Novel Phosphorylation-dependent Ubiquitination of Tristetraprolin by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase Kinase 1 (MEKK1) and Tumor Necrosis Factor Receptor-associated Factor 2 (TRAF2)^*

Plasmids and Cloning

Expression vectors for FLAG-TTP, -ZnN, and -ZnC were generated using the Expand High Fidelity^PLUS PCR system (Roche Applied Science). PCR fragments were subcloned into pcDNA3.1-FLAG. For other plasmids, general vector information, and primer sequences, see supplemental Tables S1 and S2.

Generation and Propagation of TTP Adenovirus

For TTP adenovirus generation, the human TTP coding sequence was amplified from pCMV-MycTTP using the primers BglIIMycTTP fwd/rev. The PCR fragment was subcloned into the BamHI site of pShuttleTripTetlac-BamHI. To produce the recombinant AdEasyTetOFF-MycTTP plasmid, pShuttleTripTetlac-MycTTP was linearized (PmeI) and cotransformed with pAdEasyTetOFFrev into BJ5183 bacterial cells (43). The resulting AdEasyTetOFF-MycTTP was, after quality control, linearized with PacI, transfected into HEK 293 lac cells, and grown in the presence of 4 nm doxycycline until plaque formation. A high titer adenoviral stock was obtained as described previously (44).

Reporter Gene Assay

Cells were transfected with the indicated reporter and/or expression plasmids using 4 μg of total DNA/6-well. The experiments were performed in triplicate, and luciferase values were normalized to cotransfected β-galactosidase, illustrated as mean fold induction. The error bars represent standard deviations of the mean.

Cell Culture and Transfections

HEK 293 cells and HeLa cells were obtained from ATCC; WT, TTP KO, and MEKK1 ΔKD MEF kindly provided by P. J. Blackshear and Ying Xia (45), respectively. The cells were cultured in DMEM (Bio-Whittaker) supplemented with 10% FCS (Sigma), 2 mm l-glutamine (Sigma), penicillin (100units/ml), and streptomycin (100 μg/ml). HUVEC were isolated from umbilical cords as described (46) and maintained in M199 medium (Lonza) supplemented with 20% FCS (Sigma), 2 mm l-glutamine (Sigma), penicillin (100 units/ml), streptomycin (100 μg/ml), 5 units/ml heparin, and 25 μg/ml ECGS (Promocell). HEK 293 cells were transfected using calcium phosphate (47), MEF using polyethylenimine (48). For TTP adenoviral transduction, the cells were infected with AdTTP adenovirus (multiplicity of infection = 250) for 4–6 h and analyzed 24 h after infection. For lentiviral, shRNA-mediated MEKK1 knockdown in HUVEC, 1 × 10⁵ cells were seeded and transduced with 3 × 10⁵ transducing units of MISSION lentiviral transduction particles (Sigma-Aldrich, Clone ID TRCN0000006162). 48 h post-transduction cells were induced and analyzed as described. Recombinant human (R & D systems) and mouse TNFα (BIOSOURCE) were used at a final concentration of 10 ng/ml, the proteasome inhibitor MG132 (Affinity) and the MAPK inhibitors SB203580, SP600125, and PD98059 (Tocris Bioscience) at 10 μm. The IKK2 inhibitor BMS-345541 (Sigma-Aldrich) and the deubiquitinase inhibitor N-ethylmaleimide (NEM; Sigma-Aldrich) were applied at 25 and 10 μm, respectively.

Cell Extracts, Western Blotting, and Densitometry

For cell lysates of reporter assays, calf intestine alkaline phosphatase treatment (Roche Applied Science; 20 units, 15 min, 37 °C), as well as analysis of molecular TTP patterning, cells were lysed in 1× passive lysis buffer (Promega). For detection of endogenous (p)-JNK levels in MEF, TTP in HUVEC, and virus-infected cells, the pellets were resuspended in 2× Laemmli buffer. The proteins were separated by either 10% or 5–12% gradient SDS-PAGE, transferred to nitrocellulose membranes (Hybond-C, Amersham Biosciences), and blocked in 5% nonfat dry milk in PBS and 0.1% Tween 20 (TBS used for phosphoantibodies) for 1 h at room temperature before incubation with the first antibody overnight (4 °C). Antibodies were used as indicated within the figures (see also supplemental Table S3). Densitometric analysis of MEKK1 knockdown and p-JNK1/2 levels were done using the background-corrected integrated densities of either MEKK1 or the p-JNK bands normalized to the respective loading controls (ImageJ, National Institutes of Health).

Coimmunoprecipitations

HEK 293 cells were transfected with expression plasmids containing the tagged genes of interest. 24 h later, the cells were resuspended in 1× lysis buffer containing 300 mm NaCl, 20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 0.5% Triton X-100, 0.3% Nonidet P-40, as well as protease inhibitors (Complete Mixture; Roche Applied Science). The lysates were incubated overnight with respective antibodies prebound to protein A/G-agarose (sc-2003; Santa Cruz) according to the manufacturer's instructions. For analysis of endogenous TTP-MEKK1 interaction, HUVEC were lysed (50 mm Hepes, pH 8, 150 mm NaCl, 5 mm EDTA, 0,5% Nonidet P-40, 1% Triton, 5% glycerol, protease/phosphatase inhibitors), and TTP was immunoprecipitated using protein A/G-agarose for 2,5 h (4 °C). The beads were washed three times with lysis buffer, resuspended in 2× Laemmli buffer, and finally subjected to electrophoresis on SDS-polyacrylamide gels.

In Vivo Ubiquitination Assays

HEK 293 cells were transfected with a total amount of 4 μg of DNA/6-well. 24 h later, the cells were harvested, and either ubiquitinated proteins were precipitated using nickel-nitrilotriacetic acid-agarose (Qiagen), according to the Tansey lab protocol, or tagged TTP was immunoprecipitated using FLAG matrix (anti-FLAG M2-agarose; Sigma), and ubiquitin chains were detected by specific antibodies (supplemental Table S3). Lysates were loaded on 5–12% gradient SDS-polyacrylamide gels. For ubiquitination analysis of endogenous TTP, the cells were resuspended in lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 10% glycerol, 1% Triton; protease/phosphatase inhibitors, 1 mm NEM) containing 6 m urea, RNase A (3 μg/μl; Qiagen), Benzonase® (1 units/μl; Merck), and 1.5 mm MgCl₂ (denaturing lysis buffer) for 15 min at 4 °C. The lysates were diluted with lysis buffer 1/25 and precleared (1 h, protein A/G-agarose) prior to immunoprecipitation with TTP antibody prebound to protein A/G-agarose for 2 h at 4 °C. Precipitates were washed three times with lysis buffer, resuspended in denaturing buffer again, and finally diluted with Laemmli buffer prior to loading on 7.5% SDS-polyacrylamide gels.

Data Analysis

Statistical differences between samples were analyzed using the paired Student's t test. Two-tailed probability values of <0.05, <0.01, and <0.001 were considered significant and highly significant, respectively. The p values are given within the figure legends.

MEKK1 Constitutes a Novel TTP Kinase

As we have shown previously, TTP impairs NF-κB activity in an AU-rich element-mediated decay-independent manner (42). The MAPK p38α induces TTP phosphorylation, traceable by a “molecular weight shift” of the protein in Western blots, inactivating TTP mRNA binding and degrading ability, but in contrast, p38α did not block TTP function toward NF-κB (supplemental Fig. S1A, left panel). When testing a set of other kinases, we found that coexpression of MEKK1, which has not been described as TTP kinase yet, could even counteract TTP inhibition of p65-induced NF-κB promoter activity (Fig. 1A, left panel, and supplemental Fig. S1A, right panel). As shown in Fig. 1B, the kinase domain of MEKK1 appeared sufficient for the rescue, whereas a kinase-deficient MEKK1 mutant led to the complete reconstitution of TTP-mediated NF-κB inhibition. Mutation of the PHD domain slightly attenuated TTP activity. Of note, p38α even counteracted MEKK1 function toward TTP (Fig. 1A and supplemental Fig. S1A), and we noticed a substantial difference in TTP protein “patterning” on Western blots after coexpression of either p38α or MEKK1. Whereas p38α expression resulted in the molecular weight shift of TTP already described earlier (49, 50), coexpression of MEKK1 led to an increase of “higher molecular weight species” (50–72 kDa, termed HMW) of TTP, accompanied by a different patterning (Fig. 1A, right panel). In line with that, the kinase-dead MEKK1 mutant inhibited the accumulation of this HMW-TTP (Fig. 1B, right panel). Importantly, we observed a similar patterning of endogenous TTP after TNFα stimulation in human umbilical vein endothelial cells (HUVEC): “lower molecular weight” species (∼36–50 kDa, termed LMW), for example as produced by p38α-mediated phosphorylation appeared early upon TNFα treatment, whereas HMW-TTP occurred at later time points (Fig. 1C). Pharmacological inhibition of p38α, JNK, or ERK activity did not block the MEKK1-mediated TTP protein shift (supplemental Fig. S1B), suggesting that MEKK1 phosphorylates TTP at different or additional sites.

Consequently, we analyzed the physical interaction of MEKK1 with TTP. Immunoprecipitation of HA-tagged MEKK1 led to coprecipitation of HMW-TTP as shown in Fig. 2A. Interestingly, and in line with our earlier observations, p38α coprecipitated with LMW-TTP (Fig. 2B). On the endogenous level, an interaction of TTP with full-length MEKK1 was barely detectable. Instead, a strong interaction with a ∼95-kDa fragment was detected 90 min after TNFα stimulation, concomitantly with the appearance of HMW-TTP in the input control (Fig. 2C). In this context it is important to note that cleavage of MEKK1 occurs during the induction of apoptosis, resulting in the release of a 91-kDa activated kinase domain from insoluble structures to the soluble cytoplasmic fraction (51, 52). It is therefore conceivable that TTP predominantly interacts with and becomes phosphorylated by the cytosolic kinase fragment of MEKK1. To further confirm that the observed HMW species are the result of MEKK1-mediated phosphorylation, we utilized phosphatase treatment, resulting in a complete reversal of the molecular shift and reappearance of TTP comparable with the control sample (Fig. 2D). To confine the localization of the MEKK1-mediated TTP phosphorylation, we used TTP truncation constructs (scheme in Fig. 2D). The molecular weight of a ZnC-TTP-mutant was not influenced by MEKK1 coexpression, whereas the ZnN-TTP mutant had similar “shifting” characteristics as the full-length protein (Fig. 2D, middle and bottom panels, respectively). The differences in band strength and distribution can be attributed to the varying composition of the gradient polyacrylamide gels. Together, our data suggest that MEKK1 physically interacts with TTP to phosphorylate its N terminus, thereby abrogating TTP function toward NF-κB.

MEKK1-mediated Phosphorylation Serves as Basis for TRAF2-directed, Lys-63-linked TTP Ubiquitination

MEKK1 is a key player of the TNFR1-associated cytoplasmic signaling complex and acts in concert with TRAF2 to trigger activation of the JNK signaling cascade (53). Thereby, TRAF2-mediated ubiquitination processes provide the basis for stabilization and functionality of the JNK-activating complex (54). Using in vivo ubiquitin assays, we investigated whether TTP was also part of these ubiquitination events. We observed that not only TTP, but also its mRNA binding-deficient mutant (C124R), as well as the ZnN-TTP mutant were ubiquitinated when phosphorylated by MEKK1, whereas the ZnC-TTP mutant was not (Fig. 3A). Moreover, TTP ubiquitination was confirmed endogenously in MEF and HUVEC (Fig. 3B). In line with the observed MEKK1-TTP interaction, TTP polyubiquitination was detected only after TNFα stimulation in the presence of the deubiquitinase inhibitor NEM.

Because the MEKK1 mutant M1 Kin exhibits no E3 ligase activity, we investigated whether the MEKK1 interacting adaptor protein TRAF2 could function as E3 ligase in this context. TRAF2 coexpression indeed resulted in a strongly enhanced TTP ubiquitination that was independent of proteasome inhibition by MG132 (Fig. 3C). This suggested that TRAF2 attached ubiquitin chains were Lys-63-linked, and moreover, phosphorylation by MEKK1 was important for effective ubiquitination. These observations were further supported by the use of dominant negative MEKK1, as well as TRAF2 mutants: mutation of either MEKK1 or TRAF2 impaired whereas mutation of both completely abolished TTP ubiquitination (Fig. 3D). The expression of dominant negative MEKK1 could not be detected in the input when dominant negative TRAF2 was coexpressed (Fig. 3D, bottom panels, M1dnKin/T2dn), explained by the fact that MEKK1 stabilization depends on an intact kinase domain as well as an intact TRAF2 E3 ligase domain (3, 6, 53). The use of ubiquitin mutants additionally validated the TRAF2-mediated, Lys-63-linked TTP ubiquitination (Fig. 4, A and B). Over and above, we also found Lys-63-linked ubiquitin chains attached to endogenous TTP after TNFα stimulation in HUVEC. In line with the above results, MG132 treatment had no effect, whereas the addition of NEM strongly enhanced Lys-63-linked TTP ubiquitination (Fig. 4C). The latter can be attributed to the zinc finger domain of TTP, because required ubiquitin target lysine residues occur only in this region. Further, the loss of MEKK1 function, achieved either by lentiviral, shRNA-mediated MEKK1 knockdown in HUVEC (Fig. 4D, left panel) or by the use of MEF deficient in the MEKK1 kinase domain (Fig. 4D, right panel) confirmed that endogenous TTP ubiquitination depended on phosphorylation by MEKK1. Importantly, the E3 ligase activity of MEKK1 was negligible in terms of TTP ubiquitination, proven by the respective MEKK1 mutant (data not shown), as well as the use of ΔKD MEF (Fig. 4D, right panel). Our results indicate that TTP is covalently modified by ubiquitin in vivo (although we cannot formally exclude that other proteins bound to TTP complexes are not dissociated by 6 m urea) and that attached polyubiquitin chains are predominantly Lys-63-linked. In summary, MEKK1 mediated, N-terminal TTP phosphorylation appears to be prerequisite for TRAF2-mediated, Lys-63-linked TTP polyubiquitination.

TTP Promotes JNK and AP-1 Activity and Affects Cell Viability

The molecular cross-talk between NF-κB and JNK signaling cascades is the key to account for the timed regulation of biological processes as cell growth, differentiation, and apoptosis. TNFR1-induced prolonged JNK activation results not only from blocking NF-κB activity but depends on TRAF2-MEKK1 complex formation leading to JNK and subsequent AP-1 activation (8, 53, 55). When we analyzed TTP KO MEF for TNFα-induced JNK activation, we observed that the lack of TTP caused an impairment of p-JNK levels under stimulated conditions, comparable with the situation observed in MEKK1 ΔKD MEF, whereas total JNK levels were not affected (Fig. 5A). When analyzing TTP expression in WT MEF (Fig. 5A, middle panel), we noticed that forced, prolonged JNK activation (achieved through specific IKK2 inhibition by BMS; Fig. 5A, 90⁺–180⁺) goes in line with the appearance of HMW forms of TTP (see Fig. 7, top blot). In contrast, TTP was generally less abundant in ΔKD MEF, and although HMW-TTP was somewhat detectable, which can also be attributed to phosphorylation by other kinases, it appeared visibly different. Notably, mainly JNK2 activation was impaired but restored after reconstitution of TTP expression in KO MEF (Fig. 5B). Accordingly, AP-1 activity was also affected by TTP: whereas TTP increased AP-1 promoter activity in a dose-dependent manner in HEK 293 cells (Fig. 5C, top panel), AP-1 activity was diminished in KO MEF and could be restored to WT levels after TTP reconstitution (Fig. 5C, bottom panel). This is in line with our previous findings where TTP down-regulated NF-κB promoter activity dose-dependently in HEK 293 cells, whereas MEF lacking TTP displayed a much higher basal NF-κB promoter activity (42).

In light of the results above, we analyzed the potential of TTP to influence cell growth and/or apoptosis. We generated a recombinant adenovirus for expression of Myc-tagged TTP under the control of a tetracycline responsive promoter (Tet-OFF-system). This allowed the controlled expression of TTP in terms of time and expression levels. Via titration experiments, we assured that viral infection does not affect cell viability (data not shown). Under the established conditions, we tested the impact of TTP on cell viability: HeLa cells ectopically expressing TTP (Fig. 6A, verified by Western blotting) proliferated significantly slower as compared with non-TTP-expressing control cells over a time period of 72 h. Because doxycycline itself affected the cells between 48 and 72 h of treatment (data not shown) only effects within the first 48 h were further studied (Fig. 6A, bottom graphs). Uninfected and control adenovirus-infected cells without or treated with doxycycline exhibited a doubling in cell number (100%) during the first 24 h. The cells that ectopically expressed TTP reached a cell number of ∼60% compared with the above mentioned controls after 24 h (graph 1). Remarkably, in one experiment, where we erroneously added a lower doxycycline concentration to control AdTTP-infected cells, we observed a decline in cell number (graph 2, 48 h, #), which could be correlated to the respective TTP protein levels assessed by Western blotting (#). Obviously, upcoming TTP expression, attributed to insufficient doxycycline treatment, resulted in an immediate convergency of total cell numbers from control infected and TTP-expressing cells. Besides, the coexpression of TTP or its N-terminal mutant ZnN with their modifiers MEKK1 (kinase domain), TRAF2, ubiquitin, or Lys-63-linked ubiquitin led to an alteration of cell viability in HEK 293 cells (Fig. 6, B and C). In agreement with the results in Fig. 3D, cells that coexpressed dominant negative versions of either MEKK1 or TRAF2 remained unaffected (Fig. 6B).

Together, our data provide evidence that TTP is hypermodified by the combined action of MEKK1 and the E3 ligase TRAF2 (Fig. 6D). Thus we hypothesize that the ability of TTP to affect cell viability is strongly dependent on an accurately timed, post-translational control of the protein through p38α on the one hand and MEKK1 on the other hand. The expression of TTP in combination with appropriately directed hypermodification events differentially regulate its involvement in the cross-talk between NF-κB and JNK signaling. Therefore, we propose that TTP acts as “balancer” in the coordinated control of survival- versus death-promoting signaling cascades (Fig. 7).