The MEKK1 SWIM domain is a novel substrate receptor for c-Jun ubiquitylation

Abstract

MEKK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase 1] is a MAP3K (MAPK kinase kinase) that regulates MAPK activation, and is the only known mammalian kinase that is also a ubiquitin ligase. MEKK1 contains a RING domain within its N-terminal regulatory region, and MEKK1 has been shown to ubiquitylate the AP-1 (activator protein 1) transcription factor protein c-Jun, but the mechanism by which MEKK1 interacts with c-Jun to induce ubiquitylation has not been defined. Proximal to the RING domain is a SWIM (SWI2/SNF2 and MuDR) domain of undetermined function. In the present study, we demonstrate that the MEKK1 SWIM domain, but not the RING domain, directly associates with the c-Jun DNA-binding 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 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 c-Jun ubiquitylation.

Figure 1. The MEKK1 SWIM domain mutation enhances cell viability and c-Jun expression

(A) Display of the sequences included in the MEKK1 SWIM and RING domains, including a graphical representation of the predicted secondary structures of these domains based on the NNPREDICT algorithm. The conserved cysteine residues in both domains are enlarged. (B and C) The functional consequence of MEKK1 expression was evaluated in immortalized MEKK1-deficient fibroblasts that stably express either wild type or mutant MEKK1 (add-back cell lines). (B) Mean relative fibroblast cell line viability as assessed by MTT is represented. Each bar represents the mean of three independent experiments. *p<0.05 (C) c-Jun expression is assessed by immunoblot (IB) analysis of add-back cell lysates. The upper panel is an anti-MEKK1 immunoblot showing add-back expression. The middle panel displays the corresponding c-Jun expression in each cell line, and the lower panel shows an anti-tubulin immunoblot to demonstrate equal loading of lysate proteins. (D) c-Jun phosphorylation in fibroblast cell lines using antibodies raised to phosphorylated serine residue 73. Displayed blot is representative of at least three experiments.

Figure 2. The SWIM domain is required for c-Jun ubiquitylation, but not MKK4 phosphorylation

(A) MEKK1 expression enhances prevalence of higher molecular weight c-Jun protein in the presence of proteasome inhibitor MG132. (B) MEKK1 in vitro kinase activity is assessed by immunoblot analysis detecting phosphorylated unactive MKK4 (upper panel). Middle panel shows total MKK4 from each reaction, and the lower panel shows immunoblot of purified wild type or SD MEKK1. (C) A comparison of ubiquitylation activity of MEKK1 mutant SWIM-deleted to that of wild type MEKK1. Transfected FLAG-c-Jun was co-expressed with HA –tagged ubiquitin and wild type or mutant MEKK1, and ubiquitylation of immunoprecipitated (IP) c-Jun was detected by anti-HA immunoblot (top panel). Lysates (lower panels) from transfected 293T cell lysates display loading and associated level of phosphorylated JNK in each lane. Results are representative of three or more independent experiments.

Figure 3. MEKK1 SWIM domain directly and specifically binds Jun

(A) GST-SWIM, but not GST-RING forms a complex with c-Jun from 293T cell lysates. In (A), (B) and (C), the upper panels are anti-c-Jun immunoblots, and the lower panels display coomassie stained gel to show the relative amounts of GST fusion proteins. (B) GST-SWIM directly binds recombinant c-Jun in vitro, but GST alone does not bind c-Jun. (C) The MEKK1 SWIM domain binds to c-Jun from 293T cell lysates, but the SWIM domains from proteins ZSWIM1 and ZSWIM2 do not bind c-Jun. Results are representative of at least three independent experiments. (D) Immunoblot analysis of c-Jun co-immunoprecipitation with HA-tagged full-length MEKK1. SWIM-4A is a full length MEKK1 in which SWIM signature motif cysteines and histidine have been mutated to alanines. Upper two panels display immunoblots of immunoprecipitated proteins with anti MEKK1 and anti Jun antibodies. The lower two panels are immunoblots of total cell lysates to indicate protein expression and loading. Results are representative of at least three independent experiments.

Figure 4. The Jun DNA-binding domain associates with the MEKK1 SWIM domain

(A) A graphical representation of the FLAG-tagged full length c-Jun and Jun truncation mutant proteins used in the pulldown experiments. (B) GST-SWIM domain fusion protein pulldown of full length c-Jun and truncated c-Jun mutants. Results are representative of at least three independent experiments. δ = delta domain, D = DNA-binding domain, LZ = leucine zipper domain

Figure 5. The arginine necessary for SWIM association is conserved in Jun but not Fos proteins

(A) GST-SWIM fusion proteins were used to pull down FLAG-tagged AP-1 proteins. Upper panel displays anti-FLAG immunoblot of transfected cell lysates. The middle panel shows anti-FLAG immunoblot to reveal proteins that formed complexes with GST-SWIM. The lower panel is a coomassie-stained gel that includes the GST-SWIM fusion protein to indicate the relative fusion protein levels used in each sample. (B) A sequence alignment of the Jun and Fos protein DNA-binding domains. The conserved residues are indicated in gray, and the numbered residues were selected for mutagenesis in c-Jun. UniProt accession numbers for the sequences are as follows: P05627 (c-Jun), P09450 (JunB), P15066 (JunD), P01101 (c-Fos), and P47930 (Fra2). (C) GST-SWIM pulldown assay using c-Jun and mutant c-Jun proteins from transfected 293T cells. The upper panel shows an anti-FLAG immunoblot of the total cell lysates to confirm expression of the transfected Jun proteins. The middle panel is an anti-FLAG immunoblot showing the Jun proteins pulled down by the GST-SWIM, and the lower panel shows a coomassie stain of the gel indicating fusion protein loading. (D) Graphical summary of mutant c-Jun pulldown results. Densitometry of pulldown was normalized to respective total cell lysate immunoblot and analyzed by student’s T test for statistical significance. *p<0.05. Results are representative of at least three independent experiments.