGAP transcript is truncated in some breast carcinoma cell lines and it has low expression in other breast cancer cell lines as compared to a normal breast cell line. The absence of RhoGAP transcript has been observed in a breast tumor specimen. A GST-fusion of the RhoGAP was tested for its specificity on RhoA, Cdc42, and Rac1. The protein was most active for RhoA. Transfection of RhoGAP into MCF7 cells significantly inhibits cell growth. The introduction of the RhoGAP construct into MDAMB231 cells that had previously been transfected with a p21 construct did not affect cell proliferation, indicating the involvement of p21 in Rho-mediated proliferation of cancer cells. NIH3T3 cells overexpressing RhoGAP showed considerable inhibition of stress fiber formation. Several cDNAs were identified as RhoGAP interactors by using the yeast two-hybrid assay system. These cDNAs correspond to SWI/SNF, alpha-tubulin, HMG CoA reductase, and TAX1 binding protein (TAX1BP1). The interaction with HMG CoA reductase may partially explain the growth inhibition of breast carcinoma cells by the statin class of cholesterol lowering drugs. The biological significance of the interacting proteins is discussed in the context of their involvement in tumorigenesis. These results indicate that loss of RhoGAP or its altered activity suppresses the growth of breast tumor cells. The presence of various motifs in RhoGAP and its interaction with several other proteins suggest that the protein may regulate Rho signaling in multiple ways and possibly function in a Rho-independent manner (Nagaraja, 2004).

date revised: 2 February 2023

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