Tumor necrosis factor receptor 2 (TNFR2) activates transcription factor B (NF-B) and c-Jun N-terminal kinase (JNK).The mechanisms mediating these activations are dependent on the recruitment of TNF receptor-associated factor 2 (TRAF2) to the intracellular region of the receptor. TNFR2 also induces TRAF2 degradation. We show that in addition to the well characterized TRAF2 binding motif 402-SKEE-405, the human receptor contains another sequence located at the C-terminal end (amino acids 425-439), which also recruits TRAF2 and activates NF-B. In addition to that, human TNFR2 contains a conserved region (amino acids 338 -379) which is responsible for TRAF2 degradation and therefore of terminating NF-B signaling. TRAF2 degradation and the lack of NF-B activation when both TNFR1 and TNFR2 are co-expressed results in an enhanced ability of TNFR1 to induce cell death, showing that the cross-talk between both receptors is of a great biological relevance. Induction of TRAF2 degradation appears to be independent of TRAF2 binding to the receptor. Amino acids 343-TGSSDSS-349 are essential for inducing TRAF2 degradation because deletion mutants of this region or point mutations at serine residues 345 and 346 or 348 and 349 obliterate the ability of TNFR2 to induce TRAF2 degradation. Tumor necrosis factor receptor 2 (TNFR2)5 is one of the two receptors known to bind TNF, this cytokine can be found as a transmembrane (mTNF) or a soluble (sTNF) form. Whereas both mTNF and sTNF activate TNFR1, TNFR2 is mainly activated by mTNF (1, 2), which upon binding to the receptor, causes its trimerization and activation. The fact that mTNF is the optimal activator of TNFR2 has implied serious limitations in the study of this receptor. Because the activation of TNFR2 depends on its aggregation, receptor activation can be forced by its overexpression or by the use of specific antibodies against the receptor (3, 4).TNFR2 lacks any intrinsic catalytic activity within its cytoplasmic tail, thus any signal emerging from the receptor depends on the recruitment of adaptor proteins. TNFR2 can bind directly TRAF2 and through this interaction signals for NF-B and JNK activation, as the expression of a dominant negative form of the adaptor protein (TRAF2dn) can suppress both the activation of NF-B and JNK (5).Seven different TRAF proteins have been identified so far (6). All of them share a highly conserved TRAF domain at the protein C terminus and, with the exception of TRAF1, a N-terminal-RING finger domain followed by five to seven zinc-finger motifs (7,8). TRAF proteins were initially considered as adaptor proteins between TNFRs and the kinases implicated in the activation of JNK or IB kinase IKK (9). It was then described that TRAF proteins, because of their RING finger domain, might act as E3 ubiquitin ligases, which catalyze K63-linked ubiquitination (10). In the case of TRAF2 this requires the interaction with cellular inhibitor of apoptosis 1 (cIAP1) and 2 (cIAP2) (11). More recently, it has been suggested that TRAF2 is by itself unable to ac...
Tumor Necrosis Factor (TNF) interacts with two receptors known as TNFR1 and TNFR2. TNFR1 activation may result in either cell proliferation or cell death. TNFR2 activates Nuclear Factor-kappaB (NF-kB) and c-Jun N-terminal kinase (JNK) which lead to transcriptional activation of genes related to cell proliferation and survival. This depends on the binding of TNF Receptor Associated Factor 2 (TRAF2) to the receptor. TNFR2 also induces TRAF2 degradation. In this work we have investigated the structural features of TNFR2 responsible for inducing TRAF2 degradation and have studied the biological consequences of this activity. We show that when TNFR1 and TNFR2 are co-expressed, TRAF2 depletion leads to an enhanced TNFR1 cytotoxicity which correlates with the inhibition of NF-kB. NF-kB activation and TRAF2 degradation depend of different regions of the receptor since TNFR2 mutants at amino acids 343-349 fail to induce TRAF2 degradation and have lost their ability to enhance TNFR1-mediated cell death but are still able to activate NF-kB. Moreover, whereas NF-kB activation requires TRAF2 binding to the receptor, TRAF2 degradation appears independent of TRAF2 binding. Thus, TNFR2 mutants unable to bind TRAF2 are still able to induce its degradation and to enhance TNFR1-mediated cytotoxicity. To test further this receptor crosstalk we have developed a system stably expressing in cells carrying only endogenous TNFR1 the chimeric receptor RANK-TNFR2, formed by the extracellular region of RANK (Receptor activator of NF-kB) and the intracellular region of TNFR2.This has made possible to study independently the signals triggered by TNFR1 and TNFR2. In these cells TNFR1 is selectively activated by soluble TNF (sTNF) while RANK-TNFR2 is selectively activated by RANKL. Treatment of these cells with sTNF and RANKL leads to an enhanced cytotoxicity.
Microtubule interfering agents (MIAs) are anti-tumor drugs that inhibit microtubulearrest and cell death [3,5]. Nowadays, the most used MIAs for 49 cancer treatment are vinca alkaloids (VAs) and taxanes [6]. 50VAs are drugs derived from the periwinkle Catharanthus Gel spots were subjected to in-gel digestion (http://msfacility. 161ucsf.edu/ingel.html) with trypsin (porcine, side-chain removed using a Y-10 microcone (Millipore), proteins were (Fig. 1a). induced forms are phosphorylated (Fig. 2). Spot x also disappeared 246 after λ-PPase treatment and therefore, this form is also con-247 sidered as phosphorylated (Fig. 2) (Fig. 3a). 260These phosphorylations were also confirmed by Western blot 261 analysis (see Fig. 1 of supplementary material). As expected, 262the flow cytometry analysis of these cells showed that all the 263 compounds used induced G2/M arrest and cell death (Fig. 3b). 264Furthermore, a Western blot analysis using antibodies against 265 the molecular marker phospho-histone H3 indicated that the 266 G2/M arrest induced by these agents is at M stage (Fig. 3c). (Fig. 4b) and M phase arrest (Fig. 4c). More interestingly, (Fig. 4a) (Fig. 5a). This downregulation is coupled to a reduction in the 296 number of M phase cells (Fig. 5c) Aphidicolin was added 24 h prior to MIA treatment. Then, cell 312 extracts were subjected to 2D-PAGE followed by Western blot. (Fig. 7b). This drug also induces the spots x, y and z which 337 correspond to processed forms of the protein (Fig. 7b). 338Roscovitine treatment induced p54 nrb processing but did not 339 affect its phosphorylation (Fig. 7b). Interestingly, the addition of (Fig. 8a). In addition, a Western blot analysis using antibodies 354 against phospho-histone H3 indicated that MIAs also cause M 355 arrest in this cell line (Fig. 8b). The KSP inhibitor STLC also 356 induced the phosphorylation of this nuclear factor (see Fig. 2 357 of supplementary material). 4. Discussion
Microtubule interfering agents (MIAs) are antitumor drugs that inhibit microtubule dynamics, while kinesin spindle protein (KSP) inhibitors are substances that block the formation of the bipolar spindle during mitosis. All these compounds cause the accumulation of mitotic cells and subsequently cell death. We used two-dimensional gel electrophoresis (2DE) followed by MALDI-MS analysis to demonstrate that the MIAs vinblastine (Velban) and paclitaxel (Taxol), as well as the KSP inhibitor S-tritil-L-cysteine, induce the phosphorylation of annexin A2 in human lung carcinoma A549 cells. Further tandem mass spectrometry analysis using a combination of peptide fragmentation methods (CID and ETD) and multiple reaction monitoring (MRM) analysis determined that this modification occurs mainly at threonine 19. We show that MIAs and KSP inhibitors only induce this phosphorylation in cells capable of reaching the M phase. Furthermore, we demonstrate that CDK activity is required for the phosphorylation of annexin A2 induced by MIAs and KSP inhibitors. Finally, we have used double thymidine block synchronization to demonstrate that annexin A2 is not phosphorylated during a normal mitosis, indicating that this phosphorylation of annexin A2 is a specific response to these drugs.
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