Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex - PubMed (original) (raw)

Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex

Atsushi Matsuzawa et al. Science. 2008.

Abstract

Cytokine signaling is thought to require assembly of multicomponent signaling complexes at cytoplasmic segments of membrane-embedded receptors, in which receptor-proximal protein kinases are activated. Indeed, CD40, a tumor necrosis factor receptor (TNFR) family member, forms a complex containing adaptor molecules TRAF2 and TRAF3, ubiquitin-conjugating enzyme Ubc13, cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/2), IkappaB kinase regulatory subunit IKKgamma (also called NEMO), and mitogen-activated protein kinase (MAPK) kinase kinase MEKK1 upon ligation. TRAF2, Ubc13, and IKKgamma were required for complex assembly and activation of MEKK1 and MAPK cascades. However, these kinases were not activated unless the multicomponent signaling complex translocated from CD40 to the cytosol upon c-IAP1/2-induced degradation of TRAF3. This two-stage signaling mechanism may apply to other innate immune receptors, accounting for spatial and temporal separation of MAPK and IKK signaling.

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Figures

Fig. 1

Fig. 1

Requirement of TRAF2 for CD40-mediated activation of MEKK1. (A) MEKK1 activation in B cells. Splenic B cells from mice reconstituted with WT, _Traf2_−/minus;, _Traf3_−/minus;, and _Traf6_−/minus; fetal liver were stimulated with CD40 agonistic antibody. Cell lysates were prepared when indicated and phosphorylation of MEKK1, TAK1, JNK, and p38 was analyzed by immunoblotting. The kinase activity of immunoprecipitated JNK was measured by phosphorylation of GST-c-Jun(1–79). Immunoblotting with anti-JNK served as the loading control. (B) MEKK1 activation in a B cell line. A20 B cells transduced with lentiviruses containing no insert or shRNAs to TRAF2, 3 or 6 were stimulated with anti-CD40. MEKK1, TAK1, JNK, and p38 phosphorylation and TRAF2, 3 or 6 expression were analyzed by immunoblotting. (C) Kinetics of CD40-induced phosphorylation of MEKK1 and TAK1. Phosphorylation kinetics of MEKK1 in WT and TRAF knockout (KO) B cells were determined by densitometric analysis of two separate experiments as in (A). The early kinetics of MEKK1 and TAK1 phosphorylation in control and TRAF3 knockdown (KD) A20 B cells were determined in two separate experiments similar to the one shown in (B).

Fig. 2

Fig. 2

Dependence of MEKK1 activation on Ubc13 and IKKγ and association with formation of a multi-component signaling complex. (A) A20 B cells transduced with lentiviruses with no insert or Ubc13 shRNA were stimulated with anti-CD40. Lysates were immunoblotted to detect phosphorylation of MEKK1, JNK, and p38, degradation of IκBα, and expression of Ubc13. (B) _Ikkγ_-null 1.3E2 B cells and parental 70Z3 cells were stimulated with anti-CD40. At the indicated times, lysates were prepared and analyzed by immunoblotting for MAP3K and MAPK phosphorylation and IκBα degradation. (C, D) Splenic B cells were stimulated with anti-CD40. At the indicated times, cell lysates were prepared and immunoprecipitated (IP) with anti-CD40 (C) or anti-MEKK1 (D) antibodies. The gel-separated immunecomplexes were immunoblotted (IB) with the indicated antibodies.

Fig. 3

Fig. 3

Release of the receptor-associated signaling complex to the cytosol. Splenic B cells were stimulated with anti-CD40 and membrane and cytosolic fractions were prepared at the indicated times. CD40 (A), IKKγ (B), or MEKK1 (C) were immunoprecipitated (IP) from lysates of each fraction, and the gel-separated immunecomplexes were immunoblotted (IB) with the indicated antibodies. The amounts of the analyzed proteins in total B cell lysates and the subcellular fractions are shown in Fig. S7.

Fig. 4

Fig. 4

Role of individual components in assembly of the CD40-associated signaling complex. (A) Role of MEKK1. Mekk1+/ΔKD or Mekk1ΔKD/ΔKD B cells were stimulated with anti-CD40 and the membrane fraction was immunoprecipitated with anti-CD40. (B) Role of IKKγ. 70Z3 and 1.3E2 B cells were stimulated and analyzed as above. (C) Roles of TRAF2 and Ubc13. A20 B cells transduced with lentiviruses containing no insert or shRNAs for TRAF2 or Ubc13 were stimulated and analyzed as above. After washing, the CD40 immunecomplexes in all 3 experiments were gel-separated and immunoblotted with the indicated antibodies.

Fig. 5

Fig. 5

Requirement of c-IAP and proteasome activity for cytosolic translocation of the receptor-associated signaling complex and TRAF3 degradation. (A, B) Requirement of c-IAP1/2. c-IAP-deficient and control multiple myeloma cells (A), or splenic B cells treated with or without the c-IAP inhibitor Smac mimic (SM) for 4 hrs (B) were stimulated with anti-CD40. (C) Requirement of proteasome activity. Splenic B cells were treated with or without the proteasome inhibitor MG132 for 30 min prior to anti-CD40 stimulation. All cells were divided into membrane (Mem) and cytosolic (Cyt) fractions that were immunoprecipitated with anti-CD40 and anti-MEKK1, respectively. The gel-separated immunecomplexes were immunoblotted with the indicated antibodies. (D) Degradation of membrane- and MEKK1-associated TRAF3 in a c-IAP1/2-dependent manner. Splenic B cells were treated or not with SM as in (B) and stimulated with anti-CD40. At the indicated times, the cells were divided into membrane and cytosolic fractions. MEKK1 was immunoprecipitated and presence of indicated proteins in the immunecomplexes and total cell lysate was examined. (E) Requirement of c-IAP1/2 for TRAF2 and TRAF3 ubiquitination. B cells preincubated without or with SM were stimulated with anti-CD40 for 10 min. Cells were divided into membrane and cytosol fractions. TRAF2 and TRAF3 were immunoprecipitated from both fractions, extensively washed, gel-separated, and analyzed by immunoblotting with conventional or K63-specific anti-ubiquitin antibodies.

Fig. 6

Fig. 6

Requirement of c-IAP and proteasome activity for cytosolic activation of MAP3K signaling modules. (A, B) Splenic B cells were treated with or without SM (A) or MG132 (B) for 4 hrs and 30 min, respectively, prior to anti-CD40 stimulation. Kinase phosphorylation/activation was monitored by immunoblotting. (C) Subcellular location of MEKK1 module activation. Splenic B cells were stimulated with anti-CD40 and divided at the indicated times into membrane and cytosolic fractions that were immunoprecipitated with anti-CD40 or anti-MEKK1, respectively and separated on the same gel. Kinase phosphorylation/activation was monitored by immunoblotting. (D) Requirement of c-IAP1/2 for cytosolic translocation of the TRAF6-TAK1 signaling complex. Splenic B cells treated with or without SM were stimulated with anti-CD40. Cells were divided into membrane and cytosolic fractions that were immunoprecipitated with anti-CD40 and anti-TAK1, respectively. The gel-separated immunecomplexes were blotted with the indicated antibodies. (E) Splenic B cells were pretreated with or without SM and stimulated anti-CD40. At the indicated times, membranes were isolated, lysed and immunoprecipitated with anti-CD40. The gel-separated immunecomplexes were analyzed as above.

Fig. 7

Fig. 7

MEKK1 signaling depends on c-IAP-induced TRAF3 degradation and a two-stage activation mechanism. (A) Requirement of c-IAP2 E3 ligase activity for MEKK1 activation and TRAF3 degradation. c-IAP-deficient multiple myeloma cells were transfected with vector, flag-c-IAP2(WT) and flag-c-IAP2(RM) (c-IAP2 with RING finger mutations that render the E3 ligase inactive). After G418 selection for 1 week, the cells were stimulated with anti-CD40. Lysates were analyzed by immunoblotting with the indicated antibodies. (B) TRAF3 deficiency renders MEKK1 signaling c-IAP1/2-independent. TRAF3 knockdown A20 cells were incubated with or without SM and stimulated with anti-CD40. At the indicated times, MEKK1 and JNK activation were analyzed by immunoblotting. (C) TRAF3 ubiquitination by c-IAP2. Cell lysates were prepared as well as (A). c-IAP2 WT and RM were immunoprecipitated, washed, and incubated with or without GST-TRAF3 in a reaction mixture containing ubiquitin, ATP, E1 and a mixture of E2 ubiquitin-conjugating enzymes. After 30 min, GST-TRAF3 was immunoprecipitated, gel-separated, and analyzed by immunoblotting with anti-ubiquitin and anti-TRAF3 antibodies. Amounts of c-IAP2 WT and RM were examined with anti-Flag antibody. (D) A two-stage signaling mechanism. a. In non-stimulated B cells, only CD40 is membrane-associated. b. Receptor engagement induces trimerization and recruitment of TRAF2, TRAF3, c-IAP1/2 and Ubc13. c. Next to be recruited into this complex are IKKγ and MEKK1. Interactions between IKKγ and MEKK1 and K63-linked polyubiquitin chains catalyzed by TRAF2 and Ubc13 stabilize the complex. d. c-IAP1/2 catalyze K48-linked polyubiquitination of TRAF3 whose proteasomal (26S) degradation results in translocation of the receptor-assembled signaling complex into the cytosol where MEKK1 is activated and in-turn activates downstream components of its signaling module.

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