and okadaic acid treatment all result in steady, MEKK1- dependent JNK activation until cell death, while activation of other potential MEKK1 targets such as ERK, p38 or NF-kB are unaffected by the loss of MEKK1 [2]. In other systems, prolonged JNK activation has been linked to enhancing apoptosis [1]. For example, short term activation of JNK after TNFa stimulation results in proliferation and survival, but TNFa treatment with conditions resulting in prolonged JNK activation promotes cell death [31,32]. Given that the kinase activity of MEKK1 is required for cell death, JNK is robustly activated by MEKK1, and that prolonged JNK activation has been tied to cell death, it is strongly suggested that JNK activation plays a significant role in MEKK1-dependent apoptosis in response to cytoskeletal disrupt- ing drugs. Mutations of both the PHD and kinase domains reduce JNK activation and abrogate MEKK1-dependent apoptosis after cytoskeletal disruption. While disrupting the PHD domain at physiologically relevant levels clearly results in defective JNK activation and apoptosis (Figures 5 and Figure 8–9, respectively), we and others have shown that overexpressing MEKK1 with a mutated PHD at high levels results in activation of and possibly enhanced downstream signaling [14]. Therefore, it is possible that the PHD is required for specific upstream interactions that are unnecessary in overexpression systems. There is also evidence that mutation of the PHD results in the inability to interact with MEKK1 activating proteins such as RhoA or TRAF2, which interact with MEKK1 after activin or CD40 ligand stimulation [12,15]. However in our system these particular interactions do not appear to be required for the MEKK1-dependent apoptosis. Overexpression of a dominant negative RhoA in 293T cells did not inhibit vinblastine-induced JNK activation. Furthermore, TRAF2-deficient mouse embryonic fibroblasts (MEFs) exhibit normal JNK activation after vinblastine treatment, indicating they are not upstream of MEKK1 in the vinblastine-activated pathway (data not shown). Other functions for the PHD within MEKK1 have been proposed. Lu et al. and Xia et al. have shown that the PHD is required for MEKK1 to ubiquitinate and degrade ERK and c-Jun under conditions of osmotic stress [11,13]. Although we can repeat this data with our system when under hyperosmotic conditions (data not shown), after vinblastine treatment ERK and c-Jun are not degraded (Figure 7 and Figure 5 B, C and D). While we do not detect ubiquitination and degradation of MEKK1 substrates, we do see that MEKK1mutP lacks the modification normally seen with MEKK1 (Figure 3B and Figure 5A). We have performed multiple biochemical assays which indicate this modification is ubiquitin (Figure 4C and D). Since ubiquitination has been reported to affect protein interactions as well as protein sorting and trafficking, we hypothe- sized that decreased ubiqutination of the PHD mutant results in the mislocalization of MEKK1. The localization of MEKK1 has been well characterized by immunofluorescence [21]. Our data confirms that MEKK1 is expressed diffusely through the cytosol and in small punctuate structures (Figure 3C). However, the PHD mutant presents a strikingly different localization than wild type MEKK1. We further illustrate that this localization is not due to enhanced lysosomal degradation by immunofluorescence with a lysosome marker, LAMP1 (Figure 3D). The LAMP1 protein associates with late endosomes/lysosomes and when detected in conjunction with MEKK1 or the PHD mutant, we did not observe a difference in co- localization. Interestingly, both wild type MEKK1 and kinase dead mutant shift localization to near the nucleus after vinblastine treatment, (Figure 3E), whereas the PHD mutant does not change its cellular localization pattern after cytoskeletal disruption. Thus, this alteration in cellular localization appears to be independent of MEKK1 activity, supporting the possibility that mutation of the MEKK1-Dependent Apoptosis Requires the PHD PHD results in aberrant localization due not to impaired activation after vinblastine treatment, but from a change in initial protein interactions. Since ubiquitination can alter protein localization, lack of ubiquitination may explain mislocalization caused by mutation of the PHD and the importance of the PHD for interaction with many proteins, but this requires further investigation. Finally, we have shown that MEKK1-dependent apoptosis occurs not only by MTIs, but also in apoptosis induced by actin disruption and protein phosphatase inhibition (Figure 2). Actin disruption can activate ERK and JNK and induce apoptosis in lymphocytes, airway epithelial cells and various cancer cell lines [33,34,35]. Okadaic acid and another protein phosphatase inhibitor, cantharidin, also induce apoptosis in lymphocytes and cause extensive activation of the JNK, ERK and p38 MAPK pathways [36,37]. While cytochalasins, protein phosphatase inhibitors and microtubule inhibitors have diverse effects upon cells, maximal JNK activation and apoptosis are only triggered in the presence of MEKK1. In this study we have shown that effective treatment with three different classes of drugs requires MEKK1. Therefore, screening more drug panels for prolonged MEKK1/JNK activation may result in the identification of novel chemotherapeutics or combinations of existing drugs. It is critical to understand the MEKK1-dependent apoptotic pathway, as vinblastine and other vinca alkaloid family members play a key role in chemotherapy for lymphomas, leukemia, breast, testicular and some non-small cell lung cancers. To date, most recommended chemotherapy regimens use combinations of drugs, and understanding how MEKK1 activity is affected by these drug combinations could enhance the efficacy of therapy. For example, drug combinations that result in higher MEKK1 activity may be desirable for treatment of leukemia and lymphoma, which induce rapid cell death via MEKK1. Further study of the MEKK1- dependent pathway of apoptosis will be useful in targeting new molecules for cancer therapy, and for understanding resistance to cytoskeletal disruption-induced apoptosis. Materials and Methods Ethics Statement The human cell lines used in this study are well established and were handled under the biosafety level 2 standards established by UCLA. The murine B cell lines were created from MEKK1 wild type and knockout murine bone marrow, shipped to us from the laboratory of Bing Su at Yale University, using the standard p210 BCR-ABL retroviral transformation protocol complicit with biosafety level 2 standards. As this study did not require direct work with mice or primary human tissues, no additional approval was necessary. Plasmids PBabe ratMEKK1 containing a hemagglutinin (HA) and a His tag was generated by digesting pskMEKK1+EcoR1 plasmid with EcoR1 and subcloning into the Maloney Murine Leukemia Virus- based retroviral vector pBabepuro vector. PApuro ratMEKK1 was generated by excising MEKK1 from the pCEP4HAMEKK1 vector and this insert was subcloned into the chicken actin promoter expression vector pApuro between Spe1 and Sal1 restriction enzymes sites in the polylinker. MEKK1 mutations were created by PCR mutagenesis. N-terminal flag-tagged MEKK1 was generated by subcloning into the CMV-flag construct provided generously by the lab of Steve Smale. Reagents Vinblastine sulfate was acquired from MP Biomedicals (Solon, OH). Cytochalasin D and okadaic acid were obtained from PLoS ONE | www.plosone.org 10 February 2011 | Volume 6 | Issue 2 | e17310