RalA binds to ZONAB in a GTP-dependent manner via the ZONAB CSD. (A) Specificity test for RalA (bait) and Ras-Clone4 (prey). DNA plasmid isolated from candidate clone #4 was subjected to restriction digest and the library plasmid was identified. The plasmid was used to cotransfect Cdc25-2 cells with the Met expression vector encoding the original bait, RalA (G23V), the Met expression vector (pMet) and the Met expression vector encoding full-length activated Rac, Cdc42, Chp and the (S28N) version of RalA, which is constitutively in the GDP-bound form. Transformants were selected and used to replica plate onto appropriate plates containing galactose and lacking methionine and incubated at 36°C. Growth at 36°C indicates a positive two-hybrid interaction. (B) Identification of RalA-binding domain in ZONAB. Deletion analyses were performed on ZONAB to generate the indicated Ras-ZONAB fusion proteins. The growth of CDC25-2 transformants expressing the corresponding proteins is indicated. (C) Only the ZONAB CSD binds RalA in a GTP-dependent manner. Transformants expressing the indicated small GTPases and the Ras-CSD were tested for their ability to confer growth at 36°C as described in (A). (D) Recombinant GST-ZONAB-A binds specifically to S-His-RalA-GTP in vitro. GST-ZONAB-A or the control GST-Pak (N-term regulatory domain) was bound to glutathione–Sepharose beads followed by incubation with either S-His-RalA-GTP or S-His-RalA-GDP. Data shown are representative of three separate experiments.

RalA/ZONAB complex formation is dependent on cell density. (A) MDCK cells were seeded at different densities to reach the indicated degrees of confluence on the same day and then harvested. Cleared lysates were prepared from equal numbers of MDCK cells at each cell density and incubated with Sepharose beads with covalently bound polyclonal antibody against ZONAB. The immunoprecipitates were analysed by immunoblotting with antibodies against RalA and ZONAB. Six experiments were performed; in each, the amount of RalA bound to ZONAB as a proportion of total RalA was measured by quantitative Western blotting. Data are presented as the means with standard deviations. The t-test showed that the amount of RalA bound to ZONAB differs at confluences above 30% (P<0.02). (B) MDCK cells were treated as above, and cell lysates were incubated with the Ral-binding domain (RBD) of RalBP1 immobilised on beads. Five experiments were performed; the amount of RalA-GTP bound was analysed by Western blotting. Equal amounts of cells representing 1/20 of the total were subjected to Western blot analysis for total endogenous RalA. % RalA-GTP was determined from the ratio of RalA-GTP/total RalA and is represented in the histogram below. (C) Representative autoradiograms showing levels of RalA-GTP and phospho-ERK in 100% confluent MDCK cells either infected with Raf-1-RBD-Caax adenovirus compared to control (pAd-CMV-Myc) or Raf-1 (R89L)-RBD-Caax infected cells or transiently transfected with H-Ras (V12G) or control empty vector.

RalA subcellular localisation is dependent on cell density and oncogenic Ras activation. (A) Upper panel: MDCK cells at low cell density were fixed with methanol and stained for RalA and ZO-1. At low cell density, RalA is present in small intracellular vesicles in the cytoplasm. Lower panel: cells were fixed with Triton X-100/ethanol and stained for ZONAB. (B) Upper panel: cells at high density were fixed and stained for RalA and ZONAB to show that RalA partially colocalises with ZONAB at high cell density. Lower panel: cells at high density were fixed and stained for RalA and ZO-1 and xz series prepared to demonstrate colocalisation of RalA at tight junctions. Data shown are representative of at least three separate experiments. (C) H-Ras (G12V) was transiently transfected into low-density MDCK cells and stained for endogenous RalA and Ras. The arrows show that RalA is only localised at the plasma membrane in cells expressing oncogenic Ras.

Regulation of ZONAB-mediated transcriptional repression by RalA. Low- or high-density MDCK cells were cotransfected with plasmids carrying a short version of the human ErbB-2 promoter with an intact ZONAB-binding site driving the expression of firefly luciferase and a control plasmid carrying a mutant version of the same promoter, in which the ZONAB-binding site had been inactivated, driving the expression of Renilla luciferase. (A) These reporter plasmids were transfected together with empty expression vector (vector) or the indicated vectors resulting in the expression of myc-tagged wild-type RalA, activated RalA (Q72L) or the Ral-binding domain (RBD) of RalBP1. Expression of the luciferases was assayed with a dual-luciferase assay system. The ratios obtained (firefly luciferase divided by Renilla luciferase) were normalised to those obtained by cotransfecting empty vector. (B) Myc-RalA (Q72L) was transiently transfected into low-density MDCK cells and stained for endogenous RalA or harvested for immunoprecipitation with the ZONAB antibody as described in Figure 2A. (C) The reporter plasmids were transfected into high-density cells, together with an empty expression vector (vector) or the indicated vectors resulting in the expression of either an siRNA against ZONAB, the control duplex, or an expression vector for the Ral-binding domain (RBD) of RalBP1 either alone or in combination with both the siRNA against ZONAB or control duplex. Expression of the luciferases was assayed with a dual-luciferase assay system. Inset: Extracts of cells transfected with siRNA to ZONAB, or control as above were immunoblotted with anti-ZONAB and anti-β-tubulin antibodies. (D) The reporter plasmids were transfected into low-density cells together with either an active H-Ras allele (G12V), the Ral-binding domain (RBD) of RalBP1 or both of these constructs. The values in all panels represent means±1 s.d. of at least three independent experiments performed in triplicate. P-values obtained from t-tests mark experimental conditions that resulted in significant changes from control samples.

Proposed model for regulation of inverted CCAAT box transcription by the RalA/ZONAB complex. Under conditions of low cell density when RalA/ZONAB complex levels are low, ZONAB is present in the nucleus repressing transcription through binding to inverted CCAAT boxes. When cells become more dense, the RalA–ZONAB complex increases resulting in derepression of inverted CCAAT box-dependent transcription. Oncogenic Ras activates RalA-GTP and causes localisation to the plasma membrane leading to an increase in RalA–ZONAB complex and derepression. It is also known that ZONAB is complexed to ZO-1 under conditions of high cell density leading to derepression of inverted CCAAT box transcription. At present, it is not known how or if RalA and ZO-1 might interact to control transcriptional repression by ZONAB.