Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan.
Biochem J. 2010 Mar 29;427(2):237-45. doi: 10.1042/BJ20091011.
SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) belongs to the MAPK (mitogen-activated protein kinase) family and is important in many biological contexts. JNK activation is regulated by phosphorylation of specific tyrosine and threonine residues sequentially catalysed by MKK4 and MKK7, which are both dual-specificity MAPKKs (MAPK kinases). Previously, we reported that tyrosine-phosphorylation of JNK by MKK4 precedes threonine-phosphorylation by MKK7, and that both are required for synergistic JNK activation. In the present study, we identify the actin-binding protein-280 (Filamin A) as a presumed 'binder' protein that can bind to MKK7, as well as to MKK4, connecting them in close proximity. We show that Filamin family members A, B and C interact with MKK4 and MKK7, but not with JNK. Filamin A binds to an N-terminal region (residues 31-60) present in the MKK7gamma and MKK7beta splice isoforms, but cannot bind to MKK7alpha which lacks these amino acids. This same N-terminal region is crucial for the intracellular co-localization of MKK7gamma with actin stress fibres and Filamin A. Experiments using Filamin-A-deletion mutants revealed that the MKK7-binding region of Filamin A differs from its MKK4-binding region, and that MKK7gamma (but not MKK7alpha) can form a complex with Filamin A and MKK4. Finally, we used Filamin-A-deficient cells to show that Filamin A enhances MKK7 activation and is important for synergistic stress-induced JNK activation in vivo. Thus Filamin A is a novel member of the group of scaffold proteins whose function is to link two MAPKKs together and promote JNK activation.
SAPK/JNK（应激激活蛋白激酶/c-Jun N-末端激酶）属于 MAPK（丝裂原激活蛋白激酶）家族，在许多生物学背景下都很重要。JNK 的激活受 MKK4 和 MKK7 顺序催化的特定酪氨酸和苏氨酸残基磷酸化调节，MKK4 和 MKK7 都是双特异性 MAPKK（MAPK 激酶）。以前，我们报道 MKK4 对 JNK 的酪氨酸磷酸化先于 MKK7 的苏氨酸磷酸化，并且两者都需要协同 JNK 激活。在本研究中，我们确定肌动蛋白结合蛋白-280（Filamin A）为一种假定的“结合蛋白”，可以与 MKK7 以及 MKK4 结合，将它们紧密连接在一起。我们表明，Filamin 家族成员 A、B 和 C 与 MKK4 和 MKK7 相互作用，但与 JNK 不相互作用。Filamin A 与 MKK7gamma 和 MKK7beta 剪接异构体中存在的 N 端区域（残基 31-60）结合，但不能与缺乏这些氨基酸的 MKK7alpha 结合。该相同的 N 端区域对于 MKK7gamma 与肌动蛋白应激纤维和 Filamin A 的细胞内共定位至关重要。使用 Filamin-A 缺失突变体的实验表明，Filamin A 的 MKK7 结合区域与 MKK4 结合区域不同，并且 MKK7gamma（但不是 MKK7alpha）可以与 Filamin A 和 MKK4 形成复合物。最后，我们使用 Filamin-A 缺陷细胞表明 Filamin A 增强了 MKK7 的激活，并且对于体内协同应激诱导的 JNK 激活很重要。因此，Filamin A 是连接两个 MAPKK 并促进 JNK 激活的支架蛋白组的新成员。
