MEKK1-Dependent Apoptosis Requires the PHD Figure 8. The PHD and kinase domains are required for vinblastine induced apoptosis. A. MEKK12/2 DT40 cells reconstituted with MEKK1 or mutations thereof all initiate apoptosis in response to etoposide. Each reconstituted cell line was treated with 25 mM etoposide (Et) for four hours and subjected to DNA laddering assay. B. MEKK12/2 cell lines reconstituted with MEKK1 containing the PHD or kinase mutations do not result in DNA fragmentation after vinblastine treatment. Each reconstituted cell line was treated with 1 mM vinblastine (Vin) for six hours and subjected to a DNA laddering assay. C. There is a defective the DNA laddering response in the PHD or kinase domain mutant cell lines after cytochalasin D treatment. Reconstituted cell lines were treated with 2 mg/ml cytochalasin D (Cy) for six hours and subjected to DNA laddering assays. D. There is a defective the DNA laddering response in the PHD or kinase domain mutant cell lines after okadaic acid treatment. Reconstituted cell lines were treated with 90 nM okadaic acid (OA) for six hours and subjected to DNA laddering assays. doi:10.1371/journal.pone.0017310.g008 In order to further investigate how MEKK1 functions within this apoptotic pathway, we created mutations in characterized domains within MEKK1 and reconstituted MEKK1-deficient DT40 cells at near endogenous levels, which would not independently activate MAPK pathways and confound our results. By expressing each mutation at levels that do not increase JNK or ERK activation in resting cells, we were able to clarify which domains of MEKK1 are actually involved in carrying out apoptosis after cytoskeletal disruption. Among the four mutations in our study, the caspase cleavage and UIM deletion mutants were able to compensate for MEKK1 in our readout assays, indicating that these domains are unnecessary for MEKK1 function after cytoskeletal disruption. The caspase recognition site has been postulated to be required for the release of the kinase domain from a particulate fraction to a soluble cytoplasmic fraction, which was shown to be necessary for the pro-apoptotic function of MEKK1 in an overexpression system [21]. Previously, in a 293T overexpression system, Widmann et al. showed that mutation of the caspase 3 recognition site rendered murine MEKK1 less effective in inducting apoptosis caused by overexpression of MEKK1 [17]. However, in our system the orthologous mutation in the rat MEKK1 caspase site is inconsequential for vinblastine- induced JNK activation and apoptosis (Figure 5 and 9). As seen in Figures 8 and 9, reconstituted DT40 cells expressing the MEKK1mutc at near endogenous levels undergo apoptosis after treatment with cytoskeletal disrupting drugs. In addition, down- stream signaling as detected by c-Jun phosphorylation is unaffected (Figure 5B, C and D). There are a number of possible explanations for this apparent discrepancy. First, in our system with treatment of cytoskeletal disrupting drugs the cytoskeleton structure is already disrupted and MEKK1 moves to a region near the nucleus (Figure 3E). This movement is also detected by antibodies to the N-terminal flag tag on flagMEKK1 (data not shown); therefore MEKK1 cleavage and kinase domain release from the particulate fraction are not necessary for MEKK1 movement after cytoskeletal disruption. It is also possible that in the 293T overexpression system used by Widmann et al., the caspase cleavage mutant is highly overexpressed and therefore could function in a manner different than endogenous-level MEKK1 [17]. Additionally, our studies do not rule out the possibility of alternative cleavage sites in the chicken B cells that could compensate for mutation of the characterized caspase cleavage site. Deletion of the UIM domain also does not affect MEKK1- dependent JNK activation, apoptosis or modification in our B cell reconstitution system. UIM regions contained in other proteins have been shown to directly interact with ubiquitin and can enhance the ubiquitination of these UIM-containing proteins [16,25]. For example, auto-ubiquitination of TRAC-1 requires both its RING and UIM domains, as point mutations in either domain reduce the amount of ubiquitinated TRAC-1 [25]. MEKK1 is also autoubiquitinated when overexpressed in conjunction with ubiquitin. Ubiquitination of endogenous MEKK1 occurs after CD40 stimulation composed of K63-linked (activation related) ubiquitin chains [15,23,26]. However, the function of MEKK1 ubiqutination has not been further investigated. We hypothesized that the UIM region was required for either monoubiqutination or polyubiquitination of MEKK1, and through these actions, it can influence MEKK1 activation. The previous study regarding the function of the UIM within MEKK1 used a 35 kDa isolated region of MEKK1 containing the UIM linked to yellow fluorescent protein (YFP) to examine its PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e17310