MEKK1-Dependent Apoptosis Requires the PHD Figure 7. The PHD and kinase domains of MEKK1 are not essential for maximal ERK activation. A. ERK is not activated or degraded after vinblastine treatment. Wild type, knockout and MEKK1-reconstituted MEKK12/2 cells were treated with 1 mM vinblastine for four hours and total ERK or b-tubulin were assessed by western blot. B. Activation of ERK by cytochalasin treatment is not MEKK1-dependent. Cell lines were treated with 2 mg/ml cytochalasin D for two hours and phosphorylated and total ERK were evaluated by western blot. C. Activation of ERK by okadaic acid treatment is not MEKK1-dependent. Phosphorylation of ERK and total ERK were determined by western blot after treatment with 90 nM okadaic acid for four hours. b-tubulin was used as a loading control. doi:10.1371/journal.pone.0017310.g007 The PHD domain and the kinase domain are required for apoptosis after cytoskeletal disruption To identify the domains of MEKK1 required for apoptosis induced by cytoskeletal disruption, DT40 reconstituted cell lines were stimulated with vinblastine, cytochalasin D or okadaic acid, and apoptosis was analyzed via DNA laddering (Figure 8B, C and D). While wild type DT40 cells, MEKK1Wt, MEKK1delU and MEKK1mutC reconstituted cells exhibited DNA laddering after cytoskeletal disruption, the MEKK1mutP and MEKK1mutk reconsti- tuted cells were resistant to apoptosis. This defect in apoptosis was not the result of general resistance to apoptosis, as each cell line could still induce apoptosis in response to etoposide, a MEKK1-independent stimulus (Figure 8A). The response to cytoskeleton disruption was then quantified by PI exclusion assay, which is consistent with the DNA laddering results (Figure 9A). Our results show that MEKK1, MEKK1delU and MEKK1mutC induced apoptosis at levels similar to wild type cells, but there was no significant increase in the percentage of apoptotic cells in the knockout, MEKK1mutK or MEKK1mutP cell lines with vinblastine, cytochalasin D or okadaic acid (Figure 9A panels I, II, III, respectively). Vinblastine-induced apoptosis has been shown to activate the caspase pathway and Figure 9B shows that caspase 3 activity is also significantly reduced in the MEKK1mutP and MEKK1mutK as compared to the MEKK1Wt, MEKK1delU and MEKK1mutC reconstituted cells after treatment with vinblastine, cytochalasin D or okadaic acid (Figure 9B panels I, II, III, respectively). Discussion MTIs such as vinblastine are important chemotherapy drugs, but the mechanism by which these drugs initiate apoptosis is not clear. We have previously shown that MEKK1 is required for apoptosis in DT40 B cells in response to MTIs [2]. However, the requirement of MEKK1-dependent apoptosis upon treatment with other toxic agents had not been thoroughly investigated. Here we have shown that MEKK1 activation is involved in apoptosis induced by general cytoskeleton disruption and that a functional PHD and kinase domain are required to induce apoptosis. The requirement of MEKK1 for JNK activation in response to MTIs has been consistent in published data. However, in different studies the outcome of JNK activation after treatment with microtubule inhibitors has been attributed to two opposing effects, either enhanced apoptosis or increased cell survival Our previous study with DT40 cells indicates that MEKK1 is required for JNK activation and apoptosis after vinblastine stimulation. However, it has been published that treatment of Mekk12/2 ES cells with the MTIs nocodazole or taxol result in defective JNK activation but somewhat increased sensitivity to apoptosis [6,19,24]. Whether this discrepancy was due to a cell-specific effect or a species- specific effect was unclear. Here we report that vinblastine- stimulated apoptosis is MEKK1-dependent in both chicken DT40 and murine B cell lines, indicating that the discrepancy is not specific to chicken cells. PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e17310