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Tumor Biology |
Departments of Microbiology and Immunology, Kimmel Cancer Institute [H. T., M. T., I. O., D. R. E., M. H-M., I. S. L., C. J. J., K. T. V., A. J. W.], and Pathology, Anatomy, and Cell Biology [L. C. K.], Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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versus ß) or COOH terminus (1 versus 2) to create 10 JNK isoforms (JNK11, JNK12, JNK1ß1, JNK1ß2, JNK21, JNK22, JNK2ß1, JNK2ß2, JNK31, and JNK32; Ref. 4
). The alternate splicing at the COOH terminus leads to proteins that are either Mr 46,000 (JNK11, JNK1ß1, JNK21, JNK2ß1, and JNK31) or 55,000 (JNK12, JNK1ß2, JNK22, JNK2ß2, and JNK32) in size. Like all MAP kinase pathways, the JNK proteins require phosphorylation via an upstream kinase cascade to become active. The major MAPKKs have been shown to be MKK4 and MKK7, although other kinases can also phosphorylate the JNKs (1)
. The role of the JNK pathway in mediating cellular responses to extracellular stimuli and the proapoptotic function of activated JNK have been studied extensively (1, 2, 3) . For several cell lineages, various cytokines, cellular stresses, or trophic withdrawal can induce JNK activation with subsequent apoptosis. Indeed, inhibition of the JNK kinase cascade has been proposed as a therapeutic treatment for neuronal apoptosis caused by Alzheimer’s disease or trauma. There are also several lines of evidence that suggest the JNK pathway can play a significant role in cellular transformation and tumor cell growth. JNK activation is required for cellular transformation by certain oncogenes, and inhibition of the JNK pathway can reverse a transformed phenotype (5, 6, 7) . It has also been shown that the JNK pathway is constitutively activated in various tumor cell lines (8, 9, 10) . Thus, it has been proposed that JNK inhibition can also be used as an antitumor agent.
Several studies have demonstrated biological differences among the three JNK genes (11, 12, 13, 14, 15, 16) , e.g., the JNK2 gene may play a more significant role in cancer because antisense RNA against JNK2, but not JNK1, inhibited transformation of lung carcinoma and glioblastoma cells (12 , 16) . JNK2 knockout mice develop chemically induced papillomas at a much lower rate than wild-type or JNK1 knockout mice (13) . Although some isoforms have been shown to have distinct substrate affinities, which suggests a selective role in different biological contexts (4) , no functional significance or distinct role for a specific splice variant has been demonstrated.
We have found previously that 86% of primary glial tumors show activation of the Mr 55,000 but not the Mr 46,000 form of JNK (17) , suggesting that these isoforms play a more critical role in tumors. In an attempt to understand these differences, we initiated studies on the properties of individual isoforms. Here, we report that several JNK isoforms have autophosphorylation activity. Interestingly, all four splice forms of JNK2 show autophosphorylation activity and also enhanced basal kinase activity in the absence of the upstream kinase. Using gene product-specific antibodies combined with two-dimensional electrophoresis, we show that the Mr 55,000 form of JNK2 is expressed predominantly in primary glial tumors as compared with normal brain tissue. Moreover, the predominant phosphorylated form of JNK in tumors is composed of the Mr 55,000 form of JNK2. Collectively, these results suggest that these JNK2 isoforms play a more significant role than other JNK isoforms in the progression of primary glial tumors. Aside from EGFRvIII (18) , JNK2 is the only other kinase that has been shown to be basally active in these tumors and suggests that tumors can up-regulate the JNK pathway simply through the up-regulation of specific isoforms. Moreover, the presence of constitutively active JNK isoforms should be taken into consideration in the design of therapeutics aimed at disabling this pathway.
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MATERIALS AND METHODS |
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Plasmids and Recombinant Protein Purifications.
All JNK isoforms cDNA were amplified by RT-PCR and cloned into pET42 (Novagen, Madison, WI). The JNK11, JNK12, JNK21, and JNK22 isoforms were cloned as a fusion protein with GFP into pEGFP-C1 (Clontech, Palo Alto, CA). Mutations of the T-P-Y motif (T183A and Y185F of JNK22) and cloning of the JNKß isoform were performed using the Quick Change Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA). Sequencing confirmed that each construct matched that found in the GenBank Database. For the JNK2 isoform, the sequence from RT-PCR products was different from that of accession no. L31951 at the 152th nucleotide (G versus A resulting in Ser to Asn) but completely matched that of accession no. U09759. Moreover, we screened genomic DNA from 50 normal subjects, and all sequences matched with that of ours.
GST fusion proteins were purified as described previously (19)
. GST fusion proteins were expressed in Escherichia coli and purified using immobilized glutathione beads (Pierce, Rockford, IL) using manufacturer’s protocols. The upstream MAPKKs, MKK4, and MKK7 and a synthetic constitutively active MAPKK kinase MEKK1 (
MEKK1) were amplified by RT-PCR and cloned into Pet 9a (Novagen). The expressed proteins were purified using the TALON purification kit (Clontech). The quality and quantity of purified recombinant proteins were confirmed by SDS-PAGE and Coomassie staining.
In Vitro Kinase Assay.
For the autophosphorylation assay, 1 µg of GST-JNK isoform protein was incubated in 40 µl of kinase reaction buffer [25 mM HEPES (pH 7.4), 25 mM MgCl2, 2 mM DTT, 0.1 mM NaVO4, and 25 mM ß-glycerophosphate] containing 10 µCi of [
-32p]ATP and 20 µM cold ATP at 30°C for 30 min. For the substrate kinase assay, 300 ng of GST-ATF2 or GST-c-jun were added in the kinase buffer mixture as the substrate. Reactions were terminated with Laemmli sample buffer and boiled for 5 min. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes, and the phosphorylated proteins were detected by autoradiography.
Western Blotting.
Human glial tumor tissues and corresponding normal brain tissues were obtained under Institutional Review Board-approved protocols, and the diagnoses were confirmed by histological analysis as described previously (20)
. Tissue specimens were lysed with PBS/TDS buffer as described (17)
and centrifuged at 14,000 x g to remove insoluble material. Equal amounts of protein were electrophoresed on 4–20% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBS or PBS and then incubated with primary antibody overnight at 4°C. The membranes were washed three times with PBS containing 0.3% Tween 20 or T-TBS and then incubated for 1 h with matched secondary antibodies. The membranes were detected using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ). The scanned films were quantified by NIH Image software.
Immunofluorescence Microscopic Analysis.
Immunofluorescence microscopic analysis was performed as described previously (19
, 21)
. U87MG cells grown on coverslips were transfected with pEGFP-JNK isoform expression vectors using the Effectene reagent (Qiagen, Valencia, CA). After overnight incubation, the growing media were changed to serum-free DMEM and incubated for an additional 24 h. The cells were then fixed in 4% paraformaldehyde for 10 min, followed by 0.2% Triton X-100 for 5 min. For visualization of the nuclei, 4',6-diamidino-2-phenylindole staining was used. After washing with PBS, the coverslips were mounted with antifade reagent and visualized with an immunofluorescence microscope.
Two-dimensional Electrophoresis.
Tissue lysates were prepared with the PlusOne two-dimensional cleanup kit (Amersham Biosciences) to remove materials that interfere with IEF. The prepared proteins were resolubilized with 8 M urea and 1% Triton-X and then mixed with 2 x IEF sample buffer (Invitrogen, Carlsbad, CA). Protein samples (20 µg) were electrophoresed on an IEF gel (pH 3–7) for 2.5 h according to the manufacturer’s instructions, and the gel was then incubated with fixing solution (0.14 M sulfosalicylic acid and 0.7 M trichloroacetic acid). After washing with 20% ethanol, the IEF gel was trimmed into strips, equilibrated with 2 x Laemmli sample buffer, and placed in a 4–20% SDS-PAGE gel. Western blotting was performed according to standard procedure as described above.
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RESULTS |
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MEKK1, showing that we were able to reconstitute the kinase cascade in vitro (Fig. 1a)
. Surprisingly, several JNK isoforms showed kinase activity in the absence of any activating upstream kinase, including JNK21, JNK22, JNK2ß2, and JNK32, although the activity of the latter was very weak. Because it is thought that MAP kinases require phosphorylation to become active, we asked if these enzymes showed any evidence of autophosphorylation activity or were instead unique in possessing endogenous activity without modification. GST-JNK fusion proteins were incubated in kinase buffer in the absence of any other protein. This revealed that all JNK2 isoforms, including JNK2ß1, and JNK32 were indeed capable of autophosphorylation activity (Fig. 1b)
. JNK22 showed the highest autophosphorylation activity, followed by JNK2ß2, JNK21, JNK2ß1, and JNK32. Next, we examined any relative differences in substrate activity. GST-JNK fusion proteins were incubated with either GST-ATF2 or GST-c-jun in kinase buffer. This revealed that JNK22 showed the highest kinase activity toward both substrates and then in order JNK21, JNK2ß2, and JNK2ß1 (Fig. 1c)
. Interestingly, JNK21 showed higher kinase activity than JNK2ß2, although its autophosphorylation activity appeared lower, suggesting that it has a higher specific activity for these substrates. JNK2ß1, which only shows weak activity toward c-jun, shows more substantial phosphorylation of ATF-2. All JNK1 and JNK3 isoforms, including JNK32, which did show autophosphorylation activity, did not show any substrate kinase activity in this assay. Overall, these results demonstrate that the JNK2 gene products, especially JNK22, have a high basal kinase activity that does not require phosphorylation by an upstream kinase.
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2 constructs containing mutations of threonine to alanine and tyrosine to phenylalanine (JNK22APF). This mutant recombinant protein lacking dual phosphorylation sites did not show either autophosphorylation activity or substrate kinase activity (Fig. 2)
. These results indicate that the constitutive active isoform of JNK does indeed autophosphorylate on these sites in the activation loop and that these sites alone are required for this constitutive activation. Furthermore, it appears that autophosphorylation is required for activation.
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1 and JNK12 showed a predominantly cytoplasmic localization, whereas all JNK2 isoforms showed a predominantly nuclear localization (Fig. 3a)
. We also examined subcellular localization of these JNK isoforms under normal culture conditions (with 10% serum) and after transfection into normal human astrocytes, and the results were identical (data not shown). To further test constitutive activation of these isoforms in cells, we examined whether these GFP-tagged JNK1 and JNK2 proteins are phosphorylated using Western blotting with antiphospho-JNK antibody (Fig. 3b)
. All JNK2 isoforms showed evidence of activation, but the two JNK1 isoforms did not show any phosphorylation. Collectively, these results are consistent with the in vitro kinase assays and demonstrate that the JNK2 isoforms are indeed constitutively active in vivo and localize to the nucleus.
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2 and JNK2ß2 isoforms have the highest autophosphorylation activity. In combination with our previous observation that the Mr 55,000 form is preferentially phosphorylated in glial tumors, we speculated that these specific isoforms of JNK are the prevalent forms found in primary tumors. Accordingly, we examined the level of expression of each JNK isoform in glial tumor tissue.
First, we evaluated the isoform specificity of commercially available antibodies using the 10 recombinant proteins for each JNK isoform (Fig. 4a)
. JNK2-specific antibodies were found to be specific for all four JNK2 isoforms with no cross-reactivity toward JNK1 or JNK3. JNK1-specific antibodies were relatively specific for the four JNK1 isoforms but did show some cross-reactivity with the two JNK3 proteins. The anti-JNK3 antibody could recognize all isoforms, but because the JNK2 reactivity was preabsorbed by the manufacturer, its reactivity toward this isoform is relatively lower. However, this anti-JNK3 antibody recognized the JNK1 isoform to the same extent as JNK3.
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/ß1, over the Mr 55,000 isoforms, JNK1/ß1, in 64% of tumor specimens (16 of 25) and all five normal brain tissue specimens, revealing no statistically significant difference (P > 0.1). Fig. 4b
shows 15 representative tumor cases and three normal specimens. On the other hand, JNK2 expression revealed a very different expression pattern between normal brain and glial tumors. The JNK2/ß1 (Mr 46,000) form is predominantly expressed in all five normal brain tissues, whereas the JNK2/ß2 (Mr 55,000) is predominantly expressed in all 25 tumor samples. Statistically, there is a highly significant difference with respect to Mr 46,000 versus Mr 55,000 JNK2 expression between glial tumor tissue and normal brain tissues (P < 0.05). The JNK3 expression pattern varied between normal brain and tumor, where some cases preferentially expressed the Mr 46,000 form (2 of normal brain and 9 of the tumor samples) and others expressed Mr 55,000 forms (3 of normal brain and 16 of the tumor samples).
For these specimens, we also examined the pattern of expression with antiphospho-JNK antibody, which recognizes the active form of JNK (Fig. 4b)
. The Mr 55,000 isoform of JNK isoform appeared to be the major active form present in primary glial tumor tissues, which is in agreement with our previous study (17)
. Interestingly, this expression pattern resembled that seen for JNK2. The Mr 46,000 form was the predominant phosphorylated form found in normal brain tissue, which correlates with previous findings that JNK is active in neurons (9)
. Overall, these results suggest that tumors specifically up-regulate the constitutively active Mr 55,000 form of JNK2.
The JNK2/ß2 Form Is the Primary Phosphorylated Form Found in Glial Tumors.
To directly address which isoform was phosphorylated in glial tumor tissue, we applied two-dimensional electrophoresis to analyze the relative amounts of active JNK isoforms in these specimens. These blots have the advantage of separating according to phosphorylation status and isoform type, as well as requiring less lysate than immunoprecipitation-based methods. The antiphospho-JNK antibody revealed only a Mr 46,000 spot in all three normal brain tissues, much as was found by standard Western blots (Fig. 5a)
. In glial tumors, the phospho-JNK antibody detected a relatively strong Mr 55,000 spot toward the anode side that was only found in tumor samples. A total of five glial tumor samples was examined. This Mr 55,000 spot was observed in all these samples, and its position was highly reproducible. Quantitation showed that this anode Mr 55,000 spot was 98.2% of the total phospho-JNK signal in case 2159, 96.2% in case 1729, 91.8% in case 1043, 77.2% in case 1133, and 40.3% in case 2082 (Fig. 5a)
, indicating that this spot is the major active isoform present. Next, we asked which isoform corresponded to this phospho-JNK spot. Duplicate two-dimensional gels were blotted with antibodies that recognized each JNK gene product (Fig. 5b)
. This revealed that this spot completely coincided with JNK2. On the other hand, all JNK1 and the majority of the JNK3 isoforms migrated near the cathode side, with only a faint spot for JNK3 that migrated in the same position detected by the phospho-JNK, which is most likely because of cross-reactivity of this antibody with JNK2. These results demonstrate that the Mr 55,000 form of JNK2, JNK2/ß2, is the primary phosphorylated form found in primary glial tumors.
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DISCUSSION |
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Several lines of evidence indicate that the JNK pathway plays a role in cellular transformation and tumor cell growth. JNK activation is required for cellular transformation by certain oncogenes, such as Met, human T-cell lymphotrophic virus, type I, and Bcr-Abl (5, 6, 7) . The inhibition of the JNK pathway can reverse a transformed phenotype (5 , 8) , and it has also been shown that JNK is constitutively active in a variety of tumor cell lines (8 , 9) . Human brain tumors frequently show increased expression of the normal EGF receptor and the mutant EGF receptor, EGFRVIII, but not significantly elevated ERK activity (26) . Instead, the activated form of JNK is highly prevalent in brain tumors, and JNK is constitutively activated by EGFR vIII (10 , 17) . Therefore, understanding JNK signal transduction will provide new insights into tumor formation and therapy.
In this study, we have shown that several of the JNK isoforms, particularly the JNK2 isoforms, have autophosphorylation activity and constitutive substrate kinase activity in the absence of upstream kinase. There is the possibility that these observations may result from some contaminant in the prokaryotic expression system used for some assays. However, this possibility is unlikely for several reasons. First, each isoform shows intrinsic differences with respect to autophosphorylation and substrate kinase activity, whereas all JNK1 isoforms showed no such activity. Next, GFP-tagged JNK2 proteins expressed in mammalian cells are constitutively localized to the nucleus, whereas JNK1 isoforms are not, reflecting endogenous activation of the JNK protein. Finally, JNK2 isoforms expressed in cells also showed constitutive phosphorylation. Collectively, this is the first demonstration that specific JNK isoforms, especially the Mr 55,000 isoforms of JNK2, have constitutive activation in the absence of the upstream kinase. These results are supported by a recent study where insect cells were transfected with several JNK isoforms. Although no direct comparison among the isoforms was made, the basal activity of recombinant JNK22 appeared to be 
10 times higher than JNK11 or JNK32 (27)
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Interestingly, previous work among the JNK genes has demonstrated a specific role for JNK2 in tumorigenesis. It has been reported that specifically disabling JNK2 isoforms using antisense reagents, but not JNK1 isoforms, impairs the EGF-stimulated growth of A549 cells (8) . Moreover, inhibition of JNK2 enhances apoptosis and impairs cell cycle progression in glial tumor cell lines (16) . The induction of papillomas by 12-O-tetradecanoylphorbol-13-acetate is greatly impaired in JNK2-/- mice but not in JNK1-/- mice (13) . We speculate that the JNK2 isoforms are favored for tumorigenesis because of their constitutive activation properties. However, specific differences among isoforms, especially between the Mr 46,000 and the Mr 55,000 isoforms, have not been reported.
We have found that the Mr 55,000 JNK2 isoforms have the highest activity in vitro, and this may also have an important correlation with expression in tumors. We have shown previously that the Mr 55,000 form, but not the Mr 46,000 form, of JNK is activated in primary brain tumors (17)
, suggesting that these specific isoforms of JNK play a critical role in these tumors. Therefore, we examined the expression level of each JNK isoform and found that although the JNK2/ß1 (Mr 46,000) form is predominantly expressed in all five normal brain tissue samples examined, the JNK2/ß2 (Mr 55,000) is predominantly expressed in all 25 tumor samples examined. Our in vitro kinase assay results show that the Mr 55,000 isoforms have greater autophosphorylation, and this provides a tantalizing explanation for the prevalence of the JNK2/ß2 form in primary tumors. Intriguingly, a recent study has suggested that the distribution of active forms of JNK may result from differential expression of JNK isoforms within cells and tissues as opposed to a selective activation of isoforms (28)
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It is worth noting that normal brain tissue showed detectable expression of the Mr 55,000 forms of JNK2 but did not show significant phosphorylation using antiphospho-JNK antibody. Recent studies have demonstrated the involvement of phosphatases in the down-regulation of the JNK signaling pathway (29 , 30) . We speculate that the mechanisms for inactivating JNK signaling may function more efficiently in normal tissues and/or are disrupted in tumor tissues.
The identification of constitutively active isoforms of JNK has several implications for the study of normal physiological processes and the design of therapeutics. Because up-regulation of certain isoforms can activate the JNK pathway, our results show that JNK isoform expression must be taken into account when studying JNK activation in tissues or cells. As certain isoforms are constitutively active, any decreased activation of these particular isoforms in vivo suggests that the role of phosphatases must also be considered. Certain JNK pathway inhibitors are currently in clinical trials, such as CEP-1347, for the treatment of neurodegenerative disorders, including Alzheimer’s or Parkinson’s disease (31 , 32) . Because these drugs principally target upstream kinases in the JNK cascade, our results suggest that these drugs may not be of benefit if constitutively active JNK isoforms are expressed. In general, the greatest therapeutic benefit would result from the inhibition of JNKs directly.
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FOOTNOTES |
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1 Supported by NIH Grants CA69495 and CA96539. 
2 H. T. and M. T. contributed equally to this work.
3 To whom requests for reprints should be addressed, at Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, 233 South 10th Street, 1002 BLSB, Philadelphia, PA 19107. Phone: (215) 503-4650; Fax: (215) 923-0567; E-mail: albert.wong{at}mail.tju.edu
4 The abbreviations used are: JNK, c-Jun NH2-terminal kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GST, glutathione S-transferase; MAP, mitogen-activated protein; GFP, green fluorescence protein; RT-PCR, reverse transcription-PCR; TBS, Tris-buffered saline; ATF, activating transcription factor; IEF, isoelectric focusing; ERK, extracellular signal-regulated kinase; MEKK, MKK, MAPKK, mitogen-activated protein kinase kinase.
Received 8/16/02. Accepted 10/31/02.
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REFERENCES |
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