MEKK1-Dependent Apoptosis Requires the PHD Figure 2. Detection of apoptosis after cytoskeletal disruption in wild type and MEKK1-deficient cell lines. A. DNA laddering is significantly decreased in the MEKK12/2 DT40 and murine pre-B cell lines upon treatment with vinblastine, cytochalasin D, and okadaic acid, whereas the response to etoposide is unchanged. Wild type (Wt) or MEKK12/2 DT40 cell lines were treated with 1 mM vinblastine (Vin), 2 mg/ml cytochalasin D (Cy), 90 nM okadaic acid (OA), or DMSO (DM) for six hours or 25 mM etoposide (Et) for four hours. After treatment, each line was assessed for the presence of DNA laddering. Wild type (Wt) or MEKK12/2 murine cell lines were treated with the same concentration of these drugs for nine hours and DNA laddering was assessed. B. Apoptosis was quantified by propidium iodide exclusion assay. Wild type (Wt) or MEKK12/2 DT40 were treated with 1 mM vinblastine (Vin), 2 mg/ml cytochalasin D (Cy) for DT40 cells, 90 nM okadaic acid (OA), DMSO (DM) or 15 mM etoposide (Et) for 12 hours. Wild type (Wt) or MEKK12/2 murine pre-B cells were treated with 1 mM vinblastine (Vin), 4 mg/ml cytochalasin D (Cy) for DT40 cells, 90 nM okadaic acid (OA), DMSO (DM) or 15 mM etoposide (Et) for 14 hours. After treatment, the percentage of apoptotic cells was assessed by measuring fluorescence of the sub-diploid population using flow cytometry. C. Caspase 3 activation is abrogated in the MEKK12/2 cell lines after cytoskeletal disruption. DT40 and MEKK12/2 cells (left panel) were treated with 1 mM vinblastine and 2 mg/ml cytochalasin for four hours and 90 nM okadaic acid for six hours. Murine wild type and MEKK12/2 pre-B cells (right panel) were treated with 1 mM vinblastine and 2 mg/ml cytochalasin and 90 nM okadaic acid for six hours. Cell lysates were used to determine the catalytic activity of caspase 3. doi:10.1371/journal.pone.0017310.g002 Overexpression of the PHD mutant MEKK1 affects basal modification and localization, but not induction of downstream signaling To identify specific domains of MEKK1 required for apoptosis in response to cytoskeletal disruption, mutations were created within different domains of MEKK1 (Figure 3A). Two mutations were introduced into the PHD domain of rat MEKK1 (Cys478Ala and Cys481Ala; MutP); these mutations have been shown to disrupt the zinc finger structure and destroy its E3 ligase function [11]. The putative UIM of MEKK1 (amino acid 1176 to 1191) was deleted to create delU. The critical lysine residue within the kinase active site of MEKK1 was mutated to methionine (1265K to 1265M; MutK) to disrupt activation of the kinase domain. To determine if caspase cleavage of MEKK1 is required for apoptosis in our system, we mutated the aspartic acid residues in the caspase recognition site of rat MEKK1 to glutamate (878DTLD881 to ETLE) to create MutC; the orthologous caspase mutation of murine MEKK1 has been reported to render murine MEKK1 resistant to cleavage at this site [17,20]. Each MEKK1 mutant was overexpressed in 293T cells in the highly expressing pApuro vector and analyzed via western blot to check for protein expression. As seen in Figure 3B, the mutant constructs expressed at the proper MEKK1 protein size of 196 kD. We also noted that normally MEKK1 appears to be highly modified as apparent by the presence of multiple MEKK1 bands (Figure 3B), but among our mutations, the PHD mutant was noticeably lacking this modification. Because overexpression of MEKK1 causes JNK activation and downstream c-Jun phosphorylation, we next investigated the effects of each mutant in downstream signaling through JNK in our 293T overexpression system. Each mutant could still initiate PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e17310