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TRAF2-MLK3 interaction is essential for TNF-alpha-induced MLK3 activation.

Sondarva G, Kundu CN, Mehrotra S, Mishra R, Rangasamy V, Sathyanarayana P, Ray RS, Rana B, Rana A - Cell Res. (2009)

Bottom Line: TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK.Furthermore the downstream target of MLK3, JNK was activated by TNF-alpha in a TRAF2-dependent manner.Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-alpha-induced activation of MLK3 and its downstream target, JNK.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA.

ABSTRACT
Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-alpha (TNF-alpha) and specifically activates c-Jun N-terminal kinase (JNK) on TNF-alpha stimulation. The mechanism by which TNF-alpha activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-alpha treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-alpha treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-alpha in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-alpha-induced activation of MLK3 and its downstream target, JNK.

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TNF-α induces an association between TRAF2 and MLK3 and activates MLK3 kinase activity. (A) Association of TRAF2 with MLK3 increases its kinase activity. Jurkat T cells were starved for 12 hours as indicated in Fig. 3 and were treated with 10nM of TNF-α for indicated periods of time. Cells were lysed and comparable amounts of endogenous total MLK3 and MLK3 associated with TRAF2 were individually immunoprecipitated and MLK3 kinase activities were measured using SEK1 (K-R) as substrate. (B) Interaction between TRAF2 and MLK3 is necessary for TNF-α-induced MLK3 kinase activation. HEK-293 cells were transfected with MLK3 and increasing concentrations of TRAF2 mutant constructs (i.e. TRAF2 AA 272-358) to compete off the binding between full length endogenous TRAF2 and recombined MLK3. Some of these cells were treated with TNF-α as indicated for 30 minutes. The recombined MLK3 was immunoprecipitated and kinase activity was detected as described in Fig. 2.
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Figure 5: TNF-α induces an association between TRAF2 and MLK3 and activates MLK3 kinase activity. (A) Association of TRAF2 with MLK3 increases its kinase activity. Jurkat T cells were starved for 12 hours as indicated in Fig. 3 and were treated with 10nM of TNF-α for indicated periods of time. Cells were lysed and comparable amounts of endogenous total MLK3 and MLK3 associated with TRAF2 were individually immunoprecipitated and MLK3 kinase activities were measured using SEK1 (K-R) as substrate. (B) Interaction between TRAF2 and MLK3 is necessary for TNF-α-induced MLK3 kinase activation. HEK-293 cells were transfected with MLK3 and increasing concentrations of TRAF2 mutant constructs (i.e. TRAF2 AA 272-358) to compete off the binding between full length endogenous TRAF2 and recombined MLK3. Some of these cells were treated with TNF-α as indicated for 30 minutes. The recombined MLK3 was immunoprecipitated and kinase activity was detected as described in Fig. 2.

Mentions: TRAF2 is an adaptor protein and lacks any intrinsic kinase activity and thus we wanted to know how TRAF2 activates MLK3 kinase activity. One potential way by which TRAF2 might activate MLK3 is via protein-protein interaction in response to TNF-α treatment. To test whether direct interaction of TRAF2 with MLK3 activates MLK3 kinase activity by TNF-α, Jurkat T cells were treated with TNF-α for different time points, as indicated in Fig. 5A. First, we optimized the conditions to immunoprecipitate equal amounts of total MLK3 and TRAF2 associated MLK3 by using MLK3 and TRAF2 antibodies respectively (Fig. 5A). Once we optimized how much of cell lysates were needed to immunoprecipitate equal amounts of MLK3 by both the antibodies, we immunoprecipitated comparable amounts of total and TRAF2 associated MLK3 as shown in Fig. 5A, and measured MLK3 kinase activity by using SEK1 (K-R) protein. As envisioned, our results showed about 4 fold activation of TRAF2-associated MLK3 when compared to equal amount of total MLK3 after 30 minutes of TNF-α treatment (Fig. 5A, compare lanes 5 with 6). These results clearly suggest that TNF-α-induced TRAF2-MLK3 interaction is required for MLK3 activation. To further confirm that the interaction between TRAF2-MLK3 indeed is required for induction of MLK3 kinase activity, we attempted to compete off this interaction by using the TRAF-N domain that we determined to be the minimal domain on TRAF2 for MLK3 interaction (Fig. 4B). Constant amount of MLK3 and increasing quantity of cDNA expressing TRAF-N domain (AA, 272-358) were transfected in HEK-293 cells and treated with either 10nM of TNF-α for 30 minutes, or left alone, as controls (Fig. 5B). Equal amount of MLK3 was immunoprecipitated from these cells and subjected to kinase assay. Confirming our endogenous interaction data (Fig. 5A), the MLK3 kinase activity was gradually attenuated/competed off by increasing dose of TRAF-N domain in TNF-α treated cells but not in untreated cells (Fig. 5B). These results clearly suggest that TNF-α induces interaction between TRAF2 and MLK3 and this interaction leads to MLK3 activation.


TRAF2-MLK3 interaction is essential for TNF-alpha-induced MLK3 activation.

Sondarva G, Kundu CN, Mehrotra S, Mishra R, Rangasamy V, Sathyanarayana P, Ray RS, Rana B, Rana A - Cell Res. (2009)

TNF-α induces an association between TRAF2 and MLK3 and activates MLK3 kinase activity. (A) Association of TRAF2 with MLK3 increases its kinase activity. Jurkat T cells were starved for 12 hours as indicated in Fig. 3 and were treated with 10nM of TNF-α for indicated periods of time. Cells were lysed and comparable amounts of endogenous total MLK3 and MLK3 associated with TRAF2 were individually immunoprecipitated and MLK3 kinase activities were measured using SEK1 (K-R) as substrate. (B) Interaction between TRAF2 and MLK3 is necessary for TNF-α-induced MLK3 kinase activation. HEK-293 cells were transfected with MLK3 and increasing concentrations of TRAF2 mutant constructs (i.e. TRAF2 AA 272-358) to compete off the binding between full length endogenous TRAF2 and recombined MLK3. Some of these cells were treated with TNF-α as indicated for 30 minutes. The recombined MLK3 was immunoprecipitated and kinase activity was detected as described in Fig. 2.
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Figure 5: TNF-α induces an association between TRAF2 and MLK3 and activates MLK3 kinase activity. (A) Association of TRAF2 with MLK3 increases its kinase activity. Jurkat T cells were starved for 12 hours as indicated in Fig. 3 and were treated with 10nM of TNF-α for indicated periods of time. Cells were lysed and comparable amounts of endogenous total MLK3 and MLK3 associated with TRAF2 were individually immunoprecipitated and MLK3 kinase activities were measured using SEK1 (K-R) as substrate. (B) Interaction between TRAF2 and MLK3 is necessary for TNF-α-induced MLK3 kinase activation. HEK-293 cells were transfected with MLK3 and increasing concentrations of TRAF2 mutant constructs (i.e. TRAF2 AA 272-358) to compete off the binding between full length endogenous TRAF2 and recombined MLK3. Some of these cells were treated with TNF-α as indicated for 30 minutes. The recombined MLK3 was immunoprecipitated and kinase activity was detected as described in Fig. 2.
Mentions: TRAF2 is an adaptor protein and lacks any intrinsic kinase activity and thus we wanted to know how TRAF2 activates MLK3 kinase activity. One potential way by which TRAF2 might activate MLK3 is via protein-protein interaction in response to TNF-α treatment. To test whether direct interaction of TRAF2 with MLK3 activates MLK3 kinase activity by TNF-α, Jurkat T cells were treated with TNF-α for different time points, as indicated in Fig. 5A. First, we optimized the conditions to immunoprecipitate equal amounts of total MLK3 and TRAF2 associated MLK3 by using MLK3 and TRAF2 antibodies respectively (Fig. 5A). Once we optimized how much of cell lysates were needed to immunoprecipitate equal amounts of MLK3 by both the antibodies, we immunoprecipitated comparable amounts of total and TRAF2 associated MLK3 as shown in Fig. 5A, and measured MLK3 kinase activity by using SEK1 (K-R) protein. As envisioned, our results showed about 4 fold activation of TRAF2-associated MLK3 when compared to equal amount of total MLK3 after 30 minutes of TNF-α treatment (Fig. 5A, compare lanes 5 with 6). These results clearly suggest that TNF-α-induced TRAF2-MLK3 interaction is required for MLK3 activation. To further confirm that the interaction between TRAF2-MLK3 indeed is required for induction of MLK3 kinase activity, we attempted to compete off this interaction by using the TRAF-N domain that we determined to be the minimal domain on TRAF2 for MLK3 interaction (Fig. 4B). Constant amount of MLK3 and increasing quantity of cDNA expressing TRAF-N domain (AA, 272-358) were transfected in HEK-293 cells and treated with either 10nM of TNF-α for 30 minutes, or left alone, as controls (Fig. 5B). Equal amount of MLK3 was immunoprecipitated from these cells and subjected to kinase assay. Confirming our endogenous interaction data (Fig. 5A), the MLK3 kinase activity was gradually attenuated/competed off by increasing dose of TRAF-N domain in TNF-α treated cells but not in untreated cells (Fig. 5B). These results clearly suggest that TNF-α induces interaction between TRAF2 and MLK3 and this interaction leads to MLK3 activation.

Bottom Line: TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK.Furthermore the downstream target of MLK3, JNK was activated by TNF-alpha in a TRAF2-dependent manner.Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-alpha-induced activation of MLK3 and its downstream target, JNK.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA.

ABSTRACT
Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-alpha (TNF-alpha) and specifically activates c-Jun N-terminal kinase (JNK) on TNF-alpha stimulation. The mechanism by which TNF-alpha activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-alpha treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-alpha treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-alpha in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-alpha-induced activation of MLK3 and its downstream target, JNK.

Show MeSH
Related in: MedlinePlus