Germinal Center Kinase Family Research Articles

Abstract 603: BB5523, a multiple kinase inhibitor with HPK1, VEGFR2 and TNIK inhibition, enhanced antitumor immunity in tumor models

Abstract Introduction: Immuno-suppressive micro-environment and abnormal signaling pathways are crucial for the proliferation of tumor cells. Hematopoietic progenitor kinase 1 (HPK1) inhibitor plays important roles in activation of exhausted T cells and enhancement of anti-tumor immune responses. VEGF/VEGFR2 pathway is the central therapeutic target in anti-angiogenic treatment in multiple cancers. Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family, and is reported to be essential for Wnt signaling and colorectal cancer (CRC) proliferation and progression. Blockade of critical kinases in multiple signaling pathways inhibits the proliferation, invasion and metastasis of cancer cells. Therefore, intergradation of these functions in a small molecule serves as the next generation of immune-oncology small molecules for cancer therapy. Methods: BB5523 was developed through structure-based drug design, and optimized by SAR analysis and medicinal chemistry iteration. Biochemical assays and cell-base functional assays were applied in compound evaluation. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. CDX and syngeneic tumor models in mice were conducted to evaluate the in vivo efficacy of BB5523 as a single agent. Results: BB5523 shows high inhibition potency on HPK1 with good selectivity against other TCR signaling-related kinases, such as FYN and ZAP70. Inhibition of HPK1 kinase activity (IC50 = 3.3 nM) by BB5523 strongly suppresses the phosphorylation of the downstream biomarker protein SLP-76 (IC50 = 30 nM); and boosts the IL-2 secretion in purified human T cells and human peripheral blood mononuclear cells. BB5523 also shows good activities on VEGFR2 (IC50 = 1.6 nM) and TNIK (IC50 = 0.7 nM) which are crucial for the proliferation of cancer cells. In in vitro experiments, BB5523 significantly inhibits the proliferation of various tumor cell lines at sub-micromolar concentrations. PK profile of BB5523 shows good oral bioavailability (F > 80%). In preliminary safety evaluation, no abnormal symptoms were observed in mice dosed up to 100 mpk for 14 days. BB5523 showed significant tumor-growth inhibition efficacy in TE-8, JIMT-1, B16-F10 and MC38 tumor models. Conclusion: As a potent and oral available small molecule kinase inhibitor, BB5523 boosts anti-tumor immunity through HPK1 inhibition, while also blocking crucial signaling pathways in tumor micro-environment. BB5523 shows promising in vitro and in vivo efficacy with a well-tolerated safety profile. The preclinical study of BB5523 is in progress. Citation Format: Gongping Duan, Min Li, Puyong Zhang, Wei Hua, Enlun Hao, Shipeng Wang, Xiaoyi Ma, Junheng Wang, Lijie Wei, Xingmin Zhang. BB5523, a multiple kinase inhibitor with HPK1, VEGFR2 and TNIK inhibition, enhanced antitumor immunity in tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 603.

Suppression of Th17 cell differentiation by misshapen/NIK-related kinase MINK1.

T helper type 17 cells (Th17 cells) are major contributors to many autoimmune diseases. In this study, we demonstrate that the germinal center kinase family member MINK1 (misshapen/NIK-related kinase 1) negatively regulates Th17 cell differentiation. The suppressive effect of MINK1 on induction of Th17 cells is mediated by the inhibition of SMAD2 activation through direct phosphorylation of SMAD2 at the T324 residue. The importance of MINK1 to Th17 cell differentiation was strengthened in the animal model of experimental autoimmune encephalomyelitis (EAE). Moreover, we show that the reactive oxygen species (ROS) scavenger N-acetyl cysteine boosts Th17 cell differentiation in a MINK1-dependent manner and exacerbates the severity of EAE. Thus, we have not only established MINK1 as a critical regulator of Th17 cell differentiation, but also clarified that accumulation of ROS may limit the generation of Th17 cells. The contribution of MINK1 to ROS-regulated Th17 cell differentiation may suggest an important mechanism for the development of autoimmune diseases influenced by antioxidant dietary supplements.

Deficiency of X-Linked Protein Kinase Nrk during Pregnancy Triggers Breast Tumor in Mice

The onset and/or growth of breast tumor are controlled, at least in part, by estrogen. Therefore, to prevent the development of breast tumor, estrogen-dependent proliferation of mammary epithelial cells during pregnancy needs to be suppressed once the mammary gland is fully developed to enable lactation. However, the underlying molecular mechanisms remain unknown. Nrk is an X-linked protein serine/threonine kinase in the germinal center kinase family. Herein, we demonstrate a frequent occurrence of breast tumors in homozygous and heterozygous Nrk mutant mice that have experienced pregnancy/parturition. The tumors never developed in the mutant mice without a history of pregnancy/parturition. They exhibited histopathological features of noninvasive tubular adenocarcinoma, and expressed estrogen receptor α. At late gestation when estrogen receptor α expression was significantly reduced in the wild-type mammary gland, grossly normal mammary glands in the pregnant Nrk mutant mice occasionally contained hyperplastic foci continuously expressing the receptor. Consistently, Nrk expression was induced in the wild-type mammary gland at this period of pregnancy. On the other hand, the pregnant Nrk mutant mice also showed elevated blood estrogen levels at late gestation. We suggest that Nrk suppresses the excessive proliferation of mammary epithelial cells during pregnancy, and the impairment of this regulatory system leads to frequent occurrence of breast tumor in Nrk mutant mice.

Traf2- and Nck-interacting kinase (TNIK) is involved in the anti-cancer mechanism of dovitinib in human multiple myeloma IM-9 cells.

Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family. TNIK was first identified as a kinase that is involved in regulating cytoskeletal organization in many types of cells, and it was recently proposed as a novel therapeutic target in several types of human cancers. Although previous studies suggest that TNIK plays a pivotal role in cancer cell survival and prognosis, its function in hematological cancer cell survival has not been investigated. Here we investigated the relationship between TNIK function and cell viability in multiple myeloma IM-9 cells using TNIK small interfering RNA (siRNA) transfection and dovitinib treatment. Treatment of IM-9 cells with TNIK siRNA and dovitinib treatment reduced cell proliferation. The ATP competing kinase assay and western blot analysis showed that dovitinib strongly inhibited both the interaction of TNIK with ATP (K i, 13nM) and the activation of Wnt signaling effectors such as β-catenin and TCF4. Dovitinib also induced caspase-dependent apoptosis in IM-9 cells without significant cytotoxicity in PBMCs. Our results provide new evidence that TNIK may be involved in the proliferation of multiple myeloma IM-9 cells and in the anti-cancer activity of dovitinib via inhibition of the endogenous Wnt signaling pathway.

Nuclear expression of phosphorylated TRAF2- and NCK-interacting kinase in hepatocellular carcinoma is associated with poor prognosis

Background and aimsTRAF2- and NCK-interacting kinase (TNIK) is a member of the germinal center kinase family and a transcription factor 4 (TCF4) interactor is recruited to promoters of Wnt target genes via phosphorylation of the TCF/β-catenin complex. The aim of this study was to evaluate the TNIK, the active form of TNIK (p-TNIK), and β-catenin expression in hepatocellular carcinoma (HCC), and to identify the prognostic significance of p-TNIK. MethodsWe assessed the expression status of TNIK, p-TNIK, and β-catenin by using immunohistochemical analysis of 302 HCCs in 8 tissue microarray blocks, and we evaluated their clinicopathologic features and survival rates based on their p-TNIK expression. ResultsOf 302 HCCs, 92.7% stained positive for TNIK in the cytoplasm. Nuclear expression of p-TNIK was identified in 7.9% HCCs. Aberrant cytoplasmic expression of β-catenin was identified in 77.2% and nuclear expression in 3.3%. p-TNIK nuclear staining was positively correlated to β-catenin nuclear expression (P=0.036). Cytoplasmic and nuclear expression of p-TNIK was more frequently observed in high Edmondson–Steiner (ES) nuclear grade groups (P=0.030). Nuclear p-TNIK expression was also associated with pathological M1 stage (pM1 stage) patients (P<0.0001). Aberrant cytoplasmic expression of β-catenin was more frequently identified in larger tumors (P=0.014). Univariate (DFS, P=0.049; OS, 0.037) and multivariate analysis (DFS, P=0.006; OS, P=0.003) confirmed the independent prognostic significance of nuclear p-TNIK expression. ConclusionThis is the first time that nuclear p-TNIK expression was studied in HCC, and p-TNIK nuclear expression was associated with poor prognosis and is a candidate prognostic marker for HCC.

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.

The Germinal Center Kinase TNIK Is Required for Canonical NF-κB and JNK Signaling in B-Cells by the EBV Oncoprotein LMP1 and the CD40 Receptor

Author SummaryThe germinal center kinase family member TNIK was discovered in a yeast-two-hybrid screen for interaction partners of the adapter proteins TRAF2 and Nck, and here we show it is one of the missing molecular players in two key signaling pathways in B-lymphocytes. We found that TNIK is crucial for the activities of the CD40 receptor on Bcells and its viral mimic, the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV). EBV is a human DNA tumor virus that is associated with various malignancies. It targets and transforms B-cells by hijacking the cellular signaling machinery via its oncogene LMP1. In normal Bcell physiology, the CD40 receptor is central to the immune response by mediating B-cell activation and proliferation. TNIK turns out to be an organizer of the LMP1- and CD40-induced signaling complexes by interacting with the TRAF6 adapter protein, well known for its role in linking distinct signaling pathways. Through this mechanism the two receptors depend on TNIK to activate the canonical NF-κB and JNK signal transduction pathways, which are important for the physiological activation of B-cells (a process that enables antibody production), as well as for their transformation into tumor cells. TNIK thus constitutes a key player in the transmission of physiological and pathological signals in human B-cells that might serve as a future therapeutic target against B-cell malignancies.

Nrk, an X-linked Protein Kinase in the Germinal Center Kinase Family, Is Required for Placental Development and Fetoplacental Induction of Labor

The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.

Podocyte Injury and Albuminuria in Mice with Podocyte-Specific Overexpression of the Ste20-Like Kinase, SLK

SLK expression and activity are increased during kidney development and recovery from renal ischemia-reperfusion injury. In cultured cells, SLK promotes F-actin destabilization as well as apoptosis, partially via the p38 kinase pathway. To better understand the effects of SLK in vivo, a transgenic mouse model was developed where SLK was expressed in a podocyte-specific manner using the mouse nephrin promoter. Offspring of four founder mice carried the SLK transgene. Among male transgenic mice, 66% developed albuminuria at approximately 3 months of age, and the albuminuric mice originated from three of four founders. Overall, the male transgenic mice demonstrated about fivefold greater urinary albumin/creatinine compared with male non-transgenic mice. Transgenic and non-transgenic female mice did not develop albuminuria, suggesting that females were less susceptible to glomerular filtration barrier damage than their male counterparts. In transgenic mice, electron microscopy revealed striking podocyte injury, including poorly formed or effaced foot processes, and edematous and vacuolated cell bodies. By immunoblotting, nephrin expression was decreased in glomeruli of the albuminuric transgenic mice. Activation-specific phosphorylation of p38 was increased in transgenic mice compared with non-transgenic animals. Glomeruli of SLK transgenic mice showed around 30% fewer podocytes, and a reduction in F-actin compared with control glomeruli. Thus, podocyte SLK overexpression in vivo results in injury and podocyte loss, consistent with the effects of SLK in cultured cells.

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.

Enhanced JNK activation by NESK without kinase activity upon caspase-mediated cleavage during apoptosis

Nck-interacting kinase-like embryo-specific kinase (NESK) is a protein kinase that is predominantly expressed in skeletal muscle during the late stages of mouse embryogenesis. NESK belongs to the germinal center kinase (GCK) family and selectively activates the c-Jun N-terminal kinase (JNK) pathway when overexpressed in cultured cells. Some members of the GCK family have been shown to be proteolytically cleaved and activated during apoptosis. Here, we report that NESK is also proteolytically cleaved during apoptosis. Treatment of NESK-transfected HeLa cells with TNF-α in the presence of cycloheximide or with staurosporine induced proteolytic cleavage of NESK. The cleavage of NESK occurred at two sites, generating three fragments: an N-terminal fragment containing a kinase domain, an intermediate fragment and a C-terminal fragment containing a regulatory CNH domain. These two cleavages occurred in a stepwise manner and were dependent on a caspase activity. The cleavage sites were identified as aspartic acid residues at 868 and 1091. The N-terminal fragment had less kinase activity than the full-length NESK and did not activate the JNK pathway. In contrast, the C-terminal fragment activated the JNK pathway more strongly than the full-length NESK and promoted TNF-α-induced apoptotic cell death. These results implicate NESK in the JNK pathway-mediated promotion of apoptosis through its C-terminal regulatory domain generated by proteolytic cleavage during apoptosis, in a unique manner different from other GCK family kinases.

Cofilin phosphorylation and actin polymerization by NRK/NESK, a member of the germinal center kinase family

Nck-interacting kinase (NIK)-related kinase (NRK)/NIK-like embryo-specific kinase (NESK) is a protein kinase that belongs to the germinal center kinase family, and activates the c-Jun N-terminal kinase (JNK) signaling pathway. In this study, we examined the effect of NRK/NESK on actin cytoskeletal organization. Overexpression of NRK/NESK in COS7 cells induced accumulation of polymerized actin at the perinuclear. Phosphorylation of cofilin, an actin-depolymerizing factor, was increased in NRK/NESK-expressing HEK 293T cells. In addition, in vitro phosphorylation of cofilin was observed on NRK/NESK immunoprecipitates from HEK 293T cells expressing the kinase domain of NRK/NESK. The cofilin phosphorylation occurred at the serine residue of position 3 (Ser-3). Since the phosphorylation at Ser-3 inactivates the actin-depolymerizing activity of cofilin, these results suggest that NRK/NESK induces actin polymerization through cofilin phosphorylation. The cofilin phosphorylation did not appear to be mediated through activation of LIM-kinasel, a cofilin-phosphorylating kinase, or through the activation of JNK. Thus, cofilin is likely to be a direct substrate of NRK/NESK. NRK/NESK is predominantly expressed in skeletal muscle during the late stages of mouse embryogenesis. Thus, NRK/NESK may be involved in the regulation of actin cytoskeletal organization in skeletal muscle cells through cofilin phosphorylation.

NESK, a Member of the Germinal Center Kinase Family That Activates the c-Jun N-terminal Kinase Pathway and Is Expressed during the Late Stages of Embryogenesis

The c-Jun N-terminal kinase (JNK) signaling pathway plays a crucial role in cellular responses stimulated by stress-inducing agents and proinflammatory cytokines. The group I germinal center kinase family members selectively activate the JNK pathway. In this study, we have isolated a mouse cDNA encoding a protein kinase homologous to Nck-interacting kinase (NIK), a member of the group I germinal center kinase family. This protein kinase is expressed during the late stages of embryogenesis, but not in adult tissues, and thus named NESK (NIK-like embryo-specific kinase). NESK selectively activated the JNK pathway when overexpressed in HEK 293 cells but did not stimulate the p38 kinase or extracellular signal-regulated kinase (ERK) pathways. NESK-induced JNK activation was inhibited by the dominant negative mutants of MEKK1 and MKK4. Tumor necrosis factor (TNF)-alpha or TNF receptor-associated factor 2 (TRAF2) stimulated the NESK activity. Furthermore, the dominant negative NESK mutant inhibited the JNK activation induced by TNF-alpha or TRAF2. These results suggest that NESK, a novel activator of the JNK pathway, functions in coupling TRAF2 to the MEKK1 --> MKK4 --> JNK kinase cascade during the late stages of mammalian embryogenesis.

The Ste20 kinase misshapen regulates both photoreceptor axon targeting and dorsal closure, acting downstream of distinct signals.

We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct upstream signaling systems.

HPK1 Inhibits AP-1

HPK1, a member of the germinal center kinase family, is expressed only in hematopoietic cells. Liou et al. found that T cell receptors (TCRs) and B cell receptors activate HPK1 through similar pathways. In activated T cells, HPK1 activation was dependent on the nonreceptor tyrosine kinases, Lck and ZAP-70, and the adapter proteins, linker for activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 kD (SLP-76). In activated B cells, HPK1 activation was dependent on the nonreceptor tyrosine kinases Lyn and Syk, the adapter protein, B cell linker protein (BLNK), and either of the adapter proteins Grb2 or Grap. In transfected cells, HPK1 inhibited AP-1 and extracellular signal-regulated kinase 2 (ERK2) activation by TCR stimulation. The authors suggest that HPK1 acts as a negative regulator of the AP-1 pathway in stimulated T cells. Liou, J., Kiefer, F., Dang, A., Hashimoto, A., Cobb, M.H., Kurosaki, T., and Weiss, A. (2000) HPK1 is activated by lymphocyte antigen receptors and negatively regulates AP-1. Immunity 12 : 399-408. [Online Journal]

Molecular cloning of MINK, a novel member of mammalian GCK family kinases, which is up-regulated during postnatal mouse cerebral development

A new germinal center kinase (GCK) family kinase, Misshapen/NIKs-related kinase (MINK), has been cloned and its expression has been characterized in several tissues and various developmental stages of the mouse brain. MINK encodes a 1300 amino acid polypeptide, consisting of an N-terminal kinase domain, a proline-rich intermediate region, and a C-terminal GCK homology region. The expression of MINK is up-regulated during the postnatal development of the mouse brain. MINK activates the cJun N-terminal kinase and the p38 pathways.

TNIK, a Novel Member of the Germinal Center Kinase Family That Activates the c-Jun N-terminal Kinase Pathway and Regulates the Cytoskeleton

Germinal center kinases (GCKs) compose a subgroup of the Ste20 family of kinases. Here we describe the cloning and characterization of a novel GCK family kinase, Traf2- and Nck-interacting kinase (TNIK) that interacts with both Traf2 and Nck. TNIK encodes a polypeptide of 1360 amino acids with eight spliced isoforms. It has 90% amino acid identity to the Nck-interacting kinase in both the N-terminal kinase domain and the C-terminal germinal center kinase homology region. The homology drops to 53% in the intermediate region. TNIK specifically activates the c-Jun N-terminal kinase pathway when transfected into Phoenix-A cells (derivatives of 293 cells), similar to many GCKs. However, in contrast to other GCKs, this activation is mediated solely by the GCK homology region of TNIK. In addition, in Phoenix-A, NIH-3T3, and Hela cells, overexpression of wild type TNIK, but not the kinase mutant form of TNIK, results in the disruption of F-actin structure and the inhibition of cell spreading. Furthermore, TNIK can phosphorylate Gelsolin in vitro. This is the first time that a GCK family kinase is shown to be potentially involved in the regulation of cytoskeleton.

TANK potentiates tumor necrosis factor receptor-associated factor-mediated c-Jun N-terminal kinase/stress-activated protein kinase activation through the germinal center kinase pathway.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are mediators of many members of the TNF receptor superfamily and can activate both the nuclear factor kappaB (NF-kappaB) and stress-activated protein kinase (SAPK; also known as c-Jun N-terminal kinase) signal transduction pathways. We previously described the involvement of a TRAF-interacting molecule, TRAF-associated NF-kappaB activator (TANK), in TRAF2-mediated NF-kappaB activation. Here we show that TANK synergized with TRAF2, TRAF5, and TRAF6 but not with TRAF3 in SAPK activation. TRAF2 and TANK individually formed weak interactions with germinal center kinase (GCK)-related kinase (GCKR). However, when coexpressed, they formed a strong complex with GCKR, thereby providing a potential mechanism for TRAF and TANK synergy in GCKR-mediated SAPK activation, which is important in TNF family receptor signaling. Our results also suggest that TANK can form potential intermolecular as well as intramolecular interactions between its amino terminus and carboxyl terminus. This study suggests that TANK is a regulatory molecule controlling the threshold of NF-kappaB and SAPK activities in response to activation of TNF receptors. In addition, CD40 activated endogenous GCKR in primary B cells, implicating GCK family proteins in CD40-mediated B-cell functions.