The Traf2- and Nck-interacting kinase as a putative effector of Rap2 to regulate actin cytoskeleton

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 approximately 155 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 TNIK-induced JNK activation, but promoted autophosphorylation and translocation of TNIK to the detergent-insoluble cytoskeletal fraction. These results suggest that TNIK is a specific effector of Rap2 to regulate actin cytoskeleton.     

Mitogen-activated protein kinase kinase kinase kinase 4 as a putative effector of Rap2 to activate the c-Jun N-terminal kinase

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 N-terminal 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.     

Identification and functional characterization of a novel human misshapen/Nck interacting kinase-related kinase, hMINK beta

Misshapen/NIKs-related kinase (MINK) is a member of the germinal center family of kinases that are homologous to the yeast sterile 20 (Ste20) kinases and regulate a wide variety of cellular processes, including cell morphology, cytoskeletal rearrangement, and survival. Here, we present the cloning and functional characterization of a novel human Misshapen/NIKs-related kinase beta (hMINK beta) that encodes a polypeptide of 1312 amino acids. hMINK beta is ubiquitously expressed in most tissues with at least five alternatively spliced isoforms. Similar to Nck interacting kinase (NIK) and Traf2 and Nck-interacting kinase (TNIK), hMINK beta moderately activates c-Jun N-terminal kinase (JNK) and associates with Nck via the intermediate domain in the yeast two-hybrid system and in a glutathione S-transferase (GST) pull-down assay. Interestingly, overexpression of the kinase domain deleted and kinase-inactive mutants of hMINK beta in human fibrosarcoma HT1080 cells enhanced cell spreading, actin stress fiber formation, and adhesion to extracellular matrix, as well as decreased cell motility and cell invasion. Furthermore, these mutants also promoted cell-cell adhesion in human breast carcinoma MCF7 cells, evidenced with cell growth in clusters and increased membrane localization of beta-catenin, a multifunctional protein involved in E-cadherin-mediated cell adhesion. Finally, hMINK beta protein was found to colocalize with the Golgi apparatus, implicating that hMINK beta might exert its functions, at least in part, through the modulation of intracellular protein transport. Taken together, these results suggest that hMINK beta plays an important role in cytoskeleton reorganization, cell adhesion, and cell motility.     

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-alpha 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-alpha-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.     

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.     

Endophilin regulates JNK activation through its interaction with the germinal center kinase-like kinase

The endophilin family of proteins function in clathrin-mediated endocytosis. Here, we have identified and cloned the rat germinal center kinase-like kinase (rGLK), a member of the GCK (germinal center kinase) family of c-Jun N-terminal kinase (JNK) activating enzymes, as a novel endophilin I-binding partner. The interaction occurs both in vitro and in cells and is mediated by the Src homology 3 domain of endophilin I and a region of rGLK containing the endophilin consensus-binding sequence PPRPPPPR. Overlay analysis of rat brain extracts demonstrates that endophilin I is a major Src homology 3 domain-binding partner for rGLK. Overexpression of full-length endophilin I activates rGLK-mediated JNK activation, whereas N- and C-terminal fragments of endophilin I block JNK activation. Thus, endophilin I appears to have a novel function in JNK activation.     

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

The tumor necrosis factor-receptor-associated factor 2 (TRAF2)- and Nck-interacting kinase (TNIK) is a ubiquitously expressed member of the germinal center kinase family. The TNIK functions in hematopoietic cells and the role of TNIK-TRAF interaction remain largely unknown. By functional proteomics we identified TNIK as interaction partner of the latent membrane protein 1 (LMP1) signalosome in primary human B-cells infected with the Epstein-Barr tumor virus (EBV). RNAi-mediated knockdown proved a critical role for TNIK in canonical NF-κB and c-Jun N-terminal kinase (JNK) activation by the major EBV oncoprotein LMP1 and its cellular counterpart, the B-cell co-stimulatory receptor CD40. Accordingly, TNIK is mandatory for proliferation and survival of EBV-transformed B-cells. TNIK forms an activation-induced complex with the critical signaling mediators TRAF6, TAK1/TAB2, and IKKβ, and mediates signalosome formation at LMP1. TNIK directly binds TRAF6, which bridges TNIK's interaction with the C-terminus of LMP1. Separate TNIK domains are involved in NF-κB and JNK signaling, the N-terminal TNIK kinase domain being essential for IKKβ/NF-κB and the C-terminus for JNK activation. We therefore suggest that TNIK orchestrates the bifurcation of both pathways at the level of the TRAF6-TAK1/TAB2-IKK complex. Our data establish TNIK as a novel key player in TRAF6-dependent JNK and NF-κB signaling and a transducer of activating and transforming signals in human B-cells.     

The Germinal Center Kinase GCK-1 Is a Negative Regulator of MAP Kinase Activation and Apoptosis in the C. elegans Germline

The germinal center kinases (GCK) constitute a large, highly conserved family of proteins that has been implicated in a wide variety of cellular processes including cell growth and proliferation, polarity, migration, and stress responses. Although diverse, these functions have been attributed to an evolutionarily conserved role for GCKs in the activation of ERK, JNK, and p38 MAP kinase pathways. In addition, multiple GCKs from different species promote apoptotic cell death. In contrast to these paradigms, we found that a C. elegans GCK, GCK-1, functions to inhibit MAP kinase activation and apoptosis in the C. elegans germline. In the absence of GCK-1, a specific MAP kinase isoform is ectopically activated and oocytes undergo abnormal development. Moreover, GCK-1- deficient animals display a significant increase in germ cell death. Our results suggest that individual germinal center kinases act in mechanistically distinct ways and that these functions are likely to depend on organ- and developmental-specific contexts.     

Molecular cloning and characterization of a novel human STE20-like kinase, hSLK

We have cloned a human counterpart to a guinea pig STE20-like kinase cDNA, designated human SLK (hSLK), from a human lung carcinomatous cell line A549 cDNA library. hSLK cDNA encodes a novel 1204 amino acid serine/threonine kinase for which the kinase domain located at the N-terminus shares considerable homology to that of the STE20-like kinase family. The C-terminal domain of hSLK includes both the coiled-coil structure and four Pro/Glu/Ser/Thr-rich (PEST) sequences, but not the GTPase-binding domain (GBD) that is characteristic of the p21-activated kinase (PAK) family, polyproline consensus binding sites, or the Leu-rich domain seen in the group I germinal center kinases (GCKs). Northern blot analysis indicated that hSLK was ubiquitously expressed. hSLK overexpressed in COS-7 cells phosphorylates itself as well as myelin basic protein used as a substrate. On the other hand, hSLK cannot activate any of the three well-characterized mitogen-activated protein kinase MAPK (ERK, JNK/SAPK and p38) pathways. Moreover, hSLK kinase activity is not upregulated by constitutive active forms of GTPases (RasV12, RacV12 and Cdc42V12). These structural and functional properties indicate that hSLK should be considered to be a new member of group II GCKs.     

NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain.

Nck, an adaptor protein composed of one SH2 and three SH3 domains, is a common target for a variety of cell surface receptors. We have identified a novel mammalian serine/threonine kinase that interacts with the SH3 domains of Nck, termed Nck Interacting Kinase (NIK). This kinase is most homologous to the Sterile 20 (Ste20) family of protein kinases. Of the members of this family, GCK and MSST1 are most similar to NIK in that they bind neither Cdc42 nor Rac and contain an N-terminal kinase domain with a putative C-terminal regulatory domain. Transient overexpression of NIK specifically activates the stress-activated protein kinase (SAPK) pathway. Both the kinase domain and C-terminal regulatory region of NIK are required for full activation of SAPK. NIK likely functions upstream of MEKK1 to activate this pathway; a dominant-negative MEK kinase 1 (MEKK1) blocks activation of SAPK by NIK. MEKK1 and NIK also associate in cells and this interaction is mediated by regulatory domains on both proteins. Two other members of this kinase family, GCK and HPK1, contain C-terminal regulatory domains with homology to that of NIK. These findings indicate that the C-terminal domain of these proteins encodes a new protein domain family and suggests that this domain couples these kinases to the SAPK pathway, possibly by interacting with MEKK1 or related kinases.     

Germinal center kinase is required for optimal Jun N-terminal kinase activation by Toll-like receptor agonists and is regulated by the ubiquitin proteasome system and agonist-induced, TRAF6-dependent stabilization

Germinal center kinase (GCK), a member of the Ste20 family, selectively activates the Jun N-terminal kinase (JNK) group of mitogen-activated protein kinases. Here, we show that endogenous GCK is activated by polyinosine-polycytidine [poly(IC)] and lipopolysaccharides (LPS), lipid A, interleukin-1 (IL-1), and engagement of CD40, all agonists that require TRAF6 for JNK activation. RNA interference experiments indicate that GCK is required for the maximal activation of JNK by LPS, lipid A, poly(IC), and, to a lesser extent, IL-1 and engagement of CD40. GCK is ubiquitinated in situ and stabilized by inhibitors of the proteasome, indicating that GCK is subject to proteasomal turnover. GCK is constitutively active, and the kinase activity of GCK is required for GCK ubiquitination. Agonist activation of GCK involves the TRAF6-dependent transient stabilization of the GCK polypeptide rather than an increase in intrinsic kinase activity. Our results identify a physiologic function and unexpected mode of regulation for GCK.     

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.     

Cloning of MASK, a novel member of the mammalian germinal center kinase III subfamily, with apoptosis-inducing properties.

We have cloned a novel human GCK family kinase that has been designated as MASK (Mst3 and SOK1-related kinase). MASK is widely expressed and encodes a protein of 416 amino acid residues, with an N-terminal kinase domain and a unique C-terminal region. Like other GCK-III subfamily kinases, MASK does not activate any mitogen-activated protein kinase pathways. Wild type MASK, but not a form lacking the C terminus, exhibits homophilic binding in the yeast two-hybrid system and in coimmunoprecipitation experiments. Additionally, deletion of this C-terminal region of MASK leads to an increased kinase activity toward itself as well as toward an exogenous substrate, myelin basic protein. A potential caspase 3 cleavage site (DESDS) is present in the C-terminal region of MASK, and we show that MASK is cleaved in vitro by caspase 3. Finally, wild type and C-terminally truncated forms of MASK can both induce apoptosis upon overexpression in mammalian cells that is abrogated by CrmA, suggesting involvement of MASK in the apoptotic machinery in mammalian cells.     

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.     

Identification and characterization of a novel Ste20/germinal center kinase-related kinase,polyploidy-associated protein kinase

A novel protein kinase, polyploidy-associated protein kinase (PAPK), was isolated using a subtraction cDNA library approach from a mouse erythroleukemia cell line that had been induced to polyploidy after serum withdrawal. PAPK shares homology with members of the Ste20/germinal center kinase family of protein kinases and is ubiquitously expressed as two spliced forms, PAPK-A and PAPK-B, that encode for proteins of 418 and 189 amino acids, respectively. The expression of endogenous PAPK-A protein increased after growth factor withdrawal in murine hematopoietic and fibroblast cells. When tested in an in vitro kinase assay, PAPK-A was activated in response to the stress-inducing agent hydrogen peroxide and slightly by fetal calf serum. Biochemical characterization of the PAPK-A-initiated pathway revealed that this novel kinase does not affect MAP kinase activity but can stimulate both c-Jun N-terminal kinase 1 (JNK1) and ERK6/p38 gamma. The kinase activity of PAPK appears to be required for the activation of ERK6/p38 gamma but not JNK1. When an inducible construct of PAPK-A was expressed in stably transfected NIH3T3 cells, the cells exhibited distinct cytoskeletal changes and became resistant to apoptotic cell death induced by serum withdrawal, effects of PAPK that require its kinase activity. These data suggest that PAPK is a new member of the Ste20/germinal center kinase family that modulates cytoskeletal organization and cell survival.     

Tool inhibitors and assays to interrogate the biology of the TRAF2 and NCK interacting kinase

The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology.     

Tumor necrosis factor (TNF)-induced germinal center kinase-related (GCKR) and stress-activated protein kinase (SAPK) activation depends upon the E2/E3 complex Ubc13-Uev1A/TNF receptor-associated factor 2 (TRAF2)

Tumor necrosis factor (TNF)-induced activation of apoptosis signal-regulating kinase 1 (ASK1) and germinal center kinases (GCKs) and the subsequent activation of stress-activated protein kinases (SAPKs and c-Jun NH(2)-terminal kinases) requires TNF receptor-associated factor 2 (TRAF2). Although the TRAF2 TRAF domain binds ASK1, GCK, and the highly related kinase GCKR, the RING finger domain is needed for their activation. Here, we report that TNF activates GCKR and the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits both TNF- and TRAF2-mediated GCKR and SAPK activation, but has a minimal effect on ASK1 activation. TNF signaling leads to TRAF2 polyubiquitination and oligomerization and to the oligomerization, ubiquitination, and activation of GCKR, all of which are sensitive to the disruption of Ubc13 function. These results indicate that the assembly of a TRAF2 lysine 63-linked polyubiquitin chain by Ubc13/Uev1A is required for TNF-mediated GCKR and SAPK activation, but may not be required for ASK1 activation.