Analysis of the Rap2- MAP4K signaling system

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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.