MEKK1-Dependent Apoptosis Requires the PHD Figure 5. The PHD and kinase domains are essential for MEKK1-dependent JNK activation. A. Expression of mutations in relation to endogenous MEKK1. HA-His-tagged pBabeMEKK1 was introduced into MEKK12/2 (2/2) DT40 cells by retroviral infection (reconstituted cells are referred to here as M-Wt and MEKK1Wt in the text), selected with puromycin and protein levels were assessed by western blot probed with aMEKK1 (left panel). The size difference between His-HA-tagged and endogenous MEKK1 is indicated. Each mutation was introduced into MEKK12/2 cells and expression was compared (right panel). Reconstituted cell lines are referred to as M-mutation within each figure. B. The PHD and kinase domain are required for c-Jun phosphorylation after vinblastine stimulation. Reconstituted cell lines were stimulated with 1 mM vinblastine (Vin) for four hours and lysed in modified RIPA. Phosphorylated c-Jun, total c-Jun and b-tubulin were assessed by western blot. C. There is a decreased amount of c-Jun phosphorylation in the MEKK12/2, PHD and kinase domain mutant cell lines after cytochalasin D treatment. Phosphorylation of c-Jun and total c-Jun levels of each reconstituted cell line was assessed by western blot after four hours of 2 mg/ml cytochalasin D (Cy) treatment. D. There is a decreased amount of c-Jun phosphorylation in the MEKK12/2, PHD and kinase domain mutant cell lines after okadaic acid treatment. Phosphorylation of c-Jun and total c-Jun levels of each reconstituted cell line was assessed by western blot after four hours of 90 nM okadaic acid (OA) treatment. doi:10.1371/journal.pone.0017310.g005 ylated after treatment with vinblastine within the active site of its kinase domain, which also corresponds to activation of this protein as a kinase [15,23]. We treated knockout cells reconstituted with MEKK1 mutant cell lines with vinblastine and subjected the lysates to immunoprecipitation, western blot, and detection with a phosphorylation-specific antibody indicative of MEKK1 activation [23]. We determined that within 2 hours of vinblastine treatment MEKK1Wt, MEKK1delU and MEKK1mutC are active as indicated by auto-phosphorylation, whereas MEKK1mutP and MEKK1mutK are not (Figure 6), indicating that mutation of the PHD results in an inability to be activated under physiologically relevant conditions. Because MEKK1 can degrade ERK via the activity of the PHD under conditions of osmotic stress, and ERK activation is generally linked to survival signals, ERK levels and its activation were also analyzed after cytoskeletal disruption [11,13]. As with the parental DT40 and MEKK1-deficient cells, vinblastine treatment did not change the total level of ERK (Figure 7A), and ERK phosphor- ylation was undetectable. While ERK was activated from 30 minutes to six hours after cytochalasin D and okadaic acid treatment, no differences were seen between the wild type, knockout and reconstituted lines (Figure 7, B and C). Figure 6. MEKK1mutP is not phosphorylated in the activation loop after vinblastine treatment. MEKK12/2, MEKK1Wt, MEKK1mutP or MEKK1mutK (left panel) and MEKK1delU, MEKK1mutC (right panel) cell lines were treated with 1 mM vinblastine for 2 hours. Cell lysates were immunoprecipitated with aHA-agarose, run on SDS-PAGE gel and probed with aphospho-MEKK1 followed by detection of total MEKK1 with aMEKK1 C-22. doi:10.1371/journal.pone.0017310.g006 PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e17310