The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the "activator domain" (approximately corresponding to amino acids 26 -28 and 40 -49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.Ras belongs to a family of small GTP-binding proteins and plays essential roles in the regulation of cell proliferation and differentiation (for a review, see Ref. 1). Like other GTP-binding proteins, the GTP-bound form of Ras is active and able to interact with its effectors whereas the GDP-bound form is not. X-ray crystallographic studies showed that, on the protein surface of mammalian Ha-Ras, the conformation of two regions named "switch I" (Asp ) flanking the switch I have also been shown to be critical for effector activation (6 -9). These residues have been proposed to constitute the "activator domain" (Fig. 1A) (11). The activator domain is also exposed on the surface of Ha-Ras but its conformation is not much altered by GDP/GTP exchange (10, 11). In addition, post-translational modifications of Ha-Ras have also been shown to be crucial for its biological function (for a review, see Ref. 12). However, it is presently unclear how these individual structural features are involved in effector activation.Raf-1, a 74-kDa cytoplasmic serine/threonine protein kinase regulating the mitogen-activated protein kinase cascade, is one of the major effectors of Ha-Ras (for a review, see Ref. 13). Raf-1 shares three regions of conservation, termed CR1, 1 CR2, and CR3, with other Raf isoforms and homologs (Fig. 1B) (13). CR1 and CR2 are located in the N-terminal half of Raf-1, and CR3 corresponds to the C-terminal kinase domain. Activation of Raf-1 by N-terminal truncations indicates that the N-terminal portion plays an important regulatory role (14). The minimal region of Raf-1 responsible for the interaction with Ha-Ras has been mapped into 81 amino acids in CR1, RBD (amino acids 51-131) (15), and mutational analyses have suggested that RBD interacts directly with the switch I of . Howeve...

Summary Nedd4-1 is a ‘Neuronal Precursor Cell Expressed and Developmentally Downregulated Protein’ and among the most abundant E3 ubiquitin ligases in mammalian neurons. In analyses of conventional and conditional Nedd4-1 deficient mice, we found that Nedd4-1 plays a critical role in dendrite formation. Nedd4-1, the serine/threonine kinase TNIK, and Rap2A form a complex that controls Nedd4-1-mediated ubiquitination of Rap2A. Ubiquitination by Nedd4-1 inhibits Rap2A function, which reduces the activity of Rap2 effector kinases of the TNIK family and promotes dendrite growth. We conclude that a Nedd4-1/Rap2A/TNIK signaling pathway regulates neurite growth and arborization in mammalian neurons.

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...

Disrupted in schizophrenia 1 (DISC1), a genetic risk factor for multiple serious psychiatric diseases including schizophrenia, bipolar disorder and autism, is a key regulator of multiple neuronal functions linked to both normal development and disease processes. As these diseases are thought to share a common deficit in synaptic function and architecture, we have analyzed the role of DISC1 using an approach that focuses on understanding the protein– protein interactions of DISC1 specifically at synapses. We identify the Traf2 and Nck-interacting kinase (TNIK), an emerging risk factor itself for disease, as a key synaptic partner for DISC1, and provide evidence that the DISC1–TNIK interaction regulates synaptic composition and activity by stabilizing the levels of key postsynaptic density proteins. Understanding the novel DISC1–TNIK interaction is likely to provide insights into the etiology and underlying synaptic deficits found in major psychiatric diseases.

Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Rasbinding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes several Ras functions in vivo. Recently, we identified the cysteine-rich region (CRR) of Raf-1 as another Ras-binding domain. Ha-Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to activate Raf-1. Since these mutations replace Ras residues with those of Rap1A, we examined if Rap1A lacks the ability to bind to CRR. Contrary to the expectation, Rap1A exhibited a greatly enhanced binding to CRR compared with Ha-Ras. Enhanced CRR binding was also found with Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras-dependent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR.Ras belongs to a family of small GTP-binding proteins playing essential roles in cell proliferation and differentiation. Mammalian ras genes carrying activating mutations are found in many types of neoplastic tissue and are able to induce morphological transformation in vitro when transfected into fibroblast cell lines. However, the rap1A gene (1), encoding a 21-kDa GTP-binding protein with high homology to Ras, has been shown to to induce reversion of the transformed phenotype in Ki-ras-transformed NIH3T3 cells (2). In addition to the overall structural homology, Rap1A shares two important structural features with Ras. One is that Rap1A has an identical effector domain (amino acids 32-40) to that of Ras. The effector domain of Ras is essential for the association with and activation of its effectors (3). The other is that Rap1A undergoes similar post-translational modification to Ras at its C terminus except that Ras is farnesylated and Rap1A is geranylgeranylated (4). This modification is essential for the function of Rap1A as observed for Ras (5, 6).Raf-1, a serine/threonine kinase regulating the mitogen-activated protein kinase cascade, is a major mammalian Ras effector and is thought to play a key role in Ras-induced cellular transformation (7). Although the precise mechanism of Rasdependent Raf-1 activation remains unclear, it is known that the effector domain of Ras interacts with the N-terminal RBD 1 (amino acids 51-131) of Raf-1 and that this interaction is essential for physical association between these proteins as well as for the activation of Raf-1 (7). Rap1A, too, has been shown to associate with Raf-1 N-terminal fragment in vivo (8), and a recent x-ray diffraction study of the crystal of the complex between Rap1A and Raf-1 RBD has provided evidence for this associati...