Leo E, Welsh K, Matsuzawa S, Zapata J M, Kitada S, Mitchell R S, Ely K R, Reed J C
Burnham Institute, La Jolla, California 92037, USA.
J Biol Chem. 1999 Aug 6;274(32):22414-22. doi: 10.1074/jbc.274.32.22414.
CD40 is a member of the tumor necrosis factor receptor family that mediates a number of important signaling events in B-lymphocytes and some other types of cells through interaction of its cytoplasmic (ct) domain with tumor necrosis factor receptor-associated factor (TRAF) proteins. Alanine substitution and truncation mutants of the human CD40ct domain were generated, revealing residues critical for binding TRAF2, TRAF3, or both of these proteins. In contrast to TRAF2 and TRAF3, direct binding of TRAF1, TRAF4, TRAF5, or TRAF6 to CD40 was not detected. However, TRAF5 could be recruited to wild-type CD40 in a TRAF3-dependent manner but not to a CD40 mutant (Q263A) that selectively fails to bind TRAF3. CD40 mutants with impaired binding to TRAF2, TRAF3, or both of these proteins completely retained the ability to activate NF-kappaB and Jun N-terminal kinase (JNK), implying that CD40 can stimulate TRAF2- and TRAF3-independent pathways for NF-kappaB and JNK activation. A carboxyl-truncation mutant of CD40 lacking the last 32 amino acids required for TRAF2 and TRAF3 binding, CD40(Delta32), mediated NF-kappaB induction through a mechanism that was suppressible by co-expression of TRAF6(DeltaN), a dominant-negative version of TRAF6, but not by TRAF2(DeltaN), implying that while TRAF6 does not directly bind CD40, it can participate in CD40 signaling. In contrast, TRAF6(DeltaN) did not impair JNK activation by CD40(Delta32). Taken together, these findings reveal redundancy in the involvement of TRAF family proteins in CD40-mediated NF-kappaB induction and suggest that the membrane-proximal region of CD40 may stimulate the JNK pathway through a TRAF-independent mechanism.
CD40是肿瘤坏死因子受体家族的成员，通过其胞质（ct）结构域与肿瘤坏死因子受体相关因子（TRAF）蛋白相互作用，在B淋巴细胞和其他一些类型的细胞中介导许多重要的信号事件。生成了人CD40ct结构域的丙氨酸替代和截短突变体，揭示了对结合TRAF2、TRAF3或这两种蛋白至关重要的残基。与TRAF2和TRAF3不同，未检测到TRAF1、TRAF4、TRAF5或TRAF6与CD40的直接结合。然而，TRAF5可以以TRAF3依赖的方式被募集到野生型CD40，但不能被募集到选择性地无法结合TRAF3的CD40突变体（Q263A）。与TRAF2、TRAF3或这两种蛋白结合受损的CD40突变体完全保留了激活核因子κB（NF-κB）和Jun N末端激酶（JNK）的能力，这意味着CD40可以刺激不依赖TRAF2和TRAF3的NF-κB和JNK激活途径。缺乏TRAF2和TRAF3结合所需的最后32个氨基酸的CD40羧基截短突变体CD40(Delta32)，通过一种可被TRAF6的显性负性形式TRAF6(DeltaN)共表达抑制但不能被TRAF2(DeltaN)抑制的机制介导NF-κB诱导，这意味着虽然TRAF6不直接结合CD40，但它可以参与CD40信号传导。相比之下，TRAF6(DeltaN)不损害CD40(Delta32)对JNK的激活。综上所述，这些发现揭示了TRAF家族蛋白在CD40介导的NF-κB诱导中的冗余性，并表明CD40的膜近端区域可能通过一种不依赖TRAF的机制刺激JNK途径。
