Here we show that okadaic acid-induced apoptosis and cytochalasin-induced apoptosis are both impaired by genetic deletion of MEKK1. This indicates that MEKK1-dependent apoptosis is not specifically confined to microtubule inhibition, but may play a broader role in apoptosis activated by the two other cytoskeletal structures of the cell: actin fibers and intermediate filaments. Furthermore, we reveal that MEKK1- deficient murine B cells are also resistant to apoptosis induced by cytoskeletal disruption, indicating that this phenotype is not specific to DT40 cells. To determine the role of the various domains within MEKK1, we created mutations within the PHD, UIM, caspase cleavage site and the kinase domain, and MEKK1- deficient cells were reconstituted with each mutant at near endogenous expression levels. Using this system we found that the PHD and the kinase domain are required for MEKK1- dependent JNK activation and apoptosis. Further investigation indicates that mutation of the PHD results in altered subcellular localization and reduced levels of basal ubiquitination found on MEKK1. We also found that while the PHD mutant can be phosphorylated and activate downstream signals when overex- pressed at high levels, low level reconstitution of knockout cells with the PHD mutant results in lack of MEKK1 phosphorylation after vinblastine treatment. This suggests that the defect in the PHD mutant resides upstream of MEKK1. Therefore, the mislocalization of the PHD mutant may interfere with its ability to interact with upstream proteins. Results MEKK1 is required for JNK activation and the apoptotic response to cytoskeletal disruption Drugs such as vinblastine, cytochalasin D, and okadaic acid all induce cytoskeletal disruption and strong MAPK activation. To determine whether this activation requires MEKK1, DT40 chicken bursal B cell line and its previously generated MEKK1- deficient (MEKK12/2) counterpart were treated with vinblastine, cytochalasin D and okadaic acid (Figure 1A). Upon treatment with vinblastine, cytochalasin D and okadaic acid, MEKK1-deficient cells had undetectable or decreased levels of c-Jun phosphoryla- tion, whereas ERK and p38 phosphorylation were unaffected by MEKK1 deficiency. We also noted that total c-Jun levels were elevated after treatment with these drugs, in a manner partially dependent on MEKK1. To ensure that the requirement for MEKK1 in vinblastine-induced apoptosis is not a property inherent to the chicken DT40 cell line, murine wild type or MEKK1-deficient bone marrow cells were transformed to create stable pre-B cell lines. These newly created cell lines were then analyzed for MAPK activation in response to vinblastine, cytochalasin D or okadaic acid. Similar to the DT40 MEKK1- deficient cell line, decreased levels of phosphorylated c-Jun were observed in the murine MEKK1-deficient line as compared with the wild type cells (Figure 1B). Next, we investigated whether apoptosis induced by these drugs requires MEKK1. Strong DNA laddering was observed in the wild type DT40 and murine B cell lines in response to vinblastine, cytochalasin D and okadaic acid (Figure 2A). However, reduced levels of laddering were seen in both chicken and murine knockout cell lines. This effect did not appear to be a general defect in apoptosis, as treatment with the DNA damaging agent etoposide induced apoptosis in all cell lines (lane Et, Figure 2A). Apoptosis in response to each drug was then quantified by PI exclusion assay for each cell line (Figure 2B). In addition, we assessed caspase 3 activity to show that cells were undergoing apoptosis and not just DNA damage, and found that there is an increased level of active MEKK1-Dependent Apoptosis Requires the PHD Figure 1. Characterization of MAPK activation in wild type and MEKK1-deficient cell lines. A. Phosphorylation of c-Jun phosphor- ylation is defective in the MEKK12/2 chicken DT40 cells in response to cytoskeletal disruption. Wild type (Wt) and MEKK12/2 DT40 cells were left untreated (U) or stimulated with 1 mM vinblastine (Vin), 2 mg/ml cytochalasin D (Cy), and 90 nM okadaic acid (OA) for four hours, lysed in modified RIPA buffer and run on an SDS-PAGE gel to compare wild type and knockout cell lines. Western blot membranes were probed for phosphorylated and total c-Jun, ERK and p38. B. Phosphorylation of c-Jun is defective in MEKK12/2 murine pre-B cells in response to cytoskeletal disruption. Wild type (Wt) and MEKK12/2 pre-B murine cells were stimulated with 1 mM vinblastine, 2 mg/ml cytochalasin D, and 90 nM okadaic acid for four hours, lysed in modified RIPA buffer and run on an SDS-PAGE gel. Membranes were probed for phosphorylated and total c-Jun, ERK and p38. b-tubulin is used as loading control. doi:10.1371/journal.pone.0017310.g001 caspase 3 after each drug treatment in the wild type cells when compared to the knockout cells, which is consistent with the DNA laddering and PI exclusion data (Figure 2C). Taken together, these data indicate that MEKK1 is involved in both JNK activation and the apoptotic response to a number of cytoskeletal disrupting agents. PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e17310