MEKK1 is a mitogen activated proteins kinase kinase kinase (MAP3K) that regulates MAPK activation and is the only known mammalian kinase that is also a ubiquitin ligase. domain and that the SWIM domain is required for MEKK1-dependent c-Jun ubiquitylation. We further show that this MEKK1 SWIM/Jun interaction is specific as SWIM Nexavar domains from other proteins failed to bind c-Jun. We reveal that although the Jun and Fos DNA-binding domains are highly conserved the MEKK1 SWIM domain does not bind Fos. Finally we identify the sequence unique to Jun proteins required for specific interaction with the MEKK1 SWIM domain. Therefore we propose that the MEKK1 SWIM domain represents a novel substrate-binding domain necessary for direct interaction between c-Jun and MEKK1 that promotes MEKK1-dependent Nexavar c-Jun ubiquitylation. at a level similar to that of wild type MEKK1 (Figure 2B) and thus we concluded that SD MEKK1 was Nexavar catalytically intact and that the SWIM domain was not required for MEKK1-dependent MKK4 phosphorylation. We then co-expressed wild type MEKK1 or SD MEKK1 with HA-tagged ubiquitin and FLAG-tagged c-Jun to determine whether the SD MEKK1 was capable of ubiquitylating c-Jun. Consistent with our kinase assay results we observed that expression of transfected SD MEKK1 induced activation of JNK pathway signaling resulting in JNK phosphorylation (Figure 2C) similar to that associated with expression of wild type MEKK1. Further anti-HA immunoblots of immunoprecipitated FLAG-c-Jun revealed that c-Jun co-expressed with wild type MEKK1 was strongly ubiquitylated (Figure 2C top panel). In striking contrast we observed a dramatic reduction in c-Jun ubiquitylation when c-Jun was co-expressed with the SD mutant MEKK1 (Fig. 2C) indicating that SD MEKK1 is less effective than wild type MEKK1 at inducing c-Jun ubiquitylation. It is important to note that although these experiments with transfected HA-ubiquitin did not include MG132 that would inhibit proteasomal degradation of c-Jun we did not observe a loss of transiently expressed c-Jun protein in 293T cells that mirrored our data with endogenous c-Jun stably transfected fibroblasts (Figure 1C). One possible explanation for this observation is that the transiently expressed c-Jun is produced at higher levels than in the fibroblasts that more than compensates for c-Jun protein lost to proteasomal degradation thereby masking ubiquitin-mediated c-Jun destruction. Alternatively high expression of exogenous ubiquitin may alter the kinetics of proteasome-mediated protein removal. Regardless our data confirm that wild type MEKK1 promotes c-Jun ubiquitylation and strongly suggest that the SWIM domain is necessary for efficient ubiquitylation of c-Jun by MEKK1. c-Jun is a transcription factor that binds DNA and thus is localized in the nucleus. As MEKK1 is a large kinase with established cytoplasmic functions we asked whether MEKK1 could localize to the nucleus in 293T cells. We observed that a green fluorescent protein-linked MEKK1 protein localized to the cell nucleus in a portion of transfected cells thereby colocalizing with nuclear c-Jun (Supplementary Figure 1A). To determine whether mutant MEKK1 could likewise localize to the cell nucleus we again expressed SD MEKK1 and the RING domain mutant MEKK1 C441A in cells and performed cell fractionation to examine the distribution of MEKK1 proteins to the cytoplasmic Rabbit Polyclonal to SFRS17A. and nuclear compartments. Our analysis confirmed that all MEKK1 proteins tested were distributed similarly to both the cytoplasm and the nucleus (Supplementary Figure 1B). Taken together our results strongly suggest that loss of MEKK1-dependent c-Jun ubiquitylation associated with SD MEKK1 expression was due to ineffective c-Jun ubuiquitylation and not caused by reduced kinase activity or altered localization of mutant MEKK1 proteins. MEKK1 SWIM directly associates with Jun To determine how the MEKK1 SWIM domain regulates c-Jun ubiquitylation we asked whether c-Jun physically associated with the SWIM domain. We developed and Nexavar Nexavar purified GST fusion protein including either SWIM or Band domains and combined these recominant protein with FLAG epitope-tagged c-Jun from transfected cell lysates separated the connected protein by SDS-PAGE and performed immunoblot evaluation to detect c-Jun association. We discovered that the SWIM site alone was adequate Nexavar to draw down c-Jun whereas Band did not.