MEKK1-Dependent Apoptosis Requires the PHD Figure 9. Quantification of apoptosis by propidium iodide incorporation and caspase 3 activity. A. In the MEKK1mutP and MEKK1mutK cell lines the percentage of apoptotic cells does not significantly increase after cytoskeletal disruption. To quantify the levels of DNA fragmentation, propidium iodide staining was analyzed by flow cytometry. Cell lines were treated with 1 mM vinblastine (panel I), 2 mg/ml cytochalasin D (panel II) or 90 nM okadaic acid (panel III) for twelve hours and the percentage of apoptotic cells were determined by measuring fluorescence lower than the diploid population using CellQuest software. B. Caspase 3 is not activated in the MEKK1mutP or MEKK1mutK cell lines after cytoskeletal disruption. Caspase 3 activity was determined after four hours of treatment with 1 mM vinblastine (I), 2 mg/ml cytochalasin D (II) or 90 nM okadaic acid (III) treatment. Cell lysates were used to determine the catalytic activity of caspase 3 by using the colorimetric substrate Ac-DEVD-pNA and measured at 405 nM. doi:10.1371/journal.pone.0017310.g009 ability to interact with ubiquitin [27]. It was shown that while other UIM regions such as Hrs or epsin were ubiquitinated, the MEKK1 UIM region was unable to bind ubiquitin. The lack of interaction of MEKK1 UIM and ubiquitin in this study could have been due to the fact that the overall tertiary structure of MEKK1 is necessary for the function of the MEKK1 UIM, so in our experiments, we deleted the entire 16 amino acid region containing the UIM while retaining all other domains of full length MEKK1. However, the UIM deletion mutant of MEKK1 was able to function similarly to wild type MEKK1, indicating this domain is unnecessary for MEKK1-dependent cell death by cytoskeletal disruption. We determined that the UIM mutant can be auto-ubiquitinated in an overexpression system to the same extent as wild type MEKK1 (data not shown). Our data, combined with the YFP-UIM data, suggests that while the consensus sequence for a UIM region is present in MEKK1, this domain is not absolutely required for kinase activation or autoubiquitination of MEKK1. In this study, JNK activation correlates with apoptosis after treatment with cytoskeletal disrupting drugs. In our MEKK1- deficient cells, JNK activation is abrogated, and reconstitution of these cells with MEKK1 restores JNK activation. Therefore, JNK activity is used as an indicator of MEKK1 activity. In this paper and our previous study, MEKK1-dependent JNK activation correlates strongly with apoptosis, and previous reports have shown that absence or inhibition of JNK resulted in decreased levels of apoptosis with vinblastine treatment [28,29]. We also show that the JNK substrate c-Jun upregulation after vinblastine treatment is partially dependent upon MEKK1, and upregulation of c-Jun has been shown to contribute to vinblastine-induced apoptosis [30]. The duration of JNK activation may also be important for inducing apoptosis. Here, vinblastine, cytochalasin PLoS ONE | www.plosone.org 9 February 2011 | Volume 6 | Issue 2 | e17310