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Previous Article | Next Article 
Molecular and Cellular Biology, October 2007, p. 7273-7283, Vol. 27, No. 20
0270-7306/07/$08.00+0 doi:10.1128/MCB.00025-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Programa de Oncología Translacional, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca E-37007, Spain
Received 5 January 2007/ Returned for modification 21 March 2007/ Accepted 7 August 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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c-Jun N-terminal kinase (JNK) is primarily a stress response kinase and can be activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (18). The molecular initiators and modulators of MAPK activation from endogenous signals that do not start at membrane receptors are less well known. Several studies have reported that hypoxia induces JUN and FOS gene expression in selective brain areas (7), in PC12 cells (39), and in HeLa cells (33). Hypoxia also stimulates JNK and activates the transcription of AP1-inducible genes in endothelial cells (2), cardiac myocytes (52), PC12 cells (37), and HeLa cells (33). Nevertheless, the kind of response varies depending on the magnitude of the stimulus, the type of tissue, and the cellular context. Thus, in neurons cultured from the developing rat forebrain, sequential activation of JNK and AP1 was found to be involved in the apoptosis induced by reoxygenation after hypoxia (7); but in cardiac myocytes, the JNK response improves cell survival under hypoxic conditions (12, 13). Therefore, the modularity of signaling pathways permits great flexibility and thus, depending on the molecular context, the particular biological response will be specific in each cell (23).
There are multiple MAPKs (9, 49) that can form complexes with different scaffold proteins (23), one of which is represented by the JIP family (58). JIP forms a complex with these kinases that is phosphorylated sequentially, and the third one phosphorylates the target transcription factor. Among the kinases that are representative of the three steps are MLK, which is an initial kinase; MKK7 (31, 49), which is a middle kinase; and JNK (9, 21, 53), which is the last kinase in the complex and phosphorylates the transcription factor c-Jun. JIP1 can also target MAPK to dephosphorylation, instead of activation, and thus downregulates the signaling pathway (56); but how this negative regulation is performed is not known.
A new family of kinases, known as the vaccinia-related kinase (VRK) family, has been identified in the human kinome that is composed of three members (29). Two of them, VRK1 and VRK2, are catalytically active serine-threonine kinases but diverge in their C-terminal regions (34). Very little is known regarding their functions, despite their expression in most cell types, of which VRK1 is the most characterized. VRK1 has been shown to phosphorylate p53 (3, 28, 51), forming an autoregulatory loop (50), ATF-2 (44), and c-Jun (43). All these targets are transcription factors implicated in different stress responses. Also, VRK1 phosphorylates Baf, which is implicated in the assembly of the nuclear envelope (14, 36). The structurally more distant VRK3 modulates MAPK signaling by interacting with and activating the VHR dual-phosphatase activity (20).
The human VRK2 gene generates by alternative splicing two isoforms, A and B, consisting of 508 and 397 amino acids, respectively, which differ in their C-terminal regions. The VRK2A isoform is expressed in all cell types, it is anchored to cellular membranes by a hydrophobic tail in its C terminus, and it is located mostly in the endoplasmic reticulum (6, 35) and mitochondria, although the relative distribution varies depending on cell type (6). The shorter VRK2B isoform lacks the membrane anchor region, is more restricted in its expression, and is located in both the cytosol and the nuclei, functionally replacing VRK1 in those cells where this latter protein is cytosolic (6). VRK2 interacts with JIP1 and prevents the incorporation of JNK into the JIP1 signalosome, resulting in reduced c-Jun-dependent transcription (S. Blanco, M. Sanz-Garcia, C. R. Santos, and P. A. Lazo, submitted for publication). If the cellular response to hypoxia is partly channeled via complexes of MAPKs with scaffold proteins, it is possible that the level of VRK2 protein might affect this response.
In this work, we have analyzed the role that the interaction between the atypical TAK1 and the JIP1 scaffold proteins plays in the cell response to hypoxia and the effect that VRK2 proteins play in the modulation of this response by interacting with elements of this signaling pathway.
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MATERIALS AND METHODS |
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C (residues 1 to 300) were from G. Gross (16). Plasmids expressing HA-TAK1K63W (kinase-negative mutant) were from Shunsuke Ishii (RIKEN, Tsukuba, Japan). The plasmid pFlag-JNK was made by PCR from plasmid pHA-JNK from S. Gutkind. The mammalian expression constructs pCEFL-HA-VRK2A and -VRK2B and pCEFL-GST-VRK2A and -VRK2B and inactive kinase VRK2(K169E) have been described previously (6). Glutathione S-transferase (GST) fusion proteins spanning VRK2 regions 1 to 320, 256 to 508, and 364 to 508 were made by PCR from full-length VRK2A. The plasmid p-sh-RNA-VRK1 carrying specific short hairpin RNA (shRNA) for VRK1 was reported previously (51), and plasmids p-sh-RNA-VRK2-230, p-sh-RNA-VRK2-438, and p-sh-RNA-VRK2-1335, which carry specific VRK2 shRNA with the sequences 5'-GAAGATTGGCTCTGGAGGA-3', 5'-GGTATCCGAATGTTGGATG-3', and 5'-ATACACTTCCACAGTCAGC-3', respectively, were cloned into plasmid pSUPERIOR-Retro-puro following the manufacturer's instructions (Oligoengine, Seattle, WA). The pAP1-Luc reporter was from Stratagene (San Diego, CA). The plasmid pRL-tk, from Promega Biotech (Madison, WI), was used for internal control in luciferase assays.
Cell culture, transfection, and transcriptional assays.
Cos1 and HeLa cells were grown in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal calf serum and antibiotics in 5% CO2 humidified atmosphere. For assays of transcriptional activity using a reporter plasmid, Cos1 cells were transfected with 0.8 µg of the synthetic reporter plasmid pAP1-Luc, 10 ng of pRL-tk (Promega), 50 ng of pHA-TAK1a, 50 ng of pFlag-TAB1, and the indicated amounts of the specific kinase constructs or the shRNA-expressing plasmid indicated for each experiment. The plasmid DNA was mixed with 6 µl of JetPEI transfection reagent (Polytransfection, Ilkirch, France). Twenty-four hours after transfection, the medium was changed to serum-free DMEM, cells were lysed 48 h after transfection, and luciferase activity was determined with a dual-luciferase reporter reagent from Promega. Seven hours before lysis, cells were treated with 300 µM of deferoxamine mesylate (DFO) (Sigma) or were incubated under hypoxic conditions (pO2 
0.5%) in DMEM without serum at pH 7.5, which had been previously gassed with a mixture of 95% N2 and 5% CO2.
For assays of transcriptional activity using shRNA, HeLa cells were plated in 35-mm-diameter dishes and transfected with 0.2 µg of pAP1-Luc, 10 ng of pRL-tk, and 6 µg of the specific shRNA-expressing plasmids. Cells were treated as indicated above, and luciferase activity was determined 48 h posttransfection with a dual-luciferase reporter reagent from Promega.
Antibodies and reagents. The HA epitope was detected with a monoclonal clone, HA.11, from Covance (Berkeley, CA). The Flag epitope was detected with a rabbit polyclonal antibody from Sigma. GST fusion proteins were detected with a polyclonal antibody (sc-138) from Santa Cruz Biotechnology. Actin was determined with a monoclonal antibody (clone AC-15) from Sigma. Human JIP1 protein was detected with rabbit polyclonal (M-300) antibody from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against human VRK2 have been described previously (6). Human JNK1 was detected with a monoclonal antibody from BD Pharmingen. JNK phosphorylated in Thr183 and Tyr185 was detected with a monoclonal antibody (G7) from Santa Cruz Biotechnology. Human TAK1 was detected with a monoclonal antibody (C-9) from Santa Cruz Biotechnology. Phospho-TAK1 antibody, from Cell Signaling, detects TAK1 phosphorylated in Thr184 and Thr187. Phospho-MKK7 antibody, from Cell Signaling, detects MKK7 phosphorylated in Ser271 and Thr275. A goat horseradish peroxidase-anti-mouse antibody was from GE Healthcare. A sheep horseradish peroxidase-anti-rabbit antibody was from Sigma. DFO was from Sigma.
Immunoprecipitation, pull-down experiments, and immunoblot analysis. For pull-down experiments, Cos1 cells were grown in 100-mm-diameter dishes and transfected with different fragments of fusion proteins in vectors for mammalian expression. The amounts and types of the specific plasmids are indicated for each individual experiment. All TAK1 expression plasmids, deletion mutants, and the coactivator pFlag-TAB1 were used at 50 ng, 3 µg of plasmids expressing JIP1, 4 µg of pFlag-JNK, and 4 µg of plasmids expressing full-length or deletion mutants of VRK2. Cells were harvested 48 h posttransfection and lysed with buffer containing 20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 2 mM EDTA, 25 mM ß-glycerophosphate, 10% (vol/vol) glycerol, and 1% Triton X-100, with inhibitors of proteases and phosphatases (1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM Na orthovanadate). The GST fusion proteins were brought down with 20 µl of glutathione-Sepharose beads (GE Healthcare) for 12 h at 4°C with gentle shaking. The washed beads were loaded in a sodium dodecyl sulfate-polyacrylamide gel and transferred to an Immobilon-P membrane (Millipore), and the Western blot was analyzed for the indicated proteins with the corresponding antibody, in individual experiments.
For the pull-down or immunoprecipitation assay of endogenous proteins, HeLa cells were grown in 100-mm-diameter dishes and transfected with the plasmids indicated for each experiment. Twenty-four hours after transfection, cells were grown in serum-free DMEM and then treated as indicated for each experiment. Whole-cell extracts were prepared 48 h after transfection, as described before. Immunoprecipitation and pull-downs were performed using 1 mg of the whole-cell protein extract. GST-VRK2A proteins were brought down with 20 µl of glutathione-Sepharose beads (GE Healthcare). HA-TAK1 was immunoprecipitated with the monoclonal anti-HA antibody from Covance, endogenous TAK1 was immunoprecipitated with a monoclonal anti-TAK1 antibody from Santa Cruz Biotechnology (clone C-9), and endogenous JIP1 was brought down with the polyclonal anti-JIP1 antibody from Santa Cruz Biotechnology (clone M-300). Antibodies were then brought down with GammaBind G Sepharose (GE Healthcare). The washed beads were analyzed as described before.
Detection of protein complexes by gel filtration chromatography. For isolation of endogenous protein complexes, HeLa cells were grown for 24 h in serum-free medium and then treated with 300 µM DFO at different times. Protein extracts were prepared as described before. Insoluble material was removed by centrifugation at 16,000 x g for 20 min. The supernatant, containing 1.5 mg of dissolved protein, was fractionated by high-performance liquid chromatography gel filtration through a Superose 12 10/300 GL column (GE Healthcare). High-performance liquid chromatography was performed with an HP model 1100 machine from Agilent Technologies (Germany) equipped with ChemStation software and developed with a buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 100 mM KCl at a flow rate of 0.1 ml/min. Fractions (0.2 ml) were collected, precipitated, resolved on a 10% polyacrylamide gel, and immunoblotted. Molecular weight markers used to calibrate the column were bovine thyroglobulin (670,000), apoferritin from horse spleen (440,000), alcohol dehydrogenase from yeast (150,000), bovine serum albumin (66,000) and bovine carbonic anhydrase (29,000), all from Sigma. The effluent was monitored by its absorbance at 280 nm.
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RESULTS |
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JBD) that lacks the JNK-binding domain and that functions as a dominant negative of the MAPK signaling complex (55). If the effect of DFO requires the assembly of a JNK-JIP1 complex, the cell response should be blocked by this JIP1 deletion mutant. Indeed, the cell response to DFO (and low-oxygen conditions, data not shown) in the presence of TAK1/TAB1 was blocked by the dominant negative JIP1JBD protein (Fig. 1C, top), confirming that these are steps implicated in the response to hypoxia. The correct expression of the proteins was determined by immunoblot analysis (Fig. 1C, bottom panel). The JIP1 requirement indicates that assembly of the MAPK cascade is necessary for this response to hypoxia. TAK1 and JIP1 interact to form a stable complex. The previous data suggested that the TAK1 kinase mediates a hypoxia response signal that probably implicates the assembly of JIP1 complexes. However, although TAK1 is able to activate downstream signaling by complexes assembled on the JIP1 scaffold protein, so far it is not known if TAK1 can indeed form a stable complex with JIP1 (19).
To address this issue, Cos1 cells were transfected with mammalian expression pGST-JIP1 constructs that cover different regions of the JIP1 molecule, residues 1 to 282, 282 to 660, and 471 to 660, a deletion of the JNK binding domain (JBD), and the full-length protein (55). These constructs were cotransfected with HA-TAK1 plus Flag-TAB1. Cell lysates were used for a pull-down, and the associated proteins were identified by immunoblotting (Fig. 2A), although additional cellular proteins may also be present in these complexes. JIP1 stably interacts with TAK1 by a region located between residues 283 and 471. This region of JIP1 is different from the one that interacts with other MAPKs such as MLK and JNK (54). Then, a similar assay was performed in order to determine the TAK1 region required for this interaction with JIP1. Cos1 cells were transfected with the plasmid expressing GST-JIP1 and constructs that carried the full length and the N-terminal region (residues 1 to 480) of murine HA-TAK1 (variant a) (Fig. 2B) or the full length and the N-terminal region (residues 1 to 300) of murine Flag-TAK1 (variant b) (16). These constructs were cotransfected with Flag-TAB1 (Fig. 2C). Both TAK1 variants interact by their N-terminal domains, the first 300 amino acids, with JIP1 (Fig. 2D).
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Finally, we determined whether endogenous JIP1 associates with endogenous TAK1 (levels shown in Fig. 2F, bottom panel) and whether hypoxic conditions can affect this association. For this aim, HeLa cells were exposed to DFO for different times (20 to 60 min), lysed, and immunoprecipitated with either anti-JIP1 or anti-TAK1 antibody, followed by immunoblotting with the antibody for the corresponding partner. The endogenous TAK1 interacted with endogenous JIP1 even in the absence of any stimuli (Fig. 2F, second lane). The TAK1-JIP1 interaction increases within 20 min following DFO exposure (Fig. 2F, third lane). Nevertheless, this association was transient, and TAK1 disassociated from JIP1 after 1 h of DFO treatment (Fig. 2F, fourth lane). These results confirmed that endogenous TAK1 interacts with endogenous JIP1 and that this interaction is transiently enhanced during hypoxia.
TAK1 effect on JNK activation and binding to JIP1. The following step was performed to determine how the TAK1-JIP1 interaction might affect the binding of JNK to JIP1, the last step in the cascade. To address this issue, we performed a dose-response assay of JNK binding to JIP1 in the presence of increasing amounts of TAK1. The amount of phosphorylated JNK increased in the presence of both TAK1 and TAB1 in a dose-dependent manner in the whole lysate, suggesting that TAK1 contributes to JNK activation (Fig. 3, lysate). Regarding the pulled down proteins, as the amount of TAK1/TAB1 increased, so did the JIP1-bound JNK and its phosphorylated form (Fig. 3, left box). If TAB1 was not included in the assay, the binding of JNK to JIP1 was not affected (Fig. 3, central box) and there was a reduced level of phosphorylated JNK. Thus, the presence of TAB1 is essential for the JNK-JIP1 interaction induced by TAK1. When the kinase-dead protein TAK1K63W was used, the binding of JNK to JIP1 and the phosphorylation of JNK were lower than that with wild-type TAK1 (Fig. 3, right box). These data suggest that an active TAK1 and the presence of its coactivator TAB1 are required for maximal activation, detected as a phosphorylated JNK bound to JIP1, and thus contribute to the activation of the TAK1-JIP1-JNK signaling module.
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VRK2 interferes with TAK1 signaling and with the response to hypoxia. The regulation of these signaling complexes depends on interactions of the module with other proteins (23). In order to ascertain if VRK2 could also modulate the stress signal induced by hypoxia, the AP1-dependent activation of transcription was analyzed. For this aim, Cos1 cells were transfected with a reporter, pAP1-Luc, and plasmids expressing TAK1 and TAB1 and were treated with DFO to induce hypoxia (Fig. 5A). This stimulatory effect was almost completely blocked by overexpression of either the common VRK2A or the rare VRK2B (6), as well as their kinase-dead mutants (K169E substitution). These results indicated that the effect of both VRK2 isoforms is dependent on their interaction with the signaling system and does not require the kinase activity, thus ruling out additional effects by phosphorylation of either JIP1 or other cellular proteins mediated by VRK2 proteins. The downregulatory effect on transcription induced by the maximum amount of VRK2A or VRK2B was lost if the cells were cotransfected with shRNA specific for both isoforms of VRK2 (Fig. 5A).
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The dependence of this hypoxic response on endogenous VRK2 protein levels was also studied using the same type of assay but with the human HeLa cell line. Plasmids expressing shRNA for human VRK1 or VRK2 were transfected, and their effects on the AP1-dependent transcriptional response to DFO (Fig. 5D, top) or hypoxia (Fig. 5D, middle) were analyzed. Two of the shRNA for VRK2 (sh-VRK2-230 and sh-VRK2-1335) resulted in an increase in the response to hypoxia, while the shVRK1 and one of the shVRK2 had no effect, which is consistent with their effects on protein levels as determined in an immunoblot, in which the two shRNA, one for VRK1 and the other for VRK2, that were ineffective did not alter the VRK2 protein level (Fig. 5D, bottom panel). These results indicate that the effect of VRK2 was more general, since it could also be detected in a different cell line by modulating its endogenous levels, and it depends on the protein levels instead of the kinase activity.
VRK2A and TAK1 interact to form a stable complex. It is possible that VRK2A or VRK2B, in addition to its interaction with JIP1 (Blanco et al., submitted), might also directly interact with TAK1 as part of the multiprotein complex, and that would reveal a novel interaction that can contribute to determining how the signalosome is assembled or modulated. The assembly of the signaling complex suggests that interactions might occur among more than two proteins in the complex. To further characterize the assembly of the complex formed by VRK2A or VRK2B, JIP1, and TAK1, we determined the potential interaction between VRK2A and TAK1. For this aim, different constructs of VRK2 fused to GST were used to perform a pull-down assay of cells transfected with HA-TAK1a (Fig. 6A, top panel). We clearly detected the interaction of the full-length VRK2A (508 amino acids), as well as of a carboxy-terminal construction of VRK2A (residues 364 to 508). Nonetheless, the N-terminal region (residues 1 to 320) or isoform VRKB (amino acids 1 to 396) did not interact with TAK1, suggesting that the minimum region necessary for this interaction is located between residues 397 and 508 of VRK2A, probably proximal to residue 396, since GST fusion to residue 364 weakens the interaction, probably by distorting this region (Fig. 6A, bottom panel). It is known that the VRK2 C-terminal region has a low complexity and might have alternative folding conformations, of which only some might be able to interact with TAK1. This would explain the weaker interaction observed for this construct.
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Finally, it was determined if the interaction occurs with endogenous proteins and whether DFO stimulation also affects the TAK1-VRK2 interaction, as is the case with the JIP1-TAK1 association. An immunoprecipitation of endogenous proteins could not be performed because antibodies compete for the interacting region of VRK2. Therefore, an alternative experimental approach was used to detect interactions of endogenous proteins. First we examined whether endogenous VRK2A can associate with transfected HA-TAK1a. For this aim, HeLa cells were transfected with empty vector of HA-TAK1a and stimulated or not with DFO at different times, after which cells were lysed and used for immunoprecipitations with an anti-HA antibody, followed by immunoblotting with an anti-VRK2 antibody (Fig. 6C). The endogenous VRK2A stably interacts with the transfected HA-TAK1a protein under normoxic conditions (Fig. 6C, second lane), and this association was not affected by the DFO exposure of VRK2 protein (Fig. 6C, third and fourth lanes). Next, to detect the interaction in a reciprocal way, a pull-down approach was followed. HeLa cells were transfected with plasmids carrying either GST or GST-VRK2A and then treated as described before. The endogenous TAK1 protein was brought down by GST-VRK2A (Fig. 6D, second lane) under normoxic conditions, and this interaction was not affected by DFO (Fig. 6D, third and fourth lanes).
From these data, it can be concluded that both VRK2A and TAK1 can interact with each other and with JIP1 (Blanco et al., submitted) by different regions to form a stable interaction; thus, VRK2A, by interacting with TAK1 and JIP1, or VRK2B, by interacting only with JIP1 (Blanco et al., submitted), might modulate the signaling complex formed by TAK1, JNK, and JIP1, which responds to stress signals such as DFO or hypoxia.
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DISCUSSION |
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TAK1 is able to stably interact with JIP1, as the MLK protein family does (31), which is closely related to TAK1 according to the human kinome (29). This interaction takes place without the need to be assembled with other kinases of the complex or TAB cofactors, and this interaction is a step that potentiates the response to hypoxia via JNK (Fig. 1). The interaction of TAK1 occurs by a JIP1 central region (residues 283 to 471) and is likely to lock JIP1 in a conformation that increases the binding of JNK to JIP1, making the transduction of the signal, through the JIP1-JNK module, more efficient. However, TAK1 also interacts with VRK2A, and because the regions of interaction are different, it can simultaneously form a complex with both JIP1 and VRK2A (Blanco et al., submitted). Nevertheless the VRK2A interaction with the TAK1-JIP1 complex has important functional consequences, since it downregulates the response to hypoxia by disrupting only the signaling through the TAK1-JIP1-JNK complex and therefore switching the balance among the different pathways in response to a common stimulation and determining cell fate. It has been shown in the cell response to interleukin 1 that VRK2A binding to JIP1 reduces the incorporation of JNK and thus reduces the activation of c-Jun-dependent transcription (Blanco et al., submitted).
The stress response to hypoxia has been studied mainly in the context of cardiac or neural biology (11, 60), but the contributions of JNK to apoptosis or survival after oxidative stress are still conflicting. In cardiomyocytes, apoptosis is reduced by inhibition of the JNK pathways (5, 48). On the other hand, the activation of the JNK pathway under moderate hypoxia (5% O2) also contributes to the expression of proangiogenic genes, such as VEGF (4), where there is cooperation between c-Jun and Hif1 for this activation (1). These observations are very important in the context of a hypoxic tumor mass that might trigger an angiogenic response; in human gliomas, overexpression of VEGF is dependent on JNK activation (59). Also, this response promotes the expression of MDR genes that can make the cell more resistant to chemotherapy (8).
Interestingly this JNK activation induced by hypoxia appears to be initiated at the mitochondria (12), where part of VRK2A resides (6). Furthermore, calreticulin, a molecule that colocalizes with VRK2A, also affects cardio protection that is dependent on JNK activation (27). VRK2A also interacts with the BHRF1 protein of Epstein-Barr virus, which is a Bcl2 homolog, and this interaction appears to stimulate protection against apoptosis (25). In the context of hypoxia, the upstream events that result in the activation of TAK1 by the low oxygen level shown in this work remain to be identified. Here we report that the intracellular level of VRK2 conditions the magnitude of the stress response to hypoxia. Therefore, it is possible that, depending on the expression levels of VRK2, each tissue might have different responses to hypoxia and that in pathological situations such as cancer, tumors might have higher levels of VRK2 (34) that will permit survival under hypoxic conditions, a common stress in the tumor mass as it grows. For instance, in developing brain neurons, transient hypoxia and reoxygenation after hypoxia trigger apoptosis through the JNK signaling pathway (7), whereas in cardiomyocytes, which have relatively high levels of VRK2 (34), the activation of JNK, by reoxygenation induces survival (13). Hence, the modulation of MAPK pathways by additional proteins is a central process for the determination of the type of cellular reaction to specific stimulation; the levels of these other proteins might explain the differences found among different cell types to identical stimulation, and JIP1 and VRK2 appear to be some of them. Thus, the levels of VRK2 may modulate different responses where the assembly of JIP1 complexes is required. In this context, it will be very interesting to know how, in tumor biopsy samples, VRK2 is distributed in relation to the distance of a tumor cell from vessels.
In conclusion, in this work we have shown that hypoxia activates TAK1 and results in the activation of c-Jun-dependent transcription. The magnitude of this response can be modulated by the formation of a complex in which TAK1 directly interacts with JIP1 and VRK2A.
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ACKNOWLEDGMENTS |
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S.B. and C.S. have predoctoral fellowships from the Ministerio de Educación y Ciencia (Spain) and Fundação para a Ciência e a Tecnologia (Portugal), respectively. This work was funded by grants from Ministerio de Educación y Ciencia (SAF2004-02900, SAF2007-60242), Junta de Castilla y León (CSI05A05), Federación de Cajas de Ahorro de Castilla y León, and Fundación de Investigación Médica MM to P.A.L.
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FOOTNOTES |
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Published ahead of print on 20 August 2007.
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REFERENCES |
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2. Bandyopadhyay, R. S., M. Phelan, and D. V. Faller. 1995. Hypoxia induces AP-1-regulated genes and AP-1 transcription factor binding in human endothelial and other cell types. Biochim. Biophys. Acta 1264:72-78.[Medline]
3. Barcia, R., S. Lopez-Borges, F. M. Vega, and P. A. Lazo. 2002. Kinetic properties of p53 phosphorylation by the human vaccinia-related kinase 1. Arch. Biochem. Biophys. 399:1-5.[CrossRef][Medline]
4. Berra, E., G. Pages, and J. Pouyssegur. 2000. MAP kinases and hypoxia in the control of VEGF expression. Cancer Metastasis Rev. 19:139-145.[CrossRef][Medline]
5. Bishopric, N. H., P. Andreka, T. Slepak, and K. A. Webster. 2001. Molecular mechanisms of apoptosis in the cardiac myocyte. Curr. Opin. Pharmacol. 1:141-150.[CrossRef][Medline]
6. Blanco, S., L. Klimcakova, F. M. Vega, and P. A. Lazo. 2006. The subcellular localization of vaccinia-related kinase-2 (VRK2) isoforms determines their different effect on p53 stability in tumour cell lines. FEBS J. 273:2487-2504.[CrossRef][Medline]
7. Chihab, R., C. Ferry, V. Koziel, P. Monin, and J. L. Daval. 1998. Sequential activation of activator protein-1-related transcription factors and JNK protein kinases may contribute to apoptotic death induced by transient hypoxia in developing brain neurons. Brain Res. Mol. Brain Res. 63:105-120.[Medline]
8. Comerford, K. M., E. P. Cummins, and C. T. Taylor. 2004. c-Jun NH2-terminal kinase activation contributes to hypoxia-inducible factor 1 alpha-dependent P-glycoprotein expression in hypoxia. Cancer Res. 64:9057-9061.
9. Davis, R. J. 2000. Signal transduction by the JNK group of MAP kinases. Cell 103:239-252.[CrossRef][Medline]
10. Dhanasekaran, D. N., K. Kashef, C. M. Lee, H. Xu, and E. P. Reddy. 2007. Scaffold proteins of MAP-kinase modules. Oncogene 26:3185-3202.[CrossRef][Medline]
11. Dong, Z., L. Zhou, K. Del Villar, M. Ghanevati, V. Tashjian, and C. A. Miller. 2005. JIP1 regulates neuronal apoptosis in response to stress. Brain Res. Mol. Brain Res. 134:282-293.[Medline]
12. Dougherty, C. J., L. A. Kubasiak, D. P. Frazier, H. Li, W. C. Xiong, N. H. Bishopric, and K. A. Webster. 2004. Mitochondrial signals initiate the activation of c-Jun N-terminal kinase (JNK) by hypoxia-reoxygenation. FASEB J. 18:1060-1070.
13. Dougherty, C. J., L. A. Kubasiak, H. Prentice, P. Andreka, N. H. Bishopric, and K. A. Webster. 2002. Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress. Biochem. J. 362:561-571.[CrossRef][Medline]
14. Gorjanacz, M., E. P. Klerkx, V. Galy, R. Santarella, C. Lopez-Iglesias, P. Askjaer, and I. W. Mattaj. 2007. Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly. EMBO J. 26:132-143.[CrossRef][Medline]
15. Harris, A. L. 2002. Hypoxia: a key regulatory factor in tumour growth. Nat. Rev. Cancer 2:38-47.[CrossRef][Medline]
16. Hoffmann, A., O. Preobrazhenska, C. Wodarczyk, Y. Medler, A. Winkel, S. Shahab, D. Huylebroeck, G. Gross, and K. Verschueren. 2005. Transforming growth factor-beta-activated kinase-1 (TAK1), a MAP3K, interacts with Smad proteins and interferes with osteogenesis in murine mesenchymal progenitors. J. Biol. Chem. 280:27271-27283.
17. Holtmann, H., J. Enninga, S. Kalble, A. Thiefes, A. Dorrie, M. Broemer, R. Winzen, A. Wilhelm, J. Ninomiya-Tsuji, K. Matsumoto, K. Resch, and M. Kracht. 2001. The MAPK kinase kinase TAK1 plays a central role in coupling the interleukin-1 receptor to both transcriptional and RNA-targeted mechanisms of gene regulation. J. Biol. Chem. 276:3508-3516.
18. Javelaud, D., and A. Mauviel. 2005. Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-beta: implications for carcinogenesis. Oncogene 24:5742-5750.[CrossRef][Medline]
19. Jiang, Z., J. Ninomiya-Tsuji, Y. Qian, K. Matsumoto, and X. Li. 2002. Interleukin-1 (IL-1) receptor-associated kinase-dependent IL-1-induced signaling complexes phosphorylate TAK1 and TAB2 at the plasma membrane and activate TAK1 in the cytosol. Mol. Cell. Biol. 22:7158-7167.
20. Kang, T. H., and K. T. Kim. 2006. Negative regulation of ERK activity by VRK3-mediated activation of VHR phosphatase. Nat. Cell Biol. 8:863-869.[CrossRef][Medline]
21. Karin, M., and E. Gallagher. 2005. From JNK to pay dirt: jun kinases, their biochemistry, physiology and clinical importance. IUBMB Life 57:283-295.[Medline]
22. Kishimoto, K., K. Matsumoto, and J. Ninomiya-Tsuji. 2000. TAK1 mitogen-activated protein kinase kinase kinase is activated by autophosphorylation within its activation loop. J. Biol. Chem. 275:7359-7364.
23. Kolch, W. 2005. Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat. Rev. Mol. Cell. Biol. 6:827-837.[CrossRef][Medline]
24. Laderoute, K. R., H. L. Mendonca, J. M. Calaoagan, A. M. Knapp, A. J. Giaccia, and P. J. Stork. 1999. Mitogen-activated protein kinase phosphatase-1 (MKP-1) expression is induced by low oxygen conditions found in solid tumor microenvironments. A candidate MKP for the inactivation of hypoxia-inducible stress-activated protein kinase/c-Jun N-terminal protein kinase activity. J. Biol. Chem. 274:12890-12897.
25. Li, L. Y., M. Y. Liu, H. M. Shih, C. H. Tsai, and J. Y. Chen. 2006. Human cellular protein VRK2 interacts specifically with Epstein-Barr virus BHRF1, a homologue of Bcl-2, and enhances cell survival. J. Gen. Virol. 87:2869-2878.
26. Lin, A. 2003. Activation of the JNK signaling pathway: breaking the brake on apoptosis. Bioessays 25:17-24.[CrossRef][Medline]
27. Liu, X., F. Xu, Y. Fu, F. Liu, S. Sun, and X. Wu. 2006. Calreticulin induces delayed cardioprotection through mitogen-activated protein kinases. Proteomics 6:3792-3800.[CrossRef][Medline]
28. Lopez-Borges, S., and P. A. Lazo. 2000. The human vaccinia-related kinase 1 (VRK1) phosphorylates threonine-18 within the mdm-2 binding site of the p53 tumour suppressor protein. Oncogene 19:3656-3664.[CrossRef][Medline]
29. Manning, G., D. B. Whyte, R. Martinez, T. Hunter, and S. Sudarsanam. 2002. The protein kinase complement of the human genome. Science 298:1912-1934.
30. McKay, M. M., and D. K. Morrison. 2007. Integrating signals from RTKs to ERK/MAPK. Oncogene 26:3113-3121.[CrossRef][Medline]
31. Mooney, L. M., and A. J. Whitmarsh. 2004. Docking interactions in the c-Jun N-terminal kinase pathway. J. Biol. Chem. 279:11843-11852.
32. Morrison, D. K., and R. J. Davis. 2003. Regulation of MAP kinase signaling modules by scaffold proteins in mammals. Annu. Rev. Cell Dev. Biol. 19:91-118.[CrossRef][Medline]
33. Muller, J. M., B. Krauss, C. Kaltschmidt, P. A. Baeuerle, and R. A. Rupec. 1997. Hypoxia induces c-fos transcription via a mitogen-activated protein kinase-dependent pathway. J. Biol. Chem. 272:23435-23439.
34. Nezu, J., A. Oku, M. H. Jones, and M. Shimane. 1997. Identification of two novel human putative serine/threonine kinases, VRK1 and VRK2, with structural similarity to vaccinia virus B1R kinase. Genomics 45:327-331.[CrossRef][Medline]
35. Nichols, R. J., and P. Traktman. 2004. Characterization of three paralogous members of the mammalian vaccinia related kinase family. J. Biol. Chem. 279:7934-7946.
36. Nichols, R. J., M. S. Wiebe, and P. Traktman. 2006. The vaccinia-related kinases phosphorylate the N' terminus of BAF, regulating its interaction with DNA and its retention in the nucleus. Mol. Biol. Cell 17:2451-2464.
37. Norris, M. L., and D. E. Millhorn. 1995. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene. J. Biol. Chem. 270:23774-23779.
38. Pouyssegur, J., F. Dayan, and N. M. Mazure. 2006. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature 441:437-443.[CrossRef][Medline]
39. Prabhakar, N. R., B. C. Shenoy, M. S. Simonson, and N. S. Cherniack. 1995. Cell selective induction and transcriptional activation of immediate early genes by hypoxia. Brain Res. 697:266-270.[CrossRef][Medline]
40. Scheinfeld, M. H., R. Roncarati, P. Vito, P. A. Lopez, M. Abdallah, and L. D'Adamio. 2002. Jun NH2-terminal kinase (JNK) interacting protein 1 (JIP1) binds the cytoplasmic domain of the Alzheimer's beta-amyloid precursor protein (APP). J. Biol. Chem. 277:3767-3775.
41. Semenza, G. L. 2001. HIF-1 and mechanisms of hypoxia sensing. Curr. Opin. Cell Biol. 13:167-171.[CrossRef][Medline]
42. Seta, K. A., Z. Spicer, Y. Yuan, G. Lu, and D. E. Millhorn. 2002. Responding to hypoxia: lessons from a model cell line. Sci. STKE 2002:RE11.[Medline]
43. Sevilla, A., C. R. Santos, R. Barcia, F. M. Vega, and P. A. Lazo. 2004. c-Jun phosphorylation by the human vaccinia-related kinase 1 (VRK1) and its cooperation with the N-terminal kinase of c-Jun (JNK). Oncogene 23:8950-8958.[CrossRef][Medline]
44. Sevilla, A., C. R. Santos, F. M. Vega, and P. A. Lazo. 2004. Human vaccinia-related kinase 1 (VRK1) activates the ATF2 transcriptional activity by novel phosphorylation on Thr-73 and Ser-62 and cooperates with JNK. J. Biol. Chem. 279:27458-27465.
45. Shim, J. H., C. Xiao, A. E. Paschal, S. T. Bailey, P. Rao, M. S. Hayden, K. Y. Lee, C. Bussey, M. Steckel, N. Tanaka, G. Yamada, S. Akira, K. Matsumoto, and S. Ghosh. 2005. TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in vivo. Genes Dev. 19:2668-2681.
46. Singhirunnusorn, P., S. Suzuki, N. Kawasaki, I. Saiki, and H. Sakurai. 2005. Critical roles of threonine 187 phosphorylation in cellular stress-induced rapid and transient activation of transforming growth factor-beta-activated kinase 1 (TAK1) in a signaling complex containing TAK1-binding protein TAB1 and TAB2. J. Biol. Chem. 280:7359-7368.
47. Song, J. J., and Y. J. Lee. 2005. Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop. J. Cell Biol. 170:61-72.
48. Sun, H. Y., N. P. Wang, M. Halkos, F. Kerendi, H. Kin, R. A. Guyton, J. Vinten-Johansen, and Z. Q. Zhao. 2006. Postconditioning attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways. Apoptosis 11:1583-1593.[CrossRef][Medline]
49. Tournier, C., A. J. Whitmarsh, J. Cavanagh, T. Barret, and R. J. Davis. 1997. Mitogen-activated protein kinase 7 is an activator of the jun NH2-terminal kinase. Proc. Natl. Acad. Sci. USA 94:7337-7342.
50. Valbuena, A., F. M. Vega, S. Blanco, and P. A. Lazo. 2006. p53 downregulates its activating vaccinia-related kinase 1, forming a new autoregulatory loop. Mol. Cell. Biol. 26:4782-4793.
51. Vega, F. M., A. Sevilla, and P. A. Lazo. 2004. p53 stabilization and accumulation induced by human vaccinia-related kinase 1. Mol. Cell. Biol. 24:10366-10380.
52. Webster, K. A., D. J. Discher, and N. H. Bishopric. 1993. Induction and nuclear accumulation of fos and jun proto-oncogenes in hypoxic cardiac myocytes. J. Biol. Chem. 268:16852-16858.
53. Weston, C. R., and R. J. Davis. 2002. The JNK signal transduction pathway. Curr. Opin. Genet. Dev. 12:14-21.[CrossRef][Medline]
54. Whitmarsh, A. J., J. Cavanagh, C. Tournier, J. Yasuda, and R. J. Davis. 1998. A mammalian scaffold complex that selectively mediates MAP kinase activation. Science 281:1671-1674.
55. Whitmarsh, A. J., C. Y. Kuan, N. J. Kennedy, N. Kelkar, T. F. Haydar, J. P. Mordes, M. Appel, A. A. Rossini, S. N. Jones, R. A. Flavell, P. Rakic, and R. J. Davis. 2001. Requirement of the JIP1 scaffold protein for stress-induced JNK activation. Genes Dev. 15:2421-2432.
56. Willoughby, E. A., G. R. Perkins, M. K. Collins, and A. J. Whitmarsh. 2003. The JNK-interacting protein-1 scaffold protein targets MAPK phosphatase-7 to dephosphorylate JNK. J. Biol. Chem. 278:10731-10736.
57. Woo, K. J., T. J. Lee, J. W. Park, and T. K. Kwon. 2006. Desferrioxamine, an iron chelator, enhances HIF-1alpha accumulation via cyclooxygenase-2 signaling pathway. Biochem. Biophys. Res. Commun. 343:8-14.[CrossRef][Medline]
58. Yasuda, J., A. J. Whitmarsh, J. Cavanagh, M. Sharma, and R. J. Davis. 1999. The JIP group of mitogen-activated protein kinase scaffold proteins. Mol. Cell. Biol. 19:7245-7254.
59. Yoshino, Y., M. Aoyagi, M. Tamaki, L. Duan, T. Morimoto, and K. Ohno. 2006. Activation of p38 MAPK and/or JNK contributes to increased levels of VEGF secretion in human malignant glioma cells. Int. J. Oncol. 29:981-987.[Medline]
60. Zhou, L., K. Del Villar, Z. Dong, and C. A. Miller. 2004. Neurogenesis response to hypoxia-induced cell death: map kinase signal transduction mechanisms. Brain Res. 1021:8-19.[CrossRef][Medline]
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