Rho GEF in yeast

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases. Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 have been identified in a screen for high-copy number suppressors of cik1 delta, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11 degrees C, and at 37 degrees C. rom2 delta cells exhibit morphological defects. At permissive temperatures, rom2 delta cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1 delta and kar3 delta strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. rom2 delta strains exhibit increased sensitivity to the microtubule depolymerizing drug, benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and the functions of the microtubule cytoskeleton (Manning, 1997).

ect-2, a C. elegans homolog of Pebble

A reduction-of-function mutation in ect-2 was isolated as a suppressor of the Multivulva phenotype of a lin-31 mutation. Analysis using markers indicates that this mutation causes defects in both the cytokinesis and migration of epidermal P cells, phenotypes similar to those caused by expressing a rho-1 dominant-negative construct. ect-2 encodes the Caenorhabditis elegans orthologue of the mouse Ect2 and Drosophila Pebble that function as guanine nucleotide exchange factors (GEFs) for Rho GTPases. The ect-2::GFP reporter is expressed in embryonic cells and P cells. RNA interference of ect-2 causes sterility and embryonic lethality, in addition to the P-cell defects. The lesions of two ect-2 alleles have been determined, and their differences in phenotypes in specific tissues described. A model is proposed in which ECT-2GEF not only activates RHO-1 for P-cell cytokinesis, but also collaborates with UNC-73GEF and at least two Rac proteins to regulate P-cell migration (Morita, 2005).

The mechanism by which the Rho GEF proteins are regulated for the cell migration function is at present not clear in C. elegans or other systems. The mechanism acting downstream of Rho for the P-cell migration function also remains to be understood. A loss-of-function mutation in let-502, which encodes a Rho-activated kinase, shows a partially penetrant defect in P-cell migration but not a defect in cytokinesis, suggesting that let-502 is involved in the migration process. However, its weak phenotype suggests that there are other Rho effectors acting in the process (Morita, 2005).

ECT2, a mammalian homolog of Pebble

An efficient expression cloning system has been developed that allows rapid isolation of complementary DNAs able to induce the transformed phenotype. A search has been carried out for protein expressed in epithelial cells and possessing transforming potential to fibroblasts. A novel transforming gene, ect2, has been identified. The isolated cDNA is activated by amino-terminal truncation of the normal product. The Ect2 protein has sequence similarity within a central core of 255 amino acids with the products of the breakpoint cluster gene, bcr, the yeast cell cycle gene, CDC24, and the dbl oncogene. Each of these genes encodes regulatory molecules or effectors for Rho-like small GTP-binding proteins. The baculovirus-expressed Ect2 protein can bind with high specificity to Rho and Rac proteins, whereas the dbl product shows broader binding specificity to Rho family proteins. Thus ect2 is a new member of an expanding family, whose products have transforming properties and interact with Rho-like proteins of the Ras superfamily (Miki, 1993).

Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. The human ECT2 is shown to be involved in the regulation of cytokinesis. ECT2 catalyzes guanine nucleotide exchange on the small GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibits nuclear localization in interphase, spreads throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the NH(2)-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis. Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division (Tatsumoto, 1999).

Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), it has been shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. In Rat1A cells RhoA activity remains suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibits cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibits cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 is suppressed during cytokinesis and starts increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression is essential for cytokinesis because a constitutively active mutant of Rac1 induces a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was demonstrated by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delays the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells. It is concluded that RoA acitivity during cytokinesis exhibits cell type specificity (Yoshizaki, 2004).

Other mammalian Rho GEFs

Lfc and Lsc are two recently identified oncoproteins that contain a Dbl homology domain in tandem with a pleckstrin homology domain and thus share sequence similarity with a number of other growth regulatory proteins including Dbl, Tiam-1, and Lbc. Lfc and Lsc, like their closest relative Lbc, are highly specific guanine nucleotide exchange factors (GEFs) for Rho, causing a >10-fold stimulation of [3H]GDP dissociation from Rho and a marked stimulation of GDP-[35S]GTPgammas (guanosine 5'-O-(3-thiotriphosphate) exchange. All three proteins (Lbc, Lfc, and Lsc) are able to act catalytically in stimulating the guanine nucleotide exchange activity, such that a single molecule of each of these oncoproteins can activate a number of molecules of Rho. Neither Lfc nor Lsc shows any ability to stimulate GDP dissociation from other related GTP-binding proteins such as Rac, Cdc42, or Ras. Thus Lbc, Lfc, and Lsc appear to represent a subgroup of Dbl-related proteins that function as highly specific GEFs toward Rho and can be distinguished from Dbl, Ost, and Dbs, which are less specific and show GEF activity toward both Rho and Cdc42. Consistent with these results, Lbc, Lfc, and Lsc each form tight complexes with the guanine nucleotide-depleted form of Rho and bind weakly to the GDP- and GTPgammaS-bound states. None of these oncoproteins are able to form complexes with Cdc42 or Ras. However, Lfc (but not Lbc nor Lsc) can bind to Rac, and this binding occurs equally well when Rac is nucleotide-depleted or is in the GDP- or GTPgammaS-bound state. These findings raise the possibility that in addition to acting directly as a GEF for Rho, Lfc may play other roles that influence the signaling activities of Rac and/or coordinate the activities of the Rac and Rho proteins (Glaven, 1996).

A cDNA for a novel human protein named CDEP was cloned using the subtractive hybridization method between dedifferentiated cartilage cells and overtly differentiated cartilage cells. CDEP cDNA contains an open reading frame encoding 1,045 amino acids with a total length of 3.4 kb. The deduced amino acid sequence reveals that a single polypeptide contains the ezrin-like domain, which is found in cytoskeleton-associated proteins of the band 4.1 superfamily, and the Dbl homology (DH) and pleckstrin homology (PH) domains, which are conserved in the Rho GEF (guanine nucleotide exchange factor) family. Northern blot analysis demonstrates that CDEP mRNA is expressed not only in the differentiated chondrocytes but also in various fetal and adult tissues. Since members of the band 4.1 superfamily and the Rho GEF family are crucial for microfilament organization, the novel protein CDEP may be involved in the adhesion, proliferation, and differentiation of some cell types, including chondrocytes via changes in the cytoskeleton (Koyano, 1997).

A cDNA for a novel human protein named CDEP was cloned using the subtractive hybridization method between dedifferentiated cartilage cells and overtly differentiated cartilage cells. CDEP cDNA contains an open reading frame encoding 1,045 amino acids in a total length of 3.4 kb. The deduced amino acid sequence reveals that a single polypeptide contains the ezrin-like domain, which is found in cytoskeleton-associated proteins of the band 4.1 superfamily, and the Dbl homology (DH) and pleckstrin homology (PH) domains, which are conserved in the Rho GEF (guanine nucleotide exchange factor) family. Northern blot analysis demonstrates that CDEP mRNA is expressed not only in the differentiated chondrocytes but also in various fetal and adult tissues. Since members of the band 4.1 superfamily and the Rho GEF family are crucial for microfilament organization, the novel protein CDEP may be involved in the adhesion, proliferation, and differentiation of some cell types, including chondrocytes via changes in the cytoskeleton (Koyano, 1997).

Two different families of Rho GEFs have been identified that differ in the structure of their catalytic domains. The first group is composed of Rho GDP dissociation stimulators (GDS), a family of proteins distantly related to the Cdc25 homology regions present in Ras GEFs. GDSs work at stoichiometric concentrations and have a rather broad catalytic specificity, being active on prenylated K-Ras, Rho and Rap proteins. The second subset of Rho activators comprises an extensive number of enzymes containing Dbl-homology (DH) domains with catalytic activity exclusively directed toward Rho/Rac GTPases. The majority of these GEFs are highly transforming when overexpressed either as wild-type or truncated proteins, a property that highlights their importance as regulators of mitogenic processes. Although Rho GEFs have been characterized extensively both biochemically and oncogenically, little information is available regarding the mechanism by which they become activated during signal transduction. To date, the best example for the participation of a DH-containing protein in receptor-mediated cell signaling is the product of the vav proto-oncogene, a protein preferentially expressed in the hematopoietic system. Vav-2, a member of the Vav family of oncoproteins, acts as a guanosine nucleotide exchange factor (GEF) for RhoG and RhoA-like GTPases in a phosphotyrosine-dependent manner. Vav-2 oncogenic activation correlates with the acquisition of phosphorylation-independent exchange activity. In vivo, wild-type Vav-2 is activated oncogenically by tyrosine kinases, an effect enhanced further by co-expression of RhoA. Likewise, the Vav-2 oncoprotein synergizes with RhoA and RhoB proteins in cellular transformation. Transient transfection assays in NIH-3T3 cells show that phosphorylated wild-type Vav-2 and the Vav-2 oncoprotein induce cytoskeletal changes resembling those observed by the activation of the RhoG pathway. In contrast, the constitutive expression of the Vav-2 oncoprotein in rodent fibroblasts leads to major alterations in cell morphology and to highly enlarged cells in which karyokinesis and cytokinesis frequently are uncoupled. These results identify a regulated GEF for the RhoA subfamily, provide a biochemical explanation for vav family oncogenicity, and establish a new signaling model in which specific Vav-like proteins couple tyrosine kinase signals with the activation of distinct subsets of the Rho/Rac family of GTPases (Schuebel, 1998).

The Rho-related small GTPases are critical elements involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. The Dbl-like guanine nucleotide exchange factors (GEF) have been implicated in direct activation of these GTPases. A new member of the Dbl family, GEF-H1, has been identified by screening a human HeLa cell cDNA library. GEF-H1 encodes a 100-kDa protein containing the conserved structural array of a Dbl homology domain in tandem with a pleckstrin homology domain and is most closely related to the lfc oncogene, but additionally it contains a unique coiled-coil domain at the carboxyl terminus. Biochemical analysis reveals that GEF-H1 is capable of stimulating guanine nucleotide exchange of Rac and Rho but is inactive toward Cdc42, TC10, or Ras. Moreover, GEF-H1 binds to Rac and Rho proteins in both the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound states without detectable affinity for Cdc42 or Ras. Immunofluorescence reveals that GEF-H1 colocalizes with microtubules through the carboxyl-terminal coiled-coil domain. Overexpression of GEF-H1 in COS-7 cells results in induction of membrane ruffles. These results suggest that GEF-H1 may have a direct role in activation of Rac and/or Rho and in bringing the activated GTPase to specific target sites such as microtubules (Ren, 1998).

Rho GEFs act downstream of G-proteins

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. A consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) was used to search DNA data bases, and a novel human GEF for Rho-related GTPases was identified harboring structural features indicative of its possible regulatory mechanism(s). This protein contains a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF. The repeat domain has been termed the Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activates biological and biochemical pathways specific for Rho; activation of these pathways requires an intact DH and PH domain. However, the PDZ domain is dispensable for these functions, and mutants lacking the LH domain are more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain reveals a limited homology with the catalytic region of RGS14, a newly identified GTPase-activating protein for heterotrimeric G proteins. This prompted an investigation to see whether PDZ-RhoGEF could interact with representative members of each G protein family. PDZ-RhoGEF is able to form, in vivo, stable complexes with two members of the Galpha12 family (Galpha12 and Galpha13); this interaction is mediated by the LH domain. Evidence suggests that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: they appear to do this by stimulating the activity of members of the Galpha12 family, which, in turn, activate an exchange factor acting on Rho (Fukuhara, 1999).

Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho protein control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 binds tightly to p115 RhoGEF and stimulates its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibits stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho (Hart, 1998).

Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 have specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12. A family of mammalian RGS-containing proteins has been identified, and most members stimulate the GTPase activity of mammalian G proteins. The RGS sequence therefore defines a family of GTPase-activating proteins (GAPs) capable of downregulating heterotrimeric G proteins. RhoGEF is the first identified target for the G12/13 family of G proteins and serves as a biochemical link between G-protein coupled receptors and activation of Rho (Kozasa, 1998).

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein alpha subunits have been characterized in transfection systems. Galphaq, Galpha12, and Galpha13 (but not Galphai) activate SRF through RhoA. When Galphaq, alpha12, or alpha13 are coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Galpha13 (but not Galphaq or Galpha12) shows synergistic activation of SRF with GEF115. The synergy between Galpha13 and GEF115 depends on the N-terminal part of GEF115, and there is no synergistic effect between Galpha13 and another Rho-specific exchange factor: Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibits Galpha13- and Galpha12-induced SRF activation, but not GEF115- or Galphaq-induced SRF activation. The DH-domain-deletion mutant also suppresses thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Galpha12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Galpha12 and Galpha13. Thus, the inhibition of Galpha12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Galpha13 and GEF115 indicates that GEF115 mediates Galpha13-induced activation of Rho and SRF (Mao, 1998).

Rho GEF structure and protein interactions

Inherited mutations in BRCA1 predispose to breast and ovarian cancer, but an understanding of the biological function of the BRCA1 protein has remained largely elusive. The BRCA1 C-terminal region is important for BRCA1-mediated breast cancer suppression, since this domain shows similarities with the C-terminal regions of a p53-binding protein (53BP1), the yeast RAD9 protein involved in DNA repair, and two uncharacterized, hypothetical proteins (KIAA0170 and SPAC19G10.7). The highlighted domain has been suggested to be the result of an internal duplication, each of the tandem domains being designated as a 'BRCT domain' (for BRCA1 C-terminus). Sequence analysis using hydrophobic cluster analysis reveals the presence of 50 copies of the BRCT domain in 23 different proteins, including BRCA1, 53BP1, RAD9, XRCC1, RAD4, Ect2, REV1, Crb2, RAP1, terminal deoxynucleotidyltransferases (TdT) and three eukaryotic DNA ligases. Most of these proteins are known to be involved in DNA repair. The BRCT domain is not limited to the C-termini of protein sequences and can be found in multiple copies or in a single copy as in RAP1 and TdT, suggesting that it could well constitute an autonomous folding unit of approx. 90-100 amino acids (Callebaut, 1997).

The pleckstrin homology (PH) domain is an approximately 100 amino acid structural motif found in many cellular signaling molecules, including the Dbl oncoprotein and related, putative guanine nucleotide exchange factors (GEFs). The role of the Dbl PH (dPH) domain has been examined in the activities of oncogenic Dbl. The dPH domain is not involved in the interaction of Dbl with small GTP-binding proteins and is incapable of transforming NIH 3T3 fibroblasts. In contrast, co-expression of the dPH domain with oncogenic Dbl inhibits Dbl-induced transformation. A deletion mutant of Dbl, which lacks a significant portion of the PH domain, retains full GEF activity, but is completely inactive in transformation assays. Replacement of the PH domain by the membrane-targeting sequence of Ras is not sufficient for the recovery of transforming activity. However, subcellular fractionations of Dbl and Dbl mutants reveal that the PH domain is necessary and sufficient for the association of Dbl with the Triton X-100-insoluble cytoskeletal components. Thus, these results suggest that the dPH domain mediates cellular transformation by targeting the Dbl protein to specific cytoskeletal locations to activate Rho-type small GTP-binding proteins (Zheng, 1996).

Rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of Rho bound GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase) (see Drosophila Dlar), a cDNA was isolated encoding a 2861-amino acid protein termed Trio, containing three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase that similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. Since the LAR PTPase localizes to the ends of focal adhesions, it is proposed that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration (Debant, 1996).

The DH domain protein mNET1, a Rho-family guanine nucleotide exchange factor (GEF) has been characterized. N-terminal truncation of mNET1 generates an activated transforming form of the protein, mNET1DeltaN, which acts as a GEF for RhoA but not Cdc42 or Rac1. In NIH 3T3 cells, activated mNET1 induces formation of actin stress fibers and potentiates activity of the transcription factor serum response factor. Inhibitor studies show that these processes are dependent on RhoA and independent of Cdc42 or Rac1. However, in contrast to the GTPase-deficient RhoA.V14 mutant, expression of activated mNET1 also activates the SAPK/JNK pathway. This requires mNET1 GEF activity, since activation is blocked by point mutations in mNET1's DH domain and its C-terminal pleckstrin homology (PH) domain, and by the dominant-interfering RhoA mutant RhoA.N19. Although mNET1DeltaN-induced SAPK/JNK activation requires a C3 transferase-sensitive GTPase, activation occurs independent of the generation of titratable GTP-bound RhoA. Thus, mNET1 can activate signaling pathways in addition to those directly controlled by activated RhoA (Alberts, 1998).

Epithelial Cell Transforming protein 2 (Ect2) is a guanine nucleotide exchange factor (GEF) for Rho GTPases (molecular switches essential for the control of cytokinesis in mammalian cells). Aside from the canonical DH/PH cassette found in virtually all Dbl family members, Ect2 contains N-terminal tandem BRCT domains, which are also present in a number of proteins involved in cell cycle checkpoints and DNA damage response signaling pathways. The role of the Ect2 BRCT domains in the regulation of Ect2 activity and cytokinesis has been investigated. It has been shown that the depletion of endogenous Ect2 by small interfering RNA induces multinucleation, suggesting that Ect2 is required for cytokinesis. In addition, evidence is provided that Ect2 normally exists in an inactive conformation, which is at least partially due to an intramolecular interaction between the BRCT domains and the C-terminal DH/PH domain of Ect2. This intramolecular interaction masks the catalytic domain responsible for guanine nucleotide exchange toward RhoA. Consistent with a role in regulating Ect2 GEF activity, overexpression of a N-terminal Ect2 containing the tandem BRCT domains, but not single BRCT domain or BRCT domain mutant, leads to a failure in cytokinesis. Surprisingly, although ectopically expressed wild-type Ect2 rescues the multinucleation resulting from the depletion of endogenous Ect2, expression of a BRCT mutant of Ect2 failed to restore proper cytokinesis in these cells. Taken together, this study indicates that the tandem BRCT domains of Ect2 play dual roles in the regulation of Ect2. While these domains negatively regulate Ect2 GEF activity in interphase cells, they are also required for the proper function of Ect2 during cytokinesis (Kim, 2004).

Nucleotide exchange factor ECT2 interacts with the polarity protein complex Par6/Par3/protein kinase Czeta (PKCzeta) and regulates PKCzeta activity

Regulation of cell polarity is an important biological event that governs diverse cell functions such as localization of embryonic determinants and establishment of tissue and organ architecture. The Rho family GTPases and the polarity complex Par6/Par3/atypical protein kinase C (PKC) play a key role in the signaling pathway, but the molecules that regulate upstream signaling are still not known. The guanine nucleotide exchange factor ECT2 has been identified as an activator of the polarity complex. ECT2 interacts with Par6 as well as Par3 and PKCzeta. Coexpression of Par6 and ECT2 efficiently activates Cdc42 in vivo. Overexpression of ECT2 also stimulates the PKCzeta activity, whereas dominant-negative ECT2 inhibits the increase in PKCzeta activity stimulated by Par6. ECT2 localization was detected at sites of cell-cell contact as well as in the nucleus of MDCK cells. The expression and localization of ECT2 are regulated by calcium, which is a critical regulator of cell-cell adhesion. Together, these results suggest that ECT2 regulates the polarity complex Par6/Par3/PKCzeta and possibly plays a role in epithelial cell polarity (Liu, 2004).

Ect2, an ortholog of Drosophila Pebble, regulates formation of growth cones in primary cortical neurons.

in vivo RNA interference (RNAi) genome-wide screening in Drosophila embryos has revealed that Pebble is involved in Drosophila neuronal development. Depletion of Ect2, a mammalian ortholog of Pebble, induces differentiation in NG108-15 neuronal cells. The precise role of Ect2 in neuronal development has yet to be studied. This study confirmed in PC12 pheochromocytoma cells that inhibition of Ect2 expression by RNAi stimulated neurite outgrowth, and in the mouse embryonic cortex Ect2 accumulates throughout the ventricular and subventricular zones with neuronal progenitor cells. The effects of Ect2 depletion were studied in primary cultures of mouse embryonic cortical neurons: Loss of Ect2 did not affect the differentiation stages of neuritogenesis, the number of neurites, or axon length, while the numbers of growth cones and growth cone-like structures were increased. Taken together, these results suggest that Ect2 contributes to neuronal morphological differentiation through regulation of growth cone dynamics (Tsuji, 2012).

Ect2 and transformation

Ect2 was identified originally as a transforming protein and a member of the Dbl family of Rho guanine nucleotide exchange factors (GEFs). Like all Dbl family proteins, Ect2 contains a tandem Dbl homology (DH) and pleckstrin homology (PH) domain structure. N-terminal deletion of sequences upstream of the DH domain creates a constitutively activated, transforming variant of Ect2 (designated DeltaN-Ect2 DH/PH/C), indicating that the N terminus serves as a negative regulator of DH domain function in vivo. The role of sequences C-terminal to the DH domain has not been established. Therefore, the consequences of mutation of C-terminal sequences on Ect2-transforming activity was assessed. Surprisingly, in contrast to observations with other Dbl family proteins, mutation of the invariant tryptophan residue in the PH domain does not impair DeltaN-Ect2 DH/PH/C transforming activity. Furthermore, although the sequences C-terminal to the PH domain lack any known functional domains or motifs, deletion of these sequences (DeltaN-Ect2 DH/PH) results in a dramatic reduction in transforming activity. Whereas DeltaN-Ect2 causes formation of lamellipodia, DeltaN-Ect2 DH/PH enhances actin stress fiber formation, suggesting that C-terminal sequences influence Ect2 Rho GTPase specificity. Consistent with this possibility, it was determined that DeltaN-Ect2 DH/PH activates RhoA, but not Rac1 or Cdc42, whereas DeltaN-Ect2 DH/PH/C activates all three Rho GTPases in vivo. Taken together, these observations suggest that regions of Ect2 C-terminal to the DH domain alter the profile of Rho GTPases activated in vivo and consequently may contribute to the enhanced transforming activity of DeltaN-Ect2 DH/PH/C (Solski, 2004).

pebble: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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