Cytokine-induced activation of mixed lineage kinase 3 requires TRAF2 and TRAF6
Introduction
MAPK signaling pathways function to transduce extracellular signals into a wide range of cellular responses. These pathways consist of a protein kinase cascade, where an activated MAP3K phosphorylates and activates a MAPK kinase (MAP2K), which in turn, phosphorylates and activates a MAPK [1]. MAPKs translocate to the nucleus to activate transcription factors and regulate genes involved cellular processes such as proliferation, survival, differentiation and apoptosis [1]. The three most well characterized MAPKs are extracellular signal-regulated kinase (ERK), SAPK/JNK and p38. ERK is predominantly activated by growth factors, whereas SAPK/JNK and p38 MAPK are primarily activated by environmental stresses and pro-inflammatory cytokines [1], [2].
MLK3 is a member of a family of MAP3Ks. Upon activation, MLKs directly phosphorylate and activate the MAP2Ks, MKK4/SEK1 and MKK3/6 [3], [4]. Activated MKK4/SEK1 and MKK3/6 directly phosphorylate and activate SAPK/JNK and p38, respectively [1]. MLK3 is activated by epidermal growth factor (EGF), T cell receptor co-stimulation, TNF, sorbitol, ceramide and nerve growth factor deprivation in neuronal cells [5], [6], [7], [8], [9]. RNAi studies showed that MLK3 promotes microtubule instability, is required for cell proliferation in colon epithelial and lung fibroblast cells, and is required for activation of ERK and SAPK/JNK by TNF and EGF [8], [10]. MLK3 has also recently been found to limit Rho GTPase activity [11].
Activation of MLK3 occurs through autophosphorylation on amino acid residues threonine 277 (Thr277) and serine 281 (Ser281) within the MLK3 kinase domain [9]. Autoinhibition of MLK3 kinase activity is mediated by an interaction between its SH3 domain and proline 495 (Pro495) in the C-terminus [12]. MLK3 is required for optimal TNF activation of the SAPK/JNK pathway, however, the proteins required for MLK3 activation in TNF signaling have not been identified [8], [13].
In inflammatory responses, TNF binds the TNF receptor (TNFR) to activate several signal transduction pathways including SAPK/JNK [14]. TRAFs are adaptor proteins recruited to the cytoplasmic tails of receptors in the TNFR and IL-1 receptor/Toll-like receptor (IL-1R/TLR) superfamilies [15]. Different TRAFs have different targets to facilitate the activation of multiple downstream effectors. The signaling pathways activated by TRAFs result in many different responses including cell survival, proliferation, apoptosis and differentiation [15]. TRAF proteins contain a TRAF domain at the C-terminus, which is important for interactions with upstream regulators and for mediating TRAF homo or hetero-oligomerization [16]. All TRAFs, except for TRAF1, also contain an N-terminal RING finger and several zinc finger motifs, which function in downstream signaling [17].
Multiple studies have demonstrated a requirement for TRAF2 in TNF-stimulated SAPK/JNK activation and TRAF2 has also been shown to activate MAP3Ks such as MEKK1 and apoptosis-stimulated kinase 1 (ASK1) [18], [19], [20], [21], [22], [23]. TRAF6 binds receptors in the IL-1R/TLR superfamily and cells deficient in TRAF6 fail to respond to lipopolysaccharide (LPS), IL-1 and IL-8 [24], [25]. IL-1- and IL-8-induced TRAF6 activation leads to AP-1 activation via the SAPK/JNK pathway [26]. TRAF6 is ubiquitinated with K63-linked polyubiquitin chains via the same E2 complex (Ubc13/Uev1A) as TRAF2 resulting in activation of the MAP3K, TGFβ activated kinase 1 (TAK1), and the IκB kinase complex [26], [27]. The TRAF6 RING domain is required for the activation of TAK1 or SAPK/JNK by TRAF6 [28]. Collectively, these data indicate that TRAF2 and TRAF6 play critical roles in the activation of MAP3Ks in cytokine signaling.
In this study, we wished to determine if the activation of MLK3 by TNF and IL-1β occurs through a TRAF-dependent mechanism. We show that MLK3 interacts with TRAF2 and TRAF6 in response to TNF and IL-1β, respectively. Depletion of TRAF2 or TRAF6 with RNAi impaired MLK3 activation by TNF indicating that TRAF2 and TRAF6 are required for TNF-stimulated MLK3 activation. We show that MLK3 ubiquitination occurs rapidly in response to TNF and IL-1β and that MLK3 can be conjugated with K63- and K48-linked polyubiquitin chains. In addition, our results suggest that K48-linked ubiquitination directs MLK3 for proteosomal degradation while K63-linked ubiquitination is important for MLK3 kinase activity.
Section snippets
Cell culture and reagents
Human embryonic kidney 293 (HEK293), human epithelial ovarian cancer (SKOV3) and human colon cancer cells (HT29) were obtained from the American Type Culture Collection. Cells were grown in a humidified atmosphere with 5% CO2 at 37 °C. HEK293, SKOV3 and HT29 cells were cultured in Dulbecco's Modified Eagle's Medium (Cellgro) supplemented with 10% fetal bovine serum (Hyclone). All culture media was supplemented with 25 µg/ml streptomycin and 25 I.U. penicillin (Cellgro).
Human recombinant TNF and
TNF and IL-1β activate MLK3 kinase activity
TNF activates the TNFR1 receptor and this leads to activation of SAPK/JNK and p38 MAPK signaling pathways [31]. To analyze the timing of the activation of endogenous MLK3 by TNF, SKOV3 and HT29 cells were treated with 50 ng/ml of human recombinant TNF for time periods ranging from 0 to 15 min. Cellular extracts were analyzed by Western blotting with an antibody that specifically recognizes phosphorylated residues (Thr 277/Ser 281) in the activation loop of the MLK3 kinase domain.
Discussion
Both MLK3 and TRAF2 are required for TNF-induced SAPK/JNK activation. Our data suggest that MLK3 is recruited to the TNFR via binding to TRAF2 and/or TRAF6, and this interaction is necessary for MLK3 activation by cytokines. We found that MLK3 associates with both TRAF2 and TRAF6, and MLK3 specifically interacts with the TRAF domain of TRAF2. This finding is similar to that observed with other MAP3Ks such as ASK1, which binds the TRAF domain of TRAF2 to facilitate SAPK/JNK activation [22], [23]
Conclusion
Our findings indicate that upon TNF or IL-1 receptor ligation, TRAF2 and TRAF6 interact with MLK3 and are required for TNF-stimulated activation of MLK3. TNF stimulation promotes MLK3 ubiquitination and MLK3 can be ubiquitinated with K48- or K63-linked chains. We observed that K63-linked ubiquitination is required for optimal MLK3 kinase activity and K48-linked ubiquitination is important for proteosomal degradation of MLK3. Taken together, our results suggest that the interaction with TRAF
Acknowledgements
We gratefully acknowledge Dr. J. Kyriakis for providing the GST-TRAF2 constructs, Dr. D. Leaman for the TRAF6 construct and Dr. T. Dawson for the Ubiquitin constructs. This work was supported by a National Institutes of Health grant 1 R15 CA132006-01 (to D.N.C).
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These authors contributed equally to this work.