The human ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho GTPases and regulates cytokinesis. Although the oncogenic form of ECT2 contains an N-terminal truncation, it is not clear how the structural abnormality of ECT2 causes malignant transformation. Here we show that both the removal of the negative regulatory domain and alteration of subcellular localization are required to induce the oncogenic activity of ECT2. The transforming activity of oncogenic ECT2 was strongly inhibited by dominant negative Rho GTPases, suggesting the involvement of Rho GTPases in ECT2 transformation. Although deletion of the N-terminal cell cycle regulator-related domain (N) of ECT2 did not activate its transforming activity, removal of the small central domain (S), which contains two nuclear localization signals (NLSs), significantly induced the activity. The ECT2 N domain interacted with the catalytic domain and significantly inhibited the focus formation by oncogenic ECT2. Interestingly, the introduction of the NLS mutations in the S domain of N-terminally truncated ECT2 dramatically induced the transforming activity of this otherwise nononcogenic derivative. Among the known Rho GTPases expressed in NIH 3T3 cells, RhoA was predominantly activated by oncogenic ECT2 in vivo. Therefore, the mislocalization of structurally altered ECT2 might cause the untimely activation of cytoplasmic Rho GTPases leading to the malignant transformation.The ECT2 oncogene has been isolated in a search for mitogenic signal transducers in epithelial cells, where a murine keratinocyte expression cDNA library was introduced into fibroblasts to induce foci of morphologically transformed cells (1). The ECT2 transfectants exhibit anchorage-independent cell growth and efficient tumor formation in nude mice. The transforming ECT2 cDNA encodes the C-terminal half of the full-length protein containing Dbl-homology (DH) 1 and pleckstrin homology (PH) domains, which are now found in a number of molecules involved in regulation of the Rho family GTPases. The N-terminal half of ECT2 contains domains related to cell cycle control and repair proteins, including Clb6 and Rad4/Cut5 (2, 3). CLB6 encodes a B-type cyclin of the budding yeast, which promotes the transition from G 1 into S phase (4). Fission yeast cut5, which is identical to the repair gene rad4, is required for both the onset of S phase and the restraint of M phase before the completion of S phase (5). The Cut5-related domain of ECT2 consists of two repeats (6, 7), designated BRCT (BRCA1 C-terminal) repeats, which are widespread in a number of cell-cycle checkpoint control and DNA repair proteins (7). These cell-cycle regulator-related domains of ECT2 play essential roles on the regulation of cytokinesis (2, 3).ECT2 catalyzes guanine nucleotide exchange in vitro on three representative Rho GTPases; RhoA, Rac1, and Cdc42 (2). The Rho family of small GTPases function as molecular switches of diverse biological functions, including cytoplasmic actin reorganization, cell motility, and...

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. Here we identified the guanine nucleotide exchange factor ECT2 as an activator of the polarity complex. ECT2 interacted with Par6 as well as Par3 and PKC. Coexpression of Par6 and ECT2 efficiently activated Cdc42 in vivo. Overexpression of ECT2 also stimulated the PKC activity, whereas dominant-negative ECT2 inhibited the increase in PKC 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 were regulated by calcium, which is a critical regulator of cell-cell adhesion. Together, these results suggest that ECT2 regulates the polarity complex Par6/Par3/PKC and possibly plays a role in epithelial cell polarity.

Background:Thyroid hormone receptors (TRs) are critical endocrine receptors that regulate a multitude of processes in adult and developing organisms, and thyroid hormone disruption is of high concern for neurodevelopmental and reproductive toxicities in particular. To date, only a small number of chemical classes have been identified as possible TR modulators, and the receptors appear highly selective with respect to the ligand structural diversity. Thus, the question of whether TRs are an important screening target for protection of human and wildlife health remains.Objective:Our goal was to evaluate the hypothesis that there is limited structural diversity among environmentally relevant chemicals capable of modulating TR activity via the collaborative interagency Tox21 project.Methods:We screened the Tox21 chemical library (8,305 unique structures) in a quantitative high-throughput, cell-based reporter gene assay for TR agonist or antagonist activity. Active compounds were further characterized using additional orthogonal assays, including mammalian one-hybrid assays, coactivator recruitment assays, and a high-throughput, fluorescent imaging, nuclear receptor translocation assay.Results:Known agonist reference chemicals were readily identified in the TR transactivation assay, but only a single novel, direct agonist was found, the pharmaceutical betamipron. Indirect activation of TR through activation of its heterodimer partner, the retinoid-X-receptor (RXR), was also readily detected by confirmation in an RXR agonist assay. Identifying antagonists with high confidence was a challenge with the presence of significant confounding cytotoxicity and other, non-TR-specific mechanisms common to the transactivation assays. Only three pharmaceuticals—mefenamic acid, diclazuril, and risarestat—were confirmed as antagonists.Discussion:The results support limited structural diversity for direct ligand effects on TR and imply that other potential target sites in the thyroid hormone axis should be a greater priority for bioactivity screening for thyroid axis disruptors. https://doi.org/10.1289/EHP5314

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin-coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin-coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells.