Limits...
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.

Show MeSH

Related in: MedlinePlus

TNF-α induces the endogenous association of MLK3 with TRAF2. (A) MLK3 and TRAF2 associate endogenously in response to TNF-α in Jurkat cells. The Jurkat T cells were starved for 12 hours in 0.2% serum containing media and treated with 10nM of TNF-α for indicated periods of time. The endogenous TRAF2 protein was immunoprecipitated and immune-complex protein samples were blotted with anti-MLK3 antibody. (B) The endogenous interaction between MLK3 and TRAF2 is specific. The MEFs from wild type and TRAF2 deficient mice were grown and treated with TNF-α as described above and the endogenous TRAF2 was immunoprecipitated and any associated MLK3 protein was detected by anti-MLK3 antibody.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2801772&req=5

Figure 3: TNF-α induces the endogenous association of MLK3 with TRAF2. (A) MLK3 and TRAF2 associate endogenously in response to TNF-α in Jurkat cells. The Jurkat T cells were starved for 12 hours in 0.2% serum containing media and treated with 10nM of TNF-α for indicated periods of time. The endogenous TRAF2 protein was immunoprecipitated and immune-complex protein samples were blotted with anti-MLK3 antibody. (B) The endogenous interaction between MLK3 and TRAF2 is specific. The MEFs from wild type and TRAF2 deficient mice were grown and treated with TNF-α as described above and the endogenous TRAF2 was immunoprecipitated and any associated MLK3 protein was detected by anti-MLK3 antibody.

Mentions: Interaction between TRAF2 and some MAP3K members, such as ASK1 [18, 19], TAK1 [20] and MEKK1 [21] has been shown to induce their kinase activities. We examined which of the TRAF proteins (i.e. TRAF2, 5 and 6) that showed interaction with MLK3 (Fig.1) in fact can activate MLK3 kinase activity. HEK-293 cells were transiently transfected with a constant amount of MLK3 and the three TRAF expression vectors, as shown in Fig. 2. As controls, some cells were also transfected with TRAF expression vectors alone, to rule out the contribution of any contaminating kinase during the kinase assay. Recombinant MLK3 was precipitated by GSH-pull down and subjected to kinase assay using MLK3 specific substrate, SEK1 (K-R) protein, expressed in bacteria. MLK3 was about 3.5 fold more active in the cells, where TRAF2 was transfected (Fig. 2A, compare lanes 2 with 4) where as MLK3 was not significantly activated in cells that were transfected with TRAF5 and 6 (Fig. 2A). The expression levels of MLK3 and TRAFs were almost equal in all our transfection experiments. These results suggest that TRAF2 might be the adaptor protein that recruits MLK3 to the TNFR. To further confirm that our results were not due to transfection artifact, mouse embryonic fibroblasts (MEFs) from wild type and TRAF2 deficient mice were utilized to examine whether TRAF2 is required for TNF-α-induced MLK3 activation. The wild type (TRAF2+/+) and TRAF2 deficient (TRAF2-/-) MEFs were treated with 10nM of TNF-α for 30 or 60 minutes and the endogenous MLK3 kinase activity was measured. Endogenous MLK3 was activated about 4.5 fold following 30 or 60 minutes stimulation by TNF-α in wild type and not in TRAF2 deficient MEFs (Fig. 2B, compare lanes 4 and 5 with 8 and 9). The time period for TNF-α treatment was chosen based on our results as shown in Fig. 3. Taken together these results conclusively demonstrate that TRAF2 is necessary for TNF-α mediated 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 the endogenous association of MLK3 with TRAF2. (A) MLK3 and TRAF2 associate endogenously in response to TNF-α in Jurkat cells. The Jurkat T cells were starved for 12 hours in 0.2% serum containing media and treated with 10nM of TNF-α for indicated periods of time. The endogenous TRAF2 protein was immunoprecipitated and immune-complex protein samples were blotted with anti-MLK3 antibody. (B) The endogenous interaction between MLK3 and TRAF2 is specific. The MEFs from wild type and TRAF2 deficient mice were grown and treated with TNF-α as described above and the endogenous TRAF2 was immunoprecipitated and any associated MLK3 protein was detected by anti-MLK3 antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801772&req=5

Figure 3: TNF-α induces the endogenous association of MLK3 with TRAF2. (A) MLK3 and TRAF2 associate endogenously in response to TNF-α in Jurkat cells. The Jurkat T cells were starved for 12 hours in 0.2% serum containing media and treated with 10nM of TNF-α for indicated periods of time. The endogenous TRAF2 protein was immunoprecipitated and immune-complex protein samples were blotted with anti-MLK3 antibody. (B) The endogenous interaction between MLK3 and TRAF2 is specific. The MEFs from wild type and TRAF2 deficient mice were grown and treated with TNF-α as described above and the endogenous TRAF2 was immunoprecipitated and any associated MLK3 protein was detected by anti-MLK3 antibody.
Mentions: Interaction between TRAF2 and some MAP3K members, such as ASK1 [18, 19], TAK1 [20] and MEKK1 [21] has been shown to induce their kinase activities. We examined which of the TRAF proteins (i.e. TRAF2, 5 and 6) that showed interaction with MLK3 (Fig.1) in fact can activate MLK3 kinase activity. HEK-293 cells were transiently transfected with a constant amount of MLK3 and the three TRAF expression vectors, as shown in Fig. 2. As controls, some cells were also transfected with TRAF expression vectors alone, to rule out the contribution of any contaminating kinase during the kinase assay. Recombinant MLK3 was precipitated by GSH-pull down and subjected to kinase assay using MLK3 specific substrate, SEK1 (K-R) protein, expressed in bacteria. MLK3 was about 3.5 fold more active in the cells, where TRAF2 was transfected (Fig. 2A, compare lanes 2 with 4) where as MLK3 was not significantly activated in cells that were transfected with TRAF5 and 6 (Fig. 2A). The expression levels of MLK3 and TRAFs were almost equal in all our transfection experiments. These results suggest that TRAF2 might be the adaptor protein that recruits MLK3 to the TNFR. To further confirm that our results were not due to transfection artifact, mouse embryonic fibroblasts (MEFs) from wild type and TRAF2 deficient mice were utilized to examine whether TRAF2 is required for TNF-α-induced MLK3 activation. The wild type (TRAF2+/+) and TRAF2 deficient (TRAF2-/-) MEFs were treated with 10nM of TNF-α for 30 or 60 minutes and the endogenous MLK3 kinase activity was measured. Endogenous MLK3 was activated about 4.5 fold following 30 or 60 minutes stimulation by TNF-α in wild type and not in TRAF2 deficient MEFs (Fig. 2B, compare lanes 4 and 5 with 8 and 9). The time period for TNF-α treatment was chosen based on our results as shown in Fig. 3. Taken together these results conclusively demonstrate that TRAF2 is necessary for TNF-α mediated 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