Apoptosis Induced by Cytoskeletal Disruption Requires Distinct Domains of MEKK1 Erin Tricker1, Afsane Arvand1,2, Raymond Kwan1, Gordon Y. Chen1, Ewen Gallagher3, Genhong Cheng 1 * 1 Department of Microbiology, Immunology & Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America, 2 Department of Biology, Mount Saint Mary’s College, Los Angeles, California, United States of America, 3 Department of Immunology, Imperial College London, London, United Kingdom Abstract MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1- deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD. Citation: Tricker E, Arvand A, Kwan R, Chen GY, Gallagher E, et al. (2011) Apoptosis Induced by Cytoskeletal Disruption Requires Distinct Domains of MEKK1. PLoS ONE 6(2): e17310. doi:10.1371/journal.pone.0017310 Editor: Gen Sheng Wu, Wayne State University School of Medicine, United States of America Received October 11, 2010; Accepted January 27, 2011; Published February 25, 2011 Copyright: 2011 Tricker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the grant R01 CA100210 from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gcheng@mednet.ucla.edu Introduction MEKK1 is a serine-threonine kinase that activates the c-Jun amino terminal (N-terminal) kinases (JNK) MAPK module, altering gene transcription [1]. We have previously shown that MEKK1 is essential for microtubule inhibitor (MTI)-induced JNK activation and apoptosis through genetic deletion of MEKK1 in chicken bursal B cells (DT40 cell line) [2]. Given that MTI drugs are important chemotherapeutic agents involved in treatment of leukemia, lymphomas, non-small cell lung cancer, breast cancer, testicular cancer and head and neck cancer, understanding MEKK1 activation within the context of apoptosis will improve chemotherapy regimens and outcomes [3,4]. The importance of the MEKK1-dependent pathway of apoptosis has recently been highlighted by Kan et al., who identified MEKK1 as one of the top 50 genes containing somatic missense and nonsense mutations in a panel of breast, lung, ovarian and prostate tumors, indicating that impairment of the MEKK1- dependent apoptotic pathway may enhance tumorigenesis [5]. The extent of MEKK1 involvement in apoptosis by other mechanisms of cytoskeletal disruption is currently unknown. Cytochalasin toxins and the protein phosphatase inhibitor okadaic acid are two examples of drugs that disrupt the cytoskeleton and cause JNK activation [6,7]. Cytochalasins disrupt actin filament integrity by capping the barbed end of actin, thus preventing the addition of G-actin monomers [8]. Okadaic acid inhibits protein phosphatase 1 and 2A and results in the rapid phosphorylation and disruption of intermediate filaments [9]. Whether the MEKK1-dependent apoptotic pathway is involved in the response to cytochalasins or protein phosphatase inhibition has not been investigated. MEKK1 is a 196 kDa protein comprised of a large N-terminal regulatory region and a C-terminal kinase domain. The N- terminal region contains features such as a plant homeodomain (PHD), two overlapping ubiquitin-interacting motifs (UIM), and a caspase cleavage site [10,11]. Several studies have investigated the potential functions of different regions of MEKK1. The PHD is a multifunctional domain that is involved in protein interactions and is an E3 ligase. An intact PHD is required for interaction with RhoA, a small GTPase [12]. The PHD is also an E3 ligase for ERK (Extracellular regulated Kinases) and c-Jun under hyperos- motic conditions, and is required for MEKK1 autoubiquitination in overexpression conditions and after CD40 ligand stimulation [11,13,14,15]. Another MEKK1 region with a potential role in protein modification by ubiquitin is the UIM. The UIM may aid in the polyubiquitination of MEKK1, as this domain has been required for ubiquitination of other UIM-containing proteins [16]. MEKK1 kinase activity has been correlated to apoptosis. Constitutive kinase activity has been observed when full length MEKK1 is cleaved at its caspase recognition site, and mutation of this site inhibits apoptosis normally seen with overexpression in 293T cells while leaving JNK activation unaffected [17]. In addition, overexpression of MEKK1 with a mutated kinase domain inhibits apoptosis by UV light [18]. However, UV-induced apoptosis is unaffected by the genetic loss of MEKK1, highlighting the difference between the dominant negative effects caused by overexpression studies and what occurs under normal physiological conditions [19]. By reconstituting MEKK1-deficient (MEKK12/2) cells at more physiologically relevant levels, we can better differentiate any dominant negative effects of these mutations from the role that each may play within the MEKK1-dependent pathway of apoptosis. PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e17310