The Traf2- and Nck-interacting Kinase as a Putative Effector of Rap2 to Regulate Actin Cytoskeleton

Abstract

Rap2 belongs to the Ras family of small GTP-binding proteins, but its specific roles in cell signaling remain unknown. In the present study, we have affinity-purified from rat brain a Rap2-interacting protein of ϳ155 kDa, p155. By liquid chromatography tandem mass spectrometry, we have identified p155 as Traf2-and Nck-interacting kinase (TNIK). TNIK possesses an N-terminal kinase domain homologous to STE20, the Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase kinase, and a C-terminal regulatory domain termed the citron homology (CNH) domain. TNIK induces disruption of F-actin structure, thereby inhibiting cell spreading. In addition, TNIK specifically activates the c-Jun N-terminal kinase (JNK) pathway. Among our observations, TNIK interacted with Rap2 through its CNH domain but did not interact with Rap1 or Ras. TNIK interaction with Rap2 was dependent on the intact effector region and GTP-bound configuration of Rap2. When co-expressed in cultured cells, TNIK colocalized with Rap2, while a mutant TNIK lacking the CNH domain did not. Rap2 potently enhanced the inhibitory function of TNIK against cell spreading, but this was not observed for the mutant TNIK lacking the CNH domain. Rap2 did not significantly enhance TNIKinduced JNK activation, but promoted autophosphorylation and translocation of TNIK to the detergentinsoluble cytoskeletal fraction. These results suggest that TNIK is a specific effector of Rap2 to regulate actin cytoskeleton.Rap2 is a member of the Ras family of small GTP-binding proteins, which regulate a range of cellular processes including cell proliferation, differentiation, and cytoskeletal rearrangement (for a review, see Ref. 1). To regulate these processes, Ras family proteins cycle between GTP-bound active and GDPbound inactive forms. In the GTP-bound active form, Ras family proteins physically interact with downstream effectors and thereby regulate their subcellular localization and activity (1). For instance, GTP-bound Ras interacts with effectors including Raf-1, B-raf, Ral guanine nucleotide dissociation stimulator During interaction with effectors, the effector regions of Ras family proteins (amino acids 32-40 in the case of Ras) serve as binding interfaces; thus, mutations within their effector regions impair interaction with effectors (Refs. 1-4, reviewed in Ref. 6). The effector regions are also critical for the differential recognition of effectors. For instance, the effector region of Rap1, a close relative of Rap2, is identical to that of Ras. Rap1 interacts with effectors of Ras, and sometimes counteracts Rasmediated signaling (1, 2). For example, Rap1 regulates the extracellular signal-regulated kinase (ERK) pathway, the "classical" mitogen-activated protein kinase (MAPK) pathway, through Raf-1 and B-raf. Although Rap1 interacts with Raf-1 and B-raf, it only activates B-raf. In fibroblasts, Rap1 inhibits Ras-induced cellular transformation. Rap1 exerts this action presumably by trapping Raf-1 in an inactive complex, thereby inhibiting ERK activation (1...

Attenuated adenosine-to-inosine editing of microRNA-376a* promotes invasiveness of glioblastoma cells

“…It has been shown that RAP2 regulates the actin cytoskeleton via two effectors, PARG1 and TNIK (36,37). In HEK293 cells, TNIK induces actin fiber disassembly and perturbs cell spreading, a function that is promoted by RAP2A (37). A similar regulatory role for RAP2A in cancer cell migration, dependent on actin cytoskeleton remodeling, is likely and is suggested by the current work, which demonstrates in vitro that RAP2A knockdown promotes both migration and invasion of glioma cells.…”

“…RAP2A is involved in the regulation of dendrite and axonal branches and restrains dendritic outgrowth of neurons (34,35). It has been shown that RAP2 regulates the actin cytoskeleton via two effectors, PARG1 and TNIK (36,37). In HEK293 cells, TNIK induces actin fiber disassembly and perturbs cell spreading, a function that is promoted by RAP2A (37).…”

The EphA4 Receptor Regulates Neuronal Morphology through SPAR-Mediated Inactivation of Rap GTPases

“…Active Rap1 has also been shown to influence the subcellular localization of Rac and Rho regulatory proteins, leading to localized cell spreading and the formation of cell protrusions (Arthur et al, 2004;Yamada et al, 2005), consistent with positive effects of Rap1 on growth cone and dendritic spine size. On the other hand, Rap2 but not Rap1 interacts with TNIK (Traf2-and Nck-interacting kinase), a kinase expressed in the brain Taira et al, 2004;Zhu et al, 2005). Binding of activated Rap2 enhances the ability of TNIK to decrease F-actin levels and inhibit cell spreading.…”

Crystal Structure of the RUN Domain of the RAP2-interacting Protein x

“…RPIP9 expression is activated during malignant breast epithelial transformation and increases with the progression toward an invasive phenotype (15). Tumor necrosis factor receptor-associated factor 2 (Traf2)-and Nck-interacting kinase (TNIK) is a recently identified Rap2-specific effector that is distinct from RPIP8 (16,17). TNIK interacts with Rap2 through its C-terminal regulatory domain, termed the CNH domain, and regulates the actin cytoskeleton.…”