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J. Biol. Chem., Vol. 276, Issue 29, 27152-27158, July 20, 2001

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Induction of -Transducin Repeat-containing Protein by JNK Signaling and Its Role in the Activation of NF-B^*

Vladimir S. Spiegelman, Pete Stavropoulos^§, Esther Latres^¶, Michele Pagano^¶, Ze'ev Ronai^§, Tomas J. Slaga, and Serge Y. Fuchs^**

From the  AMC Cancer Research Center, Lakewood, Colorado 80214, ^§ Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029, the ^¶ Department of Pathology, New York University, New York, New York 10016, and the  Department of Animal Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication, January 2, 2001, and in revised form, May 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Activation of Jun N-kinase (JNK) and NF-B transcription factor are the hallmarks of cellular response to stress. Phosphorylation of NF-B inhibitor (IB) by respective stress-inducible kinases (IKK) is a key event in NF-B activation. -TrCP F-box protein mediates ubiquitination of phosphorylated IB via recruitment of SCF^-TrCP-Roc1 E3 ubiquitin ligase complex. Subsequent proteasome-dependent degradation of IB results in activation of the NF-B pathway. We found that a variety of cellular stress stimuli induce an increase in the steady state levels of -TrCP mRNA and protein levels in human cells. Activation of stress-activated protein kinases JNK (and, to a lesser extent, p38) by forced expression of constitutively active mutants of JNKK2 and MKK6 (but not MEK1 or IKK) also leads to accumulation of -TrCP. Transcription of the -TrCP gene is not required for JNK-mediated induction of -TrCP. A synergistic effect of stimulation of IKK and JNK on the transcriptional activity of NF-B was observed. The mechanisms of -TrCP induction via stress and its role in NF-B activation are discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The survival of living organisms depends on the promptness and efficiency of their response to adverse changes in the environment. Eukaryotic cellular responses to stress involve immediate post-transcriptional events within the maze of signal transduction pathways (1). Activation of these pathways often leads to further changes in gene expression and de novo synthesis of proteins that exert a protective effect on the cell. Whereas tremendous progress has been achieved in identification and characterization of key regulatory elements of separate signaling pathways, the complexity of coordination between simultaneously induced and transduced signals and the role of these interactions in the outcome of signaling are largely underestimated and yet to be understood.

Activation of the stress-activated protein kinases (c-Jun N-terminal kinases (JNK)¹ and p38 kinases) and IB kinases (IKK) followed by AP1/ATF- and NF-B-dependent transcription orchestrates the rapid cellular response to a wide spectrum of exogenous and endogenous stress-induced agents. These pleiotropic mediators of stress-induced gene expression play a critical role in cell growth and differentiation, apoptosis, and adaptive responses to changes in cellular redox balance (reviewed in Refs. 2 and 3).

The JNK and p38 pathways are preferentially activated by a diverse array of cellular stresses including UV light, x-rays, hydrogen peroxide (H₂O₂), heat and osmotic shock, and withdrawal of growth factors. JNK are activated via upstream phosphorylation by two distinct JNKKs, JNKK1/MKK4/SEK1 (4, 5) and JNKK2/MKK7 (6, 7). The p38 subgroup is phosphorylated by MKK3, MKK4 (4), and MKK6 (8, 9). Eleven different kinases capable of phosphorylating JNK and p38 kinases (including MEKK1-4, ASK1 and -2, TAK1, and others) have been identified as upstream activators of the JNK/p38 pathway (reviewed in Ref. 2). The precise mechanisms of their activation by stress are not yet comprehended in full. The outcome of JNK/p38 signaling is stress-inducible phosphorylation-dependent activation of their substrate proteins, which include transcription factors (c-Jun, ATF2, Elk1, etc.), apoptosis regulators (Bcl2, Bid, etc.), and other effectors. Modulation of gene expression due to an increase in transactivation potential and protein stability of proteins phosphorylated by JNK/p38 are among the mechanisms of JNK/p38-mediated effects. Recent evidence for the role of JNK/p38 kinases in stabilization of the short lived mRNAs of cytokines and growth factors (10-14) represents yet another mode of the regulation by stress-activated protein kinases, although the effector proteins, which mediate mRNA stabilization, are largely unknown.

NF-B is a dimeric transcription factor composed of members of the Rel family. A large number of stress stimuli including UV irradiation, ionizing radiation, viral infection, and reactive oxygen species can activate NF-B and its target genes. These target genes include those involved in the immune response, inflammatory response, cell adhesion, cell growth, and apoptosis. The activity of NF-B is tightly regulated at the level of its localization by a family of inhibitory proteins, IBs, that sequester NF-B in the cytoplasm of unstimulated cells. Ubiquitin-dependent degradation of IBs in response to stress results in the nuclear translocation and transcriptional activity of NF-B (reviewed in Ref. 15).

Most of the stress stimuli, which induce NF-B, trigger a cascade of events resulting in the activation of IB kinases (IKK). IKK is a large (>700-kDa) multicomponent enzyme complex containing two closely related kinase subunits with identical structural domains, IKK (IKK1) and IKK (IKK2), and two accessory proteins, IKK and IKAP (reviewed in Ref. 3). Two members of the MAP kinase family, NF-B-inducing kinase (NIK) and MEKK1, have been shown to directly interact with IKK and activate the kinase subunits in experiments based on overexpression of the recombinant proteins. The requirements of NIK and MEKK1 for activation of IKK by the relevant physiological stimuli have not been proven, and the upstream kinases in this signaling pathway remain to be identified (reviewed in Ref. 3). As an outcome of their activation, IKK mediate phosphorylation of IB on two critical serine residues (e.g. serines 32 and 36 in IB, serines 19 and 23 in IB, and serines 18 and 22 in IB).

This phosphorylation serves as a recognition signal for seven WD40 repeats located at the carboxyl-terminal domains of -TrCP (16) and HOS/Fbw1b proteins (17). Binding of -TrCP/Fbw1a (or its close relative HOS/Fbw1b) proteins to IB mediates IB ubiquitination (and subsequent 26S proteasome-dependent degradation) through recruitment of the SCF^-TrCP-Roc1 E3 ubiquitin ligase (reviewed in Refs. 18-20). These proteins belong to a large family of ubiquitin ligase receptors containing the 42-48-amino acid F-box motif, which is required for binding to the protein Skp1 (21-23). Skp1 recruits -TrCP/Fbw1a with Cdc53/cullin 1 and Roc1/Rbx1, thereby allowing the SCF E3 ubiquitin ligase to tether ubiquitin-conjugating enzymes (reviewed in Ref. 18).

Both phosphorylation of IB and -TrCP binding are required for IB ubiquitination and degradation. Inhibition of -TrCP/HOS function by expression of a dominant negative mutant abrogates the degradation of IB phosphorylated by the activated IKK (17, 24-30). The endogenous levels of -TrCP are low. These levels can be up-regulated in some cells by the activation of the Wnt/-catenin/Tcf signaling pathway, resulting in the activation of NF-B-dependent transcription (31). Interestingly, although the functional Tcf was apparently required for up-regulation of -TrCP, we have failed to completely inhibit the effect of Wnt (but not -catenin) on -TrCP levels by co-expression of the dominant negative Tcf4. Moreover, it was consistently observed that expression of Wnt was more efficient in the increase in -TrCP levels and NF-B transcriptional activity as compared with expression of the stable -catenin mutant (31). Since it has been shown that Wnt exerts some of its effects in a -catenin-independent manner via the JNK pathway (32), we investigated whether the activation of JNK signaling can actually regulate the abundance and activity of -TrCP.

In this study, we first demonstrate that cellular stress and induction of stress-activated protein kinases increase the levels and activities of -TrCP via stabilization of -TrCP mRNA. These data provide a mechanism for the positive cooperation between activated JNK and IKK in regulating the NF-B transcriptional activity in response to cellular stress.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Plasmids-- The constitutively active IKKS177E,S181E (IKK^SE), 2x-B-luciferase and glutathione S-transferase-IB expression vectors (33, 34), constitutively active JNKK2 (JNKK^CAA) and its inactive counterpart (JNKK^AA), and constitutively active MKK6 (MKK6^D/D; Ref. 10) were a generous gift of M. Karin. Constitutively active MEKK1 (MEKK1) and dominant negative SEK1/MKK4 (SEK^AL) were kindly provided by A. Minden and J. Woodgett. The expression vector for constitutively active MEK1 (MEK^EL) was a kind gift of S. Aaronson. -TrCP expression vector containing the 3'-untranslated region (3'-UTR) was a gift of R. Benarous (16).

Antibodies-- Monoclonal antibodies against FLAG tag (M2; Sigma), MEKK1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and SEK1 (New England Biolabs) were purchased. Polyclonal antibodies against -TrCP were raised by Zymed Laboratories Inc. against synthetic peptide encompassing amino acid residues NSSEREDCNNGEPPRKIIPEKNSLRQTY of -TrCP and purified via a peptide affinity column.

Northern Blot and Nuclear Run-on Analyses-- Twenty µg of total RNA isolated from freshly harvested cells using TRI-reagent (Molecular Research Center, Cincinnati, OH) was subjected to Northern blotting analysis using random primer-labeled probes for human -TrCP. The membranes were stripped and rehybridized with a probe to 7S RNA to verify that equal amounts of RNA were loaded and transferred. A nuclear run-on assay was performed as previously described (31).

Transfections, Immunoblotting, and Reporter Assays-- HeLa epithelial cells and 293T human embryo kidney cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (Life Technologies, Inc.). Mouse NIH-3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and antibiotics (Life Technologies, Inc.). Transfections were performed by the calcium precipitate method or lipofection (LipofectAMINE Plus; Life Technologies, Inc.). The overall amount of DNA in transfection mixtures was kept constant by the addition of pCDNA3. Preparation of cell lysates and immunoblotting were performed as described elsewhere (17). Briefly, for detection of endogenous -TrCP, 1 mg of protein lysates were immunoprecipitated with -TrCP antibody (1 µg), separated on 8% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and probed with -TrCP antibody at a 1:500 dilution. Membranes were developed with the ECL kit (Amersham Pharmacia Biotech) and exposed to film.

Loading of cell lysates on gels was normalized for protein concentration. The luciferase assay was performed using a kit (Promega), and the data were normalized per transfection efficiency.

Solid Phase Protein Kinase Activity Assays-- JNK and IKK activity in whole cell extracts (10 µg) was assessed in the solid phase kinase assay with bacterially expressed glutathione S-transferase-c-Jun (amino acids 5-89) or glutathione S-transferase-IB (amino acids 1-54) in the presence of [-³²P]ATP (50 cpm/fmol) as described elsewhere (37).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cellular Stress-mediated Activation of JNK Up-regulates -TrCP mRNA and Protein Levels-- We have tested the effects of JNK on -TrCP levels by forced expression of N terminus truncated MEKK1 (MEKK1), which is known as a very potent upstream activator of JNK pathway. As is evident from Fig. 1A, transfection of 293T human embryo kidney cells with the MEKK1 construct led to a robust increase in the steady state levels of -TrCP mRNA, whereas the expression of the -TrCP close relative HOS (17) was hardly affected.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Activation of the JNK pathway elevates the steady state levels of -TrCP mRNA. The representative results of three independent experiments are shown. A, Northern blot analysis of RNA extracted from 293T cells 48 h after transfection with pCDNA3 or MEKK1 plasmids (2 µg). B, Northern blot analysis of RNA extracted from 293T cells 48 h after transfection with the indicated constructs plasmids (5 µg each). The film is overexposed compared with A in order to show a moderate effect of MKK6D/D.

In the overexpression experiments, MEKK1 was reported to activate JNK as well as IKK (38) and MEK (39). To investigate which of those pathways contribute to induction of -TrCP, we expressed the constructs encoding constitutively active IKK (IKK^SE (15)), activator of JNK (JNKK^CAA (10)), activator of p38 (MKK6^D/D (10)), and activator of mitogen-activated protein kinase (MEK1^S218E,S222L, MEK^EL (40)). We found that transfection of 293T cells with JNKK^CAA but not with IKK^SE or MEK^EL elevated the steady state levels of -TrCP. A moderate increase in -TrCP levels was observed after expression of MKK6^D/D (Fig. 1B). These data suggest that activation of JNK (and, to some extent, the p38 kinase pathway) results in the induction of -TrCP.

JNK and p38 belong to a subfamily of stress-activated protein kinases, and their activities are drastically up-regulated in response to a variety of stress stimuli including oxidative and osmotic stress. Thus, we next tested whether cellular stress affects the expression of -TrCP. An oxidative stress caused by treatment of 293T cells with H₂O₂ resulted in an approximately 4-fold increase in the steady state levels of -TrCP mRNA (Fig. 2A). This increase was observed within 30 min and lasted up to 2 h. Similar extent and kinetics of the up-regulation of -TrCP mRNA levels were achieved by incubation of cells in the presence of tumor necrosis factor  (TNF; 20 ng/ml).

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Accumulation of -TrCP mRNA in response to stress. 293T cells were treated with H2O2 (100 µM; A) or UV (40 J/m2; B) and harvested at the indicated time points after the treatment. Total RNA was extracted and analyzed by Northern blot with the -TrCP and 7S probes. The representative results of three independent experiments are shown.

Irradiated 293T cells with UV led to even more robust (up to 8-10-fold) and prolonged (up to 8 h) elevation of the steady state levels of -TrCP mRNA (Fig. 2B). Comparable results were obtained in the cells treated with sorbitol (0.6 M) to induce a hyperosmotic shock.² Altogether, these data provide the evidence that cellular stress and induction of stress-activated protein kinases up-regulate the levels of -TrCP mRNA. The induction of two major -TrCP mRNA species did not seem to be coordinate; the accumulation of a longer transcript was more pronounced in the stressed cells (Fig. 2, A and B). This observation may reflect an alteration of -TrCP mRNA processing in response to stress, although the exact nature of this phenomenon remains to be elucidated.

In order to follow up the stress-inducible changes in the -TrCP protein expression, we have developed a rabbit polyclonal antibody (see "Materials and Methods") as a probe for endogenous -TrCP. This antibody readily recognized recombinant -TrCP protein, whose expression was driven by cytomegalovirus-based vector in 293T human kidney cells or by baculovirus expression system in insect cells (Fig. 3A). The antibody specifically interacted with -TrCP but not with another F-box protein Fbx7 (Fig. 3B).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Characterization of the polyclonal antibody against -TrCP and the effects of JNK activation and stress on the levels of endogenous -TrCP. A, the lysates from 293T cells transfected with pCDNA3 or -TrCP expression vector as well as the recombinant -TrCP produced by baculovirus infection in insect cells (TrCPrec., right lane) were separated on 8% SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and analyzed by immunoblotting with polyclonal antibody against -TrCP. B, the lysates from 293T cells transfected with the FLAG-tagged Fbx7 or -TrCP expression vector were separated on 8% SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and analyzed by immunoblotting with monoclonal antibody against FLAG tag (upper panel) or polyclonal antibody against -TrCP (lower panel). C, 293T cells were transfected with MEKK1 (2 µg) and SEKAL (8 µg) as indicated. Forty-eight hours later, the cells were harvested, and the cell lysates were prepared and analyzed for the levels of -TrCP by immunoprecipitation followed by immunoblotting (panel I) and expression of MEKK1 (panel II) and SEKAL (panel III) by direct immunoblotting as well as for activities of IKK (panel IV) and JNK (panel V) as described under "Materials and Methods." A representative result of three independent experiments is shown. D, 293T cells were treated with UV (40 J/m2), H2O2 (100 µM), or TNF (20 ng/ml; R&D Systems). One hour after treatment, the cells were harvested, and the cell lysates were prepared and analyzed for the levels of -TrCP by immunoprecipitation and immunoblotting. A representative result of two independent experiments is shown.

Forced expression of MEKK1 in 293T cells resulted in a robust activation of JNK (Fig. 3C, panel V), modest (if any) activation of IKK (Fig. 3C, panel IV), and noticeable accumulation of endogenous -TrCP protein (Fig. 3C, panel I). All of those effects of MEKK1 were largely inhibited by co-expression of a dominant negative JNKK/SEK construct (SEK^AL (41)). The control immunoblot analysis demonstrated that the effects of SEK^AL could not be explained by changes in the truncated MEKK1 expression (Fig. 3C, panel II). Treatment of cells with physiological JNK inducers UV, H₂O₂, and TNF also increased the steady state levels of -TrCP (Fig. 3D). These data indicate that cellular stress and stress-mediated activation of JNK pathway leads to an increase in the levels of -TrCP protein.

mRNA Stabilization as a Mechanism of JNK- and Cellular Stress-mediated Up-regulation of -TrCP-- The Wnt pathway was shown to elevate the -TrCP levels in a manner that is not dependent on -TrCP gene transcription. To investigate whether -TrCP transcription is required for induction of -TrCP by the JNK pathway, we have carried out a run-on assay with nuclei isolated from 293T cells transfected with constitutively active MEKK1 or pCDNA3 plasmids. Expression of MEKK1 led to a robust increase in transcription of c-jun, a well known downstream target of the JNK pathway (2, 3). We did not detect any corresponding increase in the rate of -TrCP transcription (Fig. 4A). These results suggest that activation of -TrCP gene promotor is not required for up-regulation of -TrCP by stress and JNK activation.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Induction of -TrCP by JNK does not require transcription of the -TrCP gene. A, nuclear run-on analysis of -TrCP, 7S, and c-jun transcription in the nuclei isolated from 293T cells 48 h after transfection with pCDNA3 or MEKK1 vectors (10 µg each). The membranes were exposed for 3 h for c-jun or overnight for -TrCP and 7S. A representative of two independent experiments is shown. B, Northern blot analysis of RNA isolated from 293T cells transfected with 5 µg of either active JNKKCAA (3-h autoradiography exposure) or inactive JNKKAA (48-h exposure) and treated with actinomycin D (10 µg/ml) for the indicated time points. A representative of two independent experiments is shown.

Treatment of 293T cells (transfected with inactive JNKK^AA) with actinomycin D to inhibit transcription allowed us to observe degradation of endogenous -TrCP mRNA within 2-4 h (Fig. 4B). In contrast to that, little -TrCP mRNA degradation was seen in the cells expressing constitutively active JNKK^CAA (Fig. 4B). These data indicate that activation of JNK may lead to stabilization of -TrCP mRNA.

To further analyze the mechanisms underlying an increase in -TrCP levels by cellular stress and JNK activation, we utilized the expression of exogenous human cytomegalovirus-driven -TrCP construct containing an intact 3'-UTR in mouse NIH-3T3 cells. These cells did not express any amounts of endogenous -TrCP, which were detectable by Northern blot analysis with either human (Fig. 5A) or mouse (data not shown) probe. Treatment of NIH-3T3 cells, which express exogenous -TrCP, with H₂O₂ led to accumulation of -TrCP mRNA (Fig. 5A). Co-expression of constitutively active JNKK^CAA (but not of inactive JNKK^AA) also increased the steady state levels of exogenous -TrCP (Fig. 5B). Before finally concluding on a posttranscriptional mechanism of -TrCP induction, the effects of stress still needed to be tested in the absence of the cytomegalovirus-driven transcription. To this end, we have inhibited transcription by treating the transfected NIH 3T3 cells with actinomycin D and assessed the rate of degradation of the exogenously expressed -TrCP mRNA. This mRNA was found rather unstable in the nonstressed cells (with the estimated half-life of ~40 min). However, incubation of cells in the presence of both actinomycin D and H₂O₂ prevented the degradation of -TrCP-3'-UTR mRNA (Fig. 5C). Altogether, these findings strongly indicate that the cellular stress-mediated increase in -TrCP levels is realized through stabilization of -TrCP mRNA.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Stabilization of -TrCP mRNA by JNK activation and stress in NIH-3T3 cells. The representative results of two independent experiments are shown. A, Northern blot analysis of RNA extracted from NIH-3T3 cells 48 h after transfection with pCDNA3 or -TrCP-3'-UTR (4 µg) and 1 h after treatment with H2O2 (100 µM) as indicated. B, Northern blot analysis of RNA extracted from NIH-3T3 cells 30 h after co-transfection with pCDNA3, -TrCP-3'-UTR (2 µg), and either active JNKKCAA or inactive JNKKAA (2 µg each). C, NIH-3T3 cells were transfected with -TrCP-3'-UTR. Thirty hours later, the cells were treated with actinomycin D (ActD; 10 µg/ml; lanes 3-10). At the time point 0 (15 min after the addition of actinomycin D), H2O2 was added to the culture medium as indicated (100 µM). Cells were harvested at the indicated time points, and RNA was extracted and analyzed by Northern blot.

Activation of JNK Up-regulates NF-B Transcriptional Activities-- Elevation of the endogenous levels of -TrCP may result in an increase in the efficiency of IB ubiquitination and degradation, leading to the activation of NF-B-dependent transcription (31). We have indeed observed a cooperative effect of constitutively active IKK and JNKK on acceleration of IB degradation measured by pulse chase in 293T cells.² It has been previously shown that MEKK1 is capable of activating the NF-B driven reporters under overexpression conditions (38, 42). To examine the possible effect of JNK-mediated accumulation of -TrCP on NF-B activities, we chose the conditions under which JNK is activated by expression of its immediately upstream and most specific activator JNKK. In order to exclude any effects on IKK activities, we saturated IB phosphorylation by forced expression of constitutively active IKK^SE. Expression of this construct in 293T cells readily elevated the activity of NF-B-driven luciferase reporter. Remarkably, co-transfection of JNKK^CAA resulted in further increase in NF-B transcriptional activity in a dose-dependent manner (Fig. 6). Similar results were obtained in HeLa cells. We did not detect any effects of JNKK^CAA on the activity of IKK^SE measured in an immunokinase assay with recombinant bacterially expressed glutathione S-transferase-IB as a substrate (data not shown). These data suggest that JNK cooperates with IKK in induction of NF-B transcriptional activity and that JNK-mediated up-regulation of -TrCP may serve as a mechanism of this cooperation.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Activation of JNK contributes to NF-B transcriptional activities. 293T cells were transfected with 2x-B-luciferase (400 ng) and pRSV--gal (100 ng) together with the indicated (in ng) amount of IKKSE and JNKKCAA. Thirty hours later, the luciferase activity was measured. Normalized arbitrary unit values of two independent experiments (each in triplicate) are shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Activation of NF-B by stress and proinflammatory cytokines requires that NF-B be released from its cytoplasmic retention by IB. Ubiquitination and subsequent proteasome-dependent degradation of IB requires both phosphorylation of IB by IKK and recognition of phosphorylated IB by -TrCP/Fbw1a (27, 29, 30) or its relative protein HOS/Fbw1b (17, 43). Several lines of evidence suggest that the cellular levels of Fbw1 proteins are critical for IB ubiquitination. First, -TrCP basal expression is very low, and -TrCP protein is hardly detected by a direct immunoblotting analysis (Ref. 31 and this study). Second, the ubiquitination and degradation of IB is readily inhibited in living cells by squelching -TrCP with the phosphopeptides mimicking the phosphorylated IB (44) or by expression of F-box-deficient dominant negative -TrCP mutants (24, 26, 27-30). Third, an increase in -TrCP levels by Wnt signaling promotes the transcriptional activation of NF-B in response to an increase in IB phosphorylation by constitutively active IKK (31).

In this study, we have demonstrated that cellular stress and activation of JNK pathway results in accumulation of -TrCP via stabilization of -TrCP mRNA. The elevated levels of -TrCP together with activation of IKK and subsequent IB phosphorylation contributes to a rapid IB degradation and NF-B nuclear translocation in response to stress (Fig. 7). Therefore, the findings reported here identify a mechanism underlying the cooperation between stress-induced activation of IKK and JNK pathways in the activation of NF-B-dependent transcription. Moreover, activation of JNK by Dsh (32) may play a role in the induction of -TrCP by the Wnt pathway (31) in addition to -catenin/Tcf-dependent mechanisms (Fig. 7).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   A model for cooperation between stress-induced JNK and IKK in the activation of NF-B. Both phosphorylation of IB by IKK and recognition of phosphorylated IB by the elevated levels of -TrCP contribute to IB degradation and NF-B activation. Activation of JNK by Dsh may also contribute to induction of -TrCP by the Wnt pathway.

Simultaneous activation of JNK and NF-B by stress and proinflammatory cytokines has been long reported and often attributed to the common components. Although several common signaling effectors (e.g. TRAF2 (45, 46), MEKK1 (38), and CIKS (47)) are known to signal to both JNK/p38 and IKK activation, little information is available about the cooperation of those signaling pathways. Activation of p38 kinase by salicylates was reported to inhibit IB degradation and NF-B activation via suppression of IKK activities (48, 49). We observed only a very modest accumulation of -TrCP mRNA in response to the p38 pathway activation (Fig. 1B). It has been recently found that DNA binding and transcriptional activation of NF-B induced by thioredoxin requires activation of JNK and can be inhibited by overexpression of the dominant negative JNKK/JNK constructs (50). These data are in agreement with our findings that JNK activation promotes NF-B transcriptional activities. Coordinated effects of IKK and JNK activation on IB phosphorylation and the recognition of phosphorylated IB by SCF^-TrCP-Roc1 E3 ubiquitin protein ligase provide a good explanation for efficient activation of NF-B by a broad spectrum of cellular stress stimuli.

Our findings also suggest that some of the important cellular functions of JNK may be attributed to its regulation of NF-B transcriptional output. For example, there is a positive correlation between JNK and NF-B activation in the regulation of apoptosis. In T lymphocytes, activation of both signaling pathways results in induction of apoptosis (51, 52). In some other cell types, both JNK and NF-B are shown to hold antiapoptotic function. For example, X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of both stress-activated protein kinase/JNK (53) and NF-B (54-56) pathways in hepatocytes. Expression of JNK2 is required to suppress apoptosis in human tumor cells MCF-7 and HCT116 (57). Activation of NF-B was also shown to prevent apoptosis in these cells (58, 59). Both JNK and NF-B prevent apoptosis induced by c-Myc overexpression in fibroblasts (60, 61).

However, the suggested -TrCP-dependent mechanism of cooperation between JNK and NF-B pathways may not necessarily exist in all types of cells. Some cells (e.g. NIH-3T3) do not express detectable levels of endogenous -TrCP (Fig. 5), and, therefore, they are not capable of modulation of NF-B activity by JNK through induction of -TrCP. Thus, one should not expect a universal concordance in JNK and NF-B cellular functions including the regulation of apoptosis.

Cellular stress and JNK activation elevate the steady state levels of -TrCP in a transcription-independent manner. The finding that JNK up-regulates the levels of -TrCP via stabilization of its mRNA is not entirely surprising. Activation of the JNK pathway has been implicated in the stabilization of many short-lived mRNAs including interleukin-2 (10), interleukin-3 (12), vascular endothelial growth factor (14), and others. The effectors of stress, which mediate such stabilization are largely unknown. Stability of many short lived mRNAs, which contain AU-rich elements, is often controlled by HuR, the ubiquitously expressed member of the ELAV protein family (reviewed in Ref. 13). Analysis of the -TrCP mRNA sequence revealed three AU-rich elements, two of which are located in the 3'-UTR. Translocation of the nuclear HuR protein to the cytoplasm is suggested to constitute one of the mechanisms of HuR-mediated stabilization of p21^CIP/WAF mRNA in response to stress (36). In addition to three AU-rich elements, -TrCP mRNA contains an ACUACUGCCCAGTTTCC sequence in its 3'-UTR. This sequence closely resembles a distal part of the JNK response element, which was found in the 5'-UTR of interleukin-2 mRNA and mediated its stabilization by MEKK1 (11). YB-1 and nucleolin proteins were shown to bind to the JNK response element (11). Future studies will determine the role of these and other factors as well as AU-rich and JNK-response elements in the regulation of -TrCP mRNA stability.

Our data do not allow us to exclude the possibility that additional mechanisms of stress-inducible accumulation of -TrCP protein (e.g. protein stabilization) may exist. The exact modes of -TrCP induction by JNK are yet to be identified and future studies are required to further delineate the mechanisms of -TrCP-mediated cooperation between JNK and NF-B activation.

	ACKNOWLEDGEMENTS

We thank Drs. M. Karin, R. Benarous, S. Aaronson, A. Minden, and J. Woodgett for the generous gifts of reagents. We thank Drs. V. Fried and H. Furneaux for critical suggestions and Dr. K. Spiegelman for help with the manuscript preparation.

	FOOTNOTES

^* This study was supported in part by National Institutes of Health Grants CA 92900 (to S. Y. F.) and CA 76262 (to T. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Animal Biology, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce St., Rm. 161E VET, Philadelphia, PA 19104-6046. Tel.: 215-573-6949; Fax: 215-573-5188; E-mail: sfuks@vet.upenn.edu.

Published, JBC Papers in Press, May 24, 2001, DOI 10.1074/jbc.M100031200

² V. Spiegelman and S. Fuchs, unpublished data.

	ABBREVIATIONS

The abbreviations used are: JNK, c-Jun N-terminal kinase(s); MEKK, mitogen-activated protein kinase kinase kinase; NF-B, nuclear factor B; IB, inhibitor of NF-B; IKK, IB kinase(s); MKK/MEK, mitogen-activated protein kinase kinase; JNKK/SEK, JNK kinase; -TrCP, -transducin repeat-containing protein; HOS, homologue of Slimb; SCF, Skp1-cullin 1-F-box protein complex; Fbw, F-box/WD40 domain protein; TNF, tumor necrosis factor ; 3'-UTR, 3'-untranslated region; E3, ubiquitin-protein isopeptide ligase.

	REFERENCES

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