Analysis of the Rap2- MAP4K signaling system

我们最近发现Ras样小GTP结合蛋白Rap2与三种蛋白激酶特异性相互作用：（1）Nck相互作用激酶（NIK），也称为HPK/GCK样激酶（HGK）或丝裂原激活蛋白激酶激酶激酶4（MAP4K4）； (2)Traf和Nck相互作用激酶(TNIK)； (3)畸形/NIKs相关激酶(MINK)。这些激酶属于 STE20 组激酶的 GCK 家族。 STE20 组激酶共享与 STE20（芽殖酵母中的 MAP4K）同源的激酶结构域。哺乳动物中的 STE20 组激酶调节应激激活的丝裂原激活蛋白激酶 (MAPK)，例如 c-Jun N 末端激酶 (JNK) 和 p38MAPK。 NIK/HGK/MAP4K4、TNIK 和 MINK 具有相似的 N 端激酶结构域和 C 端调节结构域。 Rap2 以 GTP 依赖性方式与这些激酶的 C 末端调节结构域结合。 C 端调节结构域也称为 CNH 结构域（citron 同源结构域），因为在 citron 中发现了类似的结构，citron 是 Rho 家族小 GTP 结合蛋白（如 Cdc42 和 Rac）的效应分子。 NIK/HGK/MAP4K4、TNIK 和 MINK 不与 Ras 或 Rap1 相互作用。因此，我们认为NIK/HGK/MAP4K4、TNIK和MINK是Rap2的特异性效应分子，并且是新信号系统的组成部分，我们将其称为Rap2-MAP4K系统。为了鉴定该 Rap2-MAP4K 系统上游或下游的分子，我们利用 GS​​T-TNIK 亲和色谱与质谱联用，并鉴定了具有三肽重复、锚蛋白重复、卷曲螺旋区域和 PDZ 结合基序的支架蛋白。分析还鉴定出 TNIK 本身是 TNIK 结合蛋白，表明该系统含有 MAP4K 二聚体或寡聚体。我们还利用差分二维凝胶电泳发现了受 TNIK 调控的潜在磷蛋白。

We have recently found that a Ras-like small GTP-binding protein Rap2 interacts specificaly with three protein kinases : (1)Nck-interacting kinase (NIK), also known as HPK/GCK-like kinase (HGK) or mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) ; (2)Traf-and Nck-interacting kinase (TNIK) ; and (3)Misshapen/NIKs related kinase (MINK). These kinases belong to the GCK family of the STE20 group kinases. The STE20 group kinases share kinase domains homologous to that of STE20, a MAP4K in budding yeast. STE20 group kinases in mammals regulate stress-activated mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK) and p38MAPK. NIK/HGK/MAP4K4, TNIK, and MINK share similar N-terminal kinase domains and C-terminal regulatory domains. Rap2 binds to the C-terminal regulatory domains of these kinases in a GTP-dependent manner. The C-terminal regulatory domain is also termed the CNH domain (citron homology domain), since similar structure is found in citron, an effector molecule of Rho family small GTP-binding proteins such as Cdc42 and Rac. NIK/HGK/MAP4K4, TNIK, and MINK did not interact with Ras or Rap1. Thus, we believe that NIK/HGK/MAP4K4, TNIK, and MINK are specific effector molecules of Rap2 and are components of a new signaling system, which we term the Rap2-MAP4K system. To identify molecules that are upstream or downstream of this Rap2-MAP4K system, we utilized GST-TNIK affinity chromatography coupled with mass spectrometry, and identified a scaffold protein with tricopeptide repeats, ankyrin repeats, a coiled-coil region and a PDZ-binding motif. The analysis also identified TNIK itself as a TNIK-binding protein, suggesting that the system contains dimmer or oligomer of MAP4K. We also utilized the differential two-dimensional gel electrophoresis and found potential phosphoproteins regulated by TNIK.