Interaction of the Jun proteins with BRCA1. (A) Human HEK293T (lanes 1–6) or ES2 (lanes 7–9) cells were transfected with the various expression vectors for the FLAG-tagged AP-1 proteins. Following immunoprecipitation (IP) with an anti-FLAG antibody, the proteins in the immunoprecipitates were resolved by SDS-PAGE, and the presence of the endogenous BRCA1 proteins was detected by immunoblotting (WB) with an anti-BRCA1 antibody (bottom). Equal amounts of the cell lysates were analyzed by immunoblotting for the expression of the FLAG-tagged AP-1 proteins (top). Lane 6 shows that c-Fos does not interact with BRCA1, even in the presence of exogenous HA-tagged JunB. (B) ³⁵S-labeled BRCA1–AD was made with an in vitro translation kit (Promega), and incubated with the GST alone (lane 1) or GST fused with the bZIP region of various AP1 proteins (lanes 2–5) that were immobilized on glutathione beads. After extensive washing, the coprecipitated proteins were analyzed by SDS-PAGE and fluorography. (C) Coimmunoprecipitation of native BRCA1 and native Jun proteins in ES2 cells. Lysates of ES2 cells were immunoprecipitated with different anti-AP1 antibodies (sc-44 for pan-Jun, sc-45 and sc-1694 for c-Jun, sc-8051 for JunB, and sc-52 for c-Fos). The presence of BRCA1 in the immunoprecipitates was detected by Western blotting using an anti-BRCA1 antibody (Ab-1 from Oncogene). In lanes 3 and 5, an excess of the corresponding competing peptides was included in the immunoprecipitation reactions. (D) A reciprocal co-IP was performed in ES2 cell lysates using either anti-α-tubulin (as a negative control) or anti-BRCA1 antibodies (Ab1 and Ab3; Oncogene) in the immunoprecipitation. The blot was probed with an anti-cJun antibody (sc-1694).

BRCA1-Jun interaction correlates with AD1-mediated transcriptional activation. (A) HEK293T cells were cotransfected with the expression vectors for the FLAG-tagged JunB and HA-tagged GAL4–AD domain. Lysates from the transfected cells were immunoprecipitated (IP) with an anti-FLAG antibody, and the immunoprecipitates were probed by immunoblotting (WB) with an anti-HA antibody (bottom). As a control, expression of the wild-type and mutant GAL4–AD fusion proteins was detected by immunoblotting of the crude lysates using the anti-HA antibody (top). (B) HA-tagged full-length BRCA1 proteins were coexpressed with FLAG–JunB in HEK293T cells. Anti-FLAG immunoprecipitation was followed by immunoblotting using the anti-HA antibody. (C) ES-2 cells were cotransfected with the GAL4–AD expression vectors and a GAL4-responsive luciferase reporter plasmid. Also shown at top of the graph is a Western blot for the wild-type and mutant fusion proteins. The relative transcriptional activity of the wild-type GAL4–AD construct is set at 100.

BRCA1 interacts with JunB and JunD in yeast and mammalian two-hybrid assays. (A) Summary of the results from the yeast two-hybrid screen. The plus sign (+) indicates rapid growth of the yeast cells in the selective medium as a result of elevated expression of the HIS3 gene. Also presented is a schematic diagram of the BRCA1 protein, illustrating the location of the trans-activation domains. The solid bar within AD1 indicates the coiled-coil motif. (B) Mammalian two-hybrid assay showing the interactions between BRCA1 and the Jun proteins. HEK293T cells were transfected with the mammalian bait and prey constructs. Shown in the y axis is the fold of increase in the luciferase activity over the negative control (column 1; GAL4–DBD and VP16).

Characterization of the Jun-binding domain in BRCA1. Various HA-tagged BRCA1 fragments were ectopically coexpressed with FLAG–JunB in HEK293T cells. Immunoprecipitation was carried out using an anti-FLAG antibody. The plus sign indicates a significant signal of the BRCA1 fragments in the anti-FLAG immunoprecipitates. The hatched box indicates AD1, whereas the shaded boxes designate the two BRCT repeats in AD2. The solid bar within AD1 represents the coiled-coil region.

JunB can superactivate AD1-dependent transcriptional activation. (A) An equal amount of cell lysates from various cell lines was probed by immunoblotting with specific antibodies against c-Jun, JunB, JunD, and c-Fos. (B) HEK293T cells were cotransfected with the luciferase reporter plasmid, the GAL4 derivatives, and various AP1 expression vectors. The relative luciferase activity in the presence of GAL4–DBD alone (column 1) is set at one. (C) Luciferase reporter assay was performed in ES2 cells in the same manner as described in B. (D) Normalized matched cDNA pairs of normal and tumor ovarian (1–9) or breast (10–16) tissues were analyzed by the real-time PCR reactions for JunB mRNA expression.