Traf2- And Nck-interacting Kinase Research Articles

Identification of Phosphorylation Consensus Sequences and Endogenous Neuronal Substrates of the Psychiatric Risk Kinase TNIK.

Traf2- and Nck-interacting kinase (TNIK) is a serine/threonine kinase highly expressed in the brain and enriched in the postsynaptic density of glutamatergic synapses in the mammalian brain. Accumulating genetic evidence and functional data have implicated TNIK as a risk factor for psychiatric disorders. However, the endogenous substrates of TNIK in neurons are unknown. Here, we describe a novel selective small molecule inhibitor of the TNIK kinase family. Using this inhibitor, we report the identification of endogenous neuronal TNIK substrates by immunoprecipitation with a phosphomotif antibody followed by mass spectrometry. Phosphorylation consensus sequences were defined by phosphopeptide sequence analysis. Among the identified substrates were members of the delta-catenin family including p120-catenin, δ-catenin, and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), each of which is linked to psychiatric or neurologic disorders. Using p120-catenin as a representative substrate, we show TNIK-induced p120-catenin phosphorylation in cells requires intact kinase activity and phosphorylation of TNIK at T181 and T187 in the activation loop. Addition of the small molecule TNIK inhibitor or knocking down TNIK by two shRNAs reduced endogenous p120-catenin phosphorylation in cells. Together, using a TNIK inhibitor and phosphomotif antibody, we identify endogenous substrates of TNIK in neurons, define consensus sequences for TNIK, and suggest signaling pathways by which TNIK influences synaptic development and function linked to psychiatric and neurologic disorders.

Prognostic significance of Traf2- and Nck- interacting kinase (TNIK) in colorectal cancer.

BackgroundThe potential of expression profiling using microarray analysis as a tool to predict the prognosis for different types of cancer has been realized. This study aimed to identify a novel biomarker for colorectal cancer (CRC).MethodsThe expression profiles of cancer cells in 152 patients with stage I-III CRC were examined using microarray analysis. High expression in CRC cells, especially in patients with distant recurrences, was a prerequisite to select candidate genes. Thus, we identified seventeen candidate genes, and selected Traf2- and Nck-interacting kinase (TNIK), which was known to be associated with progression in CRC through Wnt signaling pathways. We analyzed the protein expression of TNIK using immunohistochemistry (IHC) and investigated the relationship between protein expression and patient characteristics in 220 stage I-III CRC patients.ResultsRelapse-free survival was significantly worse in the TNIK high expression group than in the TNIK low expression group in stage II (p = 0.028) and stage III (p = 0.006) patients. In multivariate analysis, high TNIK expression was identified as a significant independent risk factor of distant recurrence in stage III patients.ConclusionThis study is the first to demonstrate the prognostic significance of intratumoral TNIK protein expression in clinical tissue samples of CRC, in that high expression of TNIK protein in primary tumors was associated with distant recurrence in stage II and III CRC patients. This TNIK IHC study might contribute to practical decision-making in the treatment of these patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1783-y) contains supplementary material, which is available to authorized users.

TNIK serves as a novel biomarker associated with poor prognosis in patients with pancreatic cancer.

Traf-2 and Nck interacting kinase (TNIK) is one of the STE20/MAP4K family members implicated in carcinogenesis and progression of several human malignancies. However, its expression pattern and biological behavior in pancreatic carcinoma remains completely unclear. The present study is designed to investigate the clinical and prognostic value of TNIK in pancreatic carcinoma. TNIK mRNA and protein level was respectively detected by real-time quantitative RCR (qPCR) and Western blot in ten paired samples of pancreatic cancer. Immunohistochemical staining was also conducted to examine TNIK in the tissue microarray (TMA) consisting of 91 archived specimens of pancreatic cancer. The correlation between TNIK and prognosis was assessed by Kaplan-Meier curves and Cox regression. The mRNA and protein levels of TNIK in pancreatic cancer were both significantly higher than those in matched paratumor tissues. Immunohistochemistry analysis showed that TNIK was positively associated with pathologic T (P = 0.045) and TNM (P = 0.040) stage. In addition, The Kaplan-Meier survival curves indicated that patients with high expression of TNIK had a shorter overall survival (OS) and disease-free survival (DFS) than those with low expression. Our results demonstrated that TNIK might play a crucial role in pancreatic carcinogenesis and serve as a novel therapeutic target of pancreatic cancer.

Discovery of TRAF-2 and NCK-interacting kinase (TNIK) inhibitors by ligand-based virtual screening methods

TRAF-2 and NCK-interacting kinase (TNIK) is a serine–threonine kinase with a proposed role in Wnt/β-catenin and JNK pathways.

A novel aminothiazole KY-05009 with potential to inhibit Traf2- and Nck-interacting kinase (TNIK) attenuates TGF-β1-mediated epithelial-to-mesenchymal transition in human lung adenocarcinoma A549 cells.

Transforming growth factor (TGF)-β triggers the epithelial-to-mesenchymal transition (EMT) of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carboxamide (KY-05009) with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (K i, 100 nM). In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK) signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis.

1034-LBO: Differential microRNA expression profiling in lung allograft recipients who develop donor specific antibody and Bronchiolitis Obliterans Syndrome

Aim Bronchiolitis Obliterans Syndrome (BOS), clinically diagnosed as chronic rejection, has remained a major setback following lung transplantation (LT). However, its pathogenesis is poorly understood and no predictive biomarkers have been identified. Expression of microRNA (miRNA) in blood is deregulated in physiopathological conditions including immunological disorders. Methods In this study, we hypothesized that miRNA profiles from peripheral blood mononuclear cells (PBMCs) in LT recipients (LTxR) could identify patients at risk of BOS. MiRNA expression profiling was performed using TaqMan Human MicroRNA Array in PBMCs collected from three groups of LTxR: 10 stable, 10 who developed DSA and 10 who developed BOS. Results There were 98 miRNAs upregulated in LTxR with BOS compared to that of stable transplants (relative fold > 2.5); and 32 miRNAs downregulated with BOS (relative fold > 2.5). In addition, we found that there were 16 miRNAs upregulated (including miR-99a, miR-147, and miR-124 ∗ ) with BOS compared to that of DSA; and 5 miRNAs downregulated (including miR-34b, miR-363 ∗ , and miR-155 ∗ ) in BOS compared to DSA, indicating that these miRNAs may play a role in BOS with DSA. We validated those differential miRNAs in independent PBMCs and broncheoalveolar lavage cells using Taqman real-time PCR. In addition we focused on one miRNA, miR-99a, which caused upregulation of 2.8-fold in LTxR with BOS compared to that of DSA. Bioinformatics prediction indicated that miR-99a can mediate gene-expression regulation of TNIK (TRAF2 and NCK interacting kinase) and TRAF4 (TNF receptor-associated factor 4), which were involved in regulating NF- κ B signaling pathway. Conclusions In conclusion our results demonstrate that differential miRNA expression profile has the potential to predict BOS and development of DSA following LT.

The essential role of TNIK gene amplification in gastric cancer growth

Traf2- and Nck-interacting kinase (TNIK) is one of the germinal center kinase family members involved in cytoskeleton organization and neuronal dendrite extension. Emerging evidence supports that TNIK is essential for activation of WNT signaling pathway in colon cancer growth. To search for novel genetic aberrations that drive carcinogenesis, we performed microarray-based comparative hybridization assay for gene copy number variations in primary tumor samples. Our data showed that TNIK gene was amplified in 7% (8/106) of Chinese gastric cancer patients. Theses amplifications were confirmed by fluorescence in situ hybridization analysis. PAMC82 human gastric cancer and T47D human breast cancer cell lines with TNIK amplification were identified to further understand the function of TNIK gene amplification. RNA-interference-mediated silencing of TNIK resulted in significant inhibition of cell growth and induction of cell death in TNIK-amplified, but not in TNIK-non-amplified, cell lines tested. This selective sensitivity to the TNIK inhibition was also observed under the effect of a small-molecule TNIK inhibitor. Furthermore, our data indicated that TNIK's role in gastric cancer growth was not dependent on Wnt signaling but rather was involved in AKT activation and cell autophagy. Together, our results suggest that TNIK is a novel therapeutic target in gastric cancer and TNIK amplification can be potentially used for patient selection.

Abstract A132: Development of a small-molecule inhibitor targeting the Wnt signaling pathway.

Abstract Wnt signaling is a major force driving colorectal carcinogenesis, but only a small number of druggable target molecules in the Wnt pathway have been found. Our recent series of proteomic studies has revealed that various classes of nuclear proteins participate in the β-catenin and T-cell factor-4 (TCF-4) complex and modulate the activity of Wnt signalling. Those included fusion/translocated in liposarcoma (FUS/TLS) (1), poly(ADP-ribose) polymerase-1 (PARP-1) (2), Ku70/Ku80 (3), DNA topoisomerase IIα (Topo IIα) (4), splicing factor-1 (SF1) (5), Ran (ras-related nuclear protein), RanBP2 (Ran binding protein-2), and RanGAP1 (Ran GTPase-activating protein-1) (6), Traf2- and Nck-interacting kinase (TNIK) (7). Among these proteins, TNIK protein kinase attracted our current interest because various small-molecule kinase inhibitors have been applied successfully to cancer treatment. TNIK was an activating kinase for TCF-4, and colorectal cancer cells are highly dependent upon the expression and catalytic activity of TNIK for proliferation (7). High-throughput screening of a kinase-focused compound library (>10,000 compounds) against recombinant TNIK identified a lead candidate that inhibited the kinase activity of TNIK with an IC50 value of 8.6 nM and the transcriptional activity of TCF-4. TNIK is a feasible drug target in the Wnt signaling pathway. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A132. Citation Format: Tesshi Yamada, Mari Masuda, Masaaki Sawa. Development of a small-molecule inhibitor targeting the Wnt signaling pathway. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A132.

Discovery of 4-phenyl-2-phenylaminopyridine based TNIK inhibitors

A series of compounds based on a 4-phenyl-2-phenylaminopyridine scaffold that are potent and selective inhibitors of Traf2- and Nck-interacting kinase (TNIK) activity are described. These compounds were used as tools to test the importance of TNIK kinase activity in signaling and proliferation in Wnt-activated colorectal cancer cells. The results indicate that pharmacological inhibition of TNIK kinase activity has minimal effects on either Wnt/TCF4/β-catenin-driven transcription or viability. The findings suggest that the kinase activity of TNIK may be less important to Wnt signaling than other aspects of TNIK function, such as its putative role in stabilizing the TCF4/β-catenin transcriptional complex.

TNiK Is Required for Postsynaptic and Nuclear Signaling Pathways and Cognitive Function

Traf2 and NcK interacting kinase (TNiK) contains serine-threonine kinase and scaffold domains and has been implicated in cell proliferation and glutamate receptor regulation in vitro. Here we report its role in vivo using mice carrying a knock-out mutation. TNiK binds protein complexes in the synapse linking it to the NMDA receptor (NMDAR) via AKAP9. NMDAR and metabotropic receptors bidirectionally regulate TNiK phosphorylation and TNiK is required for AMPA expression and synaptic function. TNiK also organizes nuclear complexes and in the absence of TNiK, there was a marked elevation in GSK3β and phosphorylation levels of its cognate phosphorylation sites on NeuroD1 with alterations in Wnt pathway signaling. We observed impairments in dentate gyrus neurogenesis in TNiK knock-out mice and cognitive testing using the touchscreen apparatus revealed impairments in pattern separation on a test of spatial discrimination. Object-location paired associate learning, which is dependent on glutamatergic signaling, was also impaired. Additionally, TNiK knock-out mice displayed hyperlocomotor behavior that could be rapidly reversed by GSK3β inhibitors, indicating the potential for pharmacological rescue of a behavioral phenotype. These data establish TNiK as a critical regulator of cognitive functions and suggest it may play a regulatory role in diseases impacting on its interacting proteins and complexes.

Agonistic and Antagonistic Roles for TNIK and MINK in Non-Canonical and Canonical Wnt Signalling

Wnt signalling is a key regulatory factor in animal development and homeostasis and plays an important role in the establishment and progression of cancer. Wnt signals are predominantly transduced via the Frizzled family of serpentine receptors to two distinct pathways, the canonical ß-catenin pathway and a non-canonical pathway controlling planar cell polarity and convergent extension. Interference between these pathways is an important determinant of cellular and phenotypic responses, but is poorly understood. Here we show that TNIK (Traf2 and Nck-interacting kinase) and MINK (Misshapen/NIKs-related kinase) MAP4K signalling kinases are integral components of both canonical and non-canonical pathways in Xenopus. xTNIK and xMINK interact and are proteolytically cleaved in vivo to generate Kinase domain fragments that are active in signal transduction, and Citron-NIK-Homology (CNH) Domain fragments that are suppressive. The catalytic activity of the Kinase domain fragments of both xTNIK and xMINK mediate non-canonical signalling. However, while the Kinase domain fragments of xTNIK also mediate canonical signalling, the analogous fragments derived from xMINK strongly antagonize this signalling. Our data suggest that the proteolytic cleavage of xTNIK and xMINK determines their respective activities and is an important factor in controlling the balance between canonical and non-canonical Wnt signalling in vivo.

CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression

Chronic myelogenous leukemia (CML) results from a chromosomal translocation in hematopoietic stem or early progenitor cells that gives rise to the oncogenic BCR/ABL fusion protein. Clinically, CML has a chronic phase that eventually evolves into an accelerated stage and blast crisis. A CML-specific immune response is thought to contribute to the control of disease. Whether the immune system can also promote disease progression is not known. In the present study, we investigated the possibility that the TNF receptor family member CD27 is present on leukemia stem cells (LSCs) and mediates effects of the immune system on CML. In a mouse model of CML, BCR/ABL+ LSCs and leukemia progenitor cells were found to express CD27. Binding of CD27 by its ligand, CD70, increased expression of Wnt target genes in LSCs by enhancing nuclear localization of active β-catenin and TRAF2- and NCK-interacting kinase (TNIK). This resulted in increased proliferation and differentiation of LSCs. Blocking CD27 signaling in LSCs delayed disease progression and prolonged survival. Furthermore, CD27 was expressed on CML stem/progenitor cells in the bone marrow of CML patients, and CD27 signaling promoted growth of BCR/ABL+ human leukemia cells by activating the Wnt pathway. Since expression of CD70 is limited to activated lymphocytes and dendritic cells, our results reveal a mechanism by which adaptive immunity contributes to leukemia progression. In addition, targeting CD27 on LSCs may represent an attractive therapeutic approach to blocking the Wnt/β-catenin pathway in CML.

The enormous influence of TNIK knockdown on intracellular signals and cell survival

Basic cellular activities and coordinated cell actions are governed by intracellular signals, among which the Wnt signaling cascade plays an important role in tissue polarity and cell adhesion or movement through the activation of c-Jun N-terminal kinase (JNK) pathway. As one of the central transcriptional factors, Traf2- and Nck-interacting kinase (TNIK) mediates the transactivation of Wnt target genes and promotes the activity of c-Jun N-terminus kinase (JNK)2 when overexpressed. To further understand the function of TNIK, changes in intracellular signals were detected in colon cancer cell lines using a knockdown strategy. In this study, we found that the short-hairpin RNA-mediated knockdown of TNIK decreased the expressions of CD44, c-MYC and cyclin D1, which was consistent with the results of a TCF-4 reporter assay. Our data showed, for the first time, that the activation of both JNK1 and JNK2 by TNFα could be blocked through TNIK knockdown, which dampened the AP1 luciferase activity accordingly. In addition, adenovirus mediated the downregulation of TNIK-triggered intrinsic apoptosis in SW480 cells by activating caspase-9 and PARP-1. We conclude that TNIK is essential for the activation of both the canonical Wnt pathway and the JNK pathway, and serves as a pro-survival factor.

The psychiatric disease risk factors DISC1 and TNIK interact to regulate synapse composition and function

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.

Traf2- and Nck-interacting Kinase Is Essential for Canonical Wnt Signaling in Xenopus Axis Formation

Wnt signaling pathways play important roles in various stages of developmental events and several aspects of adult homeostasis. Aberrant activation of Wnt signaling has also been associated with several types of cancer. We have recently identified Traf2- and Nck-interacting kinase (TNIK) as a novel activator of Wnt signaling through a comprehensive proteomic approach in human colorectal cancer cell lines. TNIK is an activating kinase for T-cell factor-4 (TCF4) and essential for the beta-catenin-TCF4 transactivation and colorectal cancer growth. Here, we report the essential role of TNIK in Wnt signaling during Xenopus development. We found that Xenopus TNIK (XTNIK) was expressed maternally and that the functional knockdown of XTNIK by catalytically inactive XTNIK (K54R) or antisense morpholino oligonucleotides resulted in significant malformations with a complete loss of head and axis structures. XTNIK enhanced beta-catenin-induced axis duplication and the expression of beta-catenin-TCF target genes, whereas knockdown of XTNIK inhibited it. XTNIK was recruited to the promoter region of beta-catenin-TCF target genes in a beta-catenin-dependent manner. These results demonstrate that XTNIK is an essential factor for the transcriptional activity of the beta-catenin-TCF complex and dorsal axis determination in Xenopus embryos.

Traf2- and Nck-Interacting Kinase Is Essential for Wnt Signaling and Colorectal Cancer Growth

T-cell factor-4 (TCF4) is a transcription factor essential for maintaining the undifferentiated status and self-renewal of intestinal epithelial cells. It has therefore been considered that constitutive activation of TCF4 by aberrant Wnt signaling is a major force driving colorectal carcinogenesis. We previously identified Traf2- and Nck-interacting kinase (TNIK) as one of the proteins that interact with TCF4 in colorectal cancer cells, but its functional significance has not been elucidated. Here, we report that TNIK is an activating kinase for TCF4 and essential for colorectal cancer growth. TNIK, but not its catalytically inactive mutant, phosphorylated the conserved serine 154 residue of TCF4. Small interfering RNA targeting TNIK inhibited the proliferation of colorectal cancer cells and the growth of tumors produced by injecting colorectal cancer cells s.c. into immunodeficient mice. The growth inhibition was abolished by restoring the catalytic domain of TNIK, thus confirming that its enzyme activity is essential for the maintenance of colorectal cancer growth. Several ATP-competing kinase inhibitors have been applied to cancer treatment and have shown significant activity. Our findings suggest TNIK as a feasible target for pharmacologic intervention to ablate aberrant Wnt signaling in colorectal cancer.

Abstract 1224: TNIK is a protein kinase essential for Wnt signaling and colorectal carcinogenesis

Abstract Aberrant transactivation of a certain set of target genes of T-cell factor-4 (TCF4), a member of the TCF/lymphoid enhancer factor (LEF) family of transcription factors, by Wnt signaling has been considered crucial in colorectal carcinogenesis. Through a comprehensive proteome approach we identified Traf2- and Nck-interacting kinase (TNIK) as a novel activator for TCF4. Here we report the functional involvement of TNIK in Wnt signaling and human colon cancer growth in vitro and in vivo. Methods: Proteins of two colorectal cancer cell lines, HCT-116 and DLD1, were immunoprecipitated with anti-TCF-4 antibody and analyzed directly by nano-flow liquid chromatography and mass spectrometry. Results: TNIK was detected in the immunoprecipitates with anti-TCF4 or anti-β-catenin antibody, but not with control IgG. Conversely, β-catenin and TCF4 proteins were immunoprecipitated with anti-TNIK antibody, indicating that TCF4, β-catenin, and TNIK proteins form a complex in colorectal cancer cells. TCF4 protein was phosphorylated by TNIK, but not by the catalytically inactive mutant of TNIK with substitution (K54R) of the conserved lysine 54 residue in the ATP-binding pocket of the kinase domain. Knockdown of TNIK by siRNA against TNIK suppressed the TCF/LEF transcriptional activity and proliferation of DLD1 and HCT-116 cells. The expression of known target genes of the β-catenin and TCF/LEF complexes, such as axis inhibitor-2 (AXIN2), c-myc (MYC), c-jun (JUN), and matrilysin (MMP7), except for cyclin D1 (CCND1), was significantly reduced by transient transfection with siRNA against TNIK. siRNA targeting TNIK inhibited the growth of tumors produced by injecting colorectal cancer cells subcutaneously into immunodeficient mice, and the growth inhibition was abolished by restoring the catalytic domain of TNIK. Conclusions: Wnt signaling is a major force driving colorectal carcinogenesis. More than 80% of colorectal cancers show mutation of the APC gene, resulting in accumulation of β-catenin and constitutive activation of Wnt signaling. Because restoration of the loss of function resulting from mutation of the APC gene is not a realistic approach, we have been searching for molecules involved in Wnt signaling downstream of APC, especially in the nucleus, that could be potential drug targets. We identified TNIK as the activating kinase of the TCF4 and β-catenin transcriptional complex. Several ATP-competing kinase inhibitors have been applied to cancer treatment and have shown significant activity. A new drug targeting TNIK might be effective for suppressing aberrant Wnt signaling and colorectal cancer growth.

TRAF2 and NCK interacting kinase (TNIK)

TRAF2 and NCK interacting kinase (TNIK)

Rap2 function requires palmitoylation and recycling endosome localization

Rap2A, Rap2B, and Rap2C are Ras-like small G proteins. The role of their post-translational processing has not been investigated due to the lack of information on their downstream signaling. We have recently identified the Traf2- and Nck-interacting kinase (TNIK), a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases, as a specific Rap2 effector. Here we report that, in HEK293T cells, Rap2A (farnesylated) and Rap2C (likely farnesylated), but not Rap2B (geranylgeranylated), require palmitoylation for membrane-association and TNIK activation, whereas all Rap2 proteins, including Rap2B, require palmitoylation for induction of TNIK-mediated phenotype, the suppression of cell spreading. Furthermore, we report for the first time that, in COS-1 cells, Rap2 proteins localize, and recruit TNIK, to the recycling endosomes, but not the Golgi nor the endoplasmic reticulum, in a palmitoylation-dependent manner. These observations implicate the involvement of palmitoylation and recycling endosome localization in cellular functions of Rap2 proteins.

MINK is a Rap2 effector for phosphorylation of the postsynaptic scaffold protein TANC1

Rap1 and Rap2 are similar Ras-like G proteins but perform distinct functions. By the affinity chromatography/mass-spectrometry approach and the yeast two-hybrid screening, we identified Misshapen/NIKs-related kinase (MINK) as a novel Rap2-interacting protein that does not interact with Rap1 or Ras. MINK is a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases. The interaction between MINK and Rap2 was GTP-dependent and required Phe39 within the effector region of Rap2; the corresponding residue in Rap1 and Ras is Ser. MINK was enriched in the brain, and both MINK and its close relative, Traf2- and Nck-interacting kinase (TNIK), interacted with a postsynaptic scaffold protein containing tetratricopeptide repeats, ankyrin repeats and a coiled-coil region (TANC1) and induced its phosphorylation, under control of Rap2 in cultured cells. These are novel actions of MINK and TNIK, and consistent with a role of MINK as a Rap2 effector in the brain.