(Figure 3E). Within one hour of treatment both wild type MEKK1 and the kinase dead mutant changed localization, from diffuse/ punctate in the periphery to aggregates in the peri-nuclear region. However, the appearance and location of the PHD mutant did not change after vinblastine treatment. The PHD mutant exhibits decreased ubiquitination The difference in subcellular localization between wild type MEKK1 and the PHD mutant prompted us to investigate possible differences in post-translational modifications between the two proteins. Deak et al. have previously shown that overexpressed MEKK1 is auto-phosphorylated [22]. We hypothesized that the modified form of MEKK1 that we see in our assays (Figure 3B, MEKK1 upper bands) may be due to phosphorylation of multiple sites in MEKK1. To test this hypothesis, we set out to determine whether the loss of basal modification on the PHD mutant (lack of upper band in MutP lane, Figure 3B) correlated with the loss of MEKK1 phosphorylation. We found that both wild type MEKK1 and the PHD mutant were phosphorylated in the kinase domain when overexpressed (Figure 4A). Furthermore, treatment of DT40 cells with calf-alkaline phosphatase shifted the size of vinblastine treated (and therefore phosphorylated), but not untreated DT40 cells (Figure 4B), indicating that the observed basal modification on endogenous MEKK1 is not phosphorylation. However, the PHD also has intrinsic E3 ligase activity and destroying this domain results in defective auto-polyubiquitination [14]. There- fore, it is possible that the PHD is required for ubiquitination of MEKK1. Thus, we asked whether this modification can be due to Figure 4. Identifying the post-translational modifications of the MEKK1 protein. A. The PHD mutant can be phosphorylated. Vector, wild type flag-MEKK1 and flag-PHD mutant (mutP) were transfected into 293T cells and immunoprecipitated with aflag M2 conjugated beads. Membranes were probed with total or phospho- MEKK1. B. CIP treatment does not affect basal MEKK1 modification, but does shift the molecular weight of vinblastine treated (phosphorylated) endogenous MEKK1. DT40 cells were treated with vinblastine for six hours and lysates were immunoprecipitated with aMEKK1. Half of each lysate was treated with calf alkaline phosphatase and all were incubated at 37u, run on SDS-PAGE gel and membranes were probed with aMEKK1. C. Inhibition of de-ubiquitinating enzymes via N-ethylmalei- mide (NEM) stabilizes the higher molecular weight form of MEKK1. Wild type DT40 cells were lysed in modified RIPA. Lysates were treated with or without 20 mM NEM for 30 minutes at room temperature. Lysates were run on SDS-PAGE and probed with a MEKK1. D. MEKK1 is ubiquitinated whereas the PHD mutant is not. 293T cells were transiently transfected with vector, flag-MEKK1 or mutP. Cell lysates were immunoprecipitated with flag-conjugated beads, run on an SDS- PAGE gel and probed with aubiquitin or aMEKK1. doi:10.1371/journal.pone.0017310.g004 MEKK1-Dependent Apoptosis Requires the PHD ubiquitination. To determine if MEKK1 is ubiquitinated, we questioned whether the modification found on MEKK1 would be stabilized by a deubiquitinase inhibitor, such as N-ethylmaleimide (NEM). We found that the high molecular weight band of MEKK1 is enhanced by treatment of cell lysates with NEM when compared to untreated cells (Figure 4C). Finally, overexpression and immunoprecipitation of empty vector, MEKK1 or the PHD mutant followed by detection with ubiquitin antibody indicate that only wild type MEKK1 is strongly ubiquitinated (Figure 4D). These biochemical assays indicate that while the PHD mutant can be phosphorylated when overexpressed, modification by ubiquitin is abrogated in the PHD mutant. Endogenous level reconstitutions of MEKK1-deficient cells indicate that the PHD and the kinase domain of MEKK1 are required for JNK activation after cytoskeletal disruption To determine if any candidate domains were required for JNK activation in response to cytoskeletal disrupting drugs, each MEKK1 mutant was expressed in MEKK1-deficient DT40 cells. However, because the increased JNK and ERK activation caused by MEKK1 overexpression may interfere with the response to cytoskeletal disruption, we sought to first establish a low-level expression system using the pBabe retroviral vector, which would not significantly increase JNK or ERK activation in resting cells. After retroviral infection and selection with puromycin, the polyclonal populations of knockout cells reconstituted with MEKK1 were checked for expression levels in comparison to the DT40 cell line by western blot (Figure 5A, left panel). Murine MEKK1-deficient cells were not used due to inherent puromycin resistance that prevented the analysis of puromycin-selected polyclonal populations. While the level of expression was slightly higher in the reconstituted cell line, it was far less than traditional overexpression vectors. Furthermore, all reconstituted cells are shown to have relatively equivalent levels of MEKK1 mutant expression (Figure 5A, right panel). Additionally, reconstitution of the MEKK1-deficient cells did not show the increased basal MAPK activity seen when MEKK1 is overexpressed in the 293T system (Figure 5B, 5C, 5D). Next, the MEKK1-deficient reconstituted cells were treated with cytoskeletal disrupting drugs, and the effects of these mutations on downstream signaling were determined. Cytoskeletal structure was disrupted with either 1 mM vinblastine, 2 mg/ml cytochalasin D or 90 nM okadaic acid for 4 hours and phosphorylation of c-Jun was analyzed by western blot. Robust phosphorylation of c-Jun was observed in the MEKK1Wt, MEKK1mutC and MEKK1delU reconstituted cell lines, but MEKK1mutP, MEKK1mutK (MEKK1-deficient cells reconstituted with each mutation are referred to as MEKK1mut) and MEKK1- deficient cell lines demonstrate abrogated c-Jun phosphorylation in response to cytoskeletal disruption. Total levels of c-Jun were also analyzed and levels were enhanced after cytoskeletal disruption (Figure 5B, C and D). This increase appears to be partially dependent on MEKK1 activation, but the extent of MEKK1 dependence seems to vary depending on the stimuli. After determining that the MEKK1mutP cell line exhibits decreased amounts of c-Jun phosphorylation after cytoskeletal disruption, the cause of this defect was investigated. We had already observed that MEKK1mutP can activate downstream signaling when overexpressed (Figure 3B), so we hypothesized that the defect in activation of the MEKK1mutP cell line (where MEKK1 is expressed at near endogenous levels and therefore cannot auto- activate downstream signaling) lies in receiving upstream signals. To test this we investigated whether MEKK1 can become phosphor- PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e17310