Nucleotide Exchange Factor ECT2 Interacts with the Polarity Protein Complex Par6/Par3/Protein Kinase Cζ (PKCζ) and Regulates PKCζ 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. 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 PKCζ. Coexpression of Par6 and ECT2 efficiently activated Cdc42 in vivo. Overexpression of ECT2 also stimulated the PKCζ activity, whereas dominant-negative ECT2 inhibited the increase in PKCζ 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/PKCζ and possibly plays a role in epithelial cell polarity

Novel Kelch-like Protein, KLEIP, Is Involved in Actin Assembly at Cell-Cell Contact Sites of Madin-Darby Canine Kidney Cells

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

Novel biosensor for high-throughput detection of progesterone receptor-interacting endocrine disruptors

Abstract Progesterone receptor (PR)-interacting compounds in the environment are associated with serious health hazards. However, methods for their detection in environmental samples are cumbersome. We report a sensitive activity-based biosensor for rapid and reliable screening of progesterone receptor (PR)-interacting endocrine disrupting chemicals (EDCs). The biosensor is a cell line which expresses nuclear mCherry-NF1 and a green fluorescent protein (GFP)-tagged chimera of glucocorticoid receptor (GR) N terminus fused to the ligand binding domain (LBD) of PR (GFP-GR-PR). As this LBD is shared by the PRA and PRB, the biosensor reports on the activation of both PR isoforms. This GFP-GR-PR chimera is cytoplasmic in the absence of hormone and translocates rapidly to the nucleus in response to PR agonists or antagonists in concentration- and time-dependent manner. In live cells, presence of nuclear NF1 label eliminates cell fixation and nuclear staining resulting in efficient screening. The assay can be used in screens for novel PR ligands and PR-interacting contaminants in environmental samples. A limited screen of river water samples indicated a widespread, low-level contamination with PR-interacting contaminants in all tested samples