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...

Little is known about the specific signaling roles of Rap2, a Ras family small GTP-binding protein. In a search for novel Rap2-interacting proteins by the yeast two-hybrid system, we isolated isoform 3 of the human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a previously described but uncharacterized isoform. Other isoforms of MAP4K4 in humans and mice are known as hematopoietic progenitor kinase (HPK)/ germinal center kinase (GCK)-like kinase and Nck-interacting kinase, respectively. MAP4K4 belongs to the STE20 group of protein kinases and regulates c-Jun Nterminal kinase (JNK). MAP4K4 interacted with Rap2 through its C-terminal citron homology domain but did not interact with Rap1 or Ras. Interaction with Rap2 required the intact effector region of Rap2. MAP4K4 interacted preferentially with GTP-bound Rap2 over GDP-bound Rap2 in vitro. In cultured cells, MAP4K4 colocalized with Rap2, while a mutant MAP4K4 lacking the citron homology domain failed to do so. Furthermore, Rap2 enhanced MAP4K4-induced activation of JNK. These results suggest that MAP4K4 is a putative effector of Rap2 mediating the activation of JNK by Rap2.Rap2 belongs to the Ras family of small GTP-binding proteins. The mammalian Ras family consists of Ras proteins (Ha-Ras, Ki-Ras, and N-Ras), Rap proteins (Rap1A, Rap1B, Rap2A, and Rap2B), R-Ras, R-Ras2/TC21, R-Ras3/M-Ras, Ral proteins (RalA and RalB), Rheb, Rin, and Rit (for a review, see Ref.

S U M M A R YThe Cytochrome b (Cyt b) gene has proved to be useful for identification and classification of many mammals and plants. In order to evaluate the utility of this gene for discrimination of Leishmania parasites as well as for exploring their phylogenetic relationships, we determined the nucleotide sequences of the Cyt b gene from 13 human-infecting Leishmania species (14 strains) from the New and Old Worlds. The Cyt b genes, approximately 1080 base pairs, were found to be A/T rich, and their 5k terminal-editing regions were highly conserved. The nucleotide sequence variation among them was enough to discriminate parasite species; 245 nucleotide positions were polymorphic and 190 positions were parsimony informative. The phylogenetic relationships based on this gene, showed good agreement with the classification of Lainson & Shaw (1987) ) tropica complex and the placement of L. tarentolae in another genus. These data show that the Cyt b gene is useful for phylogenetic study of Leishmania parasites.

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