Human Tribbles, a Protein Family Controlling Mitogen-activated Protein Kinase Cascades*

Control of mitogen-activated protein kinase (MAPK) cascades is central to regulation of many cellular responses. We describe here human tribbles homologues (Htrbs) that control MAPK activity. MAPK kinases interact with Trbs and regulate their steady state levels. Further, Trbs selectively regulate the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 MAPK with different relative levels of activity for the three classes of MAPK observed depending on the level of Trb expression. These results suggest that Trbs control both the extent and the specificity of MAPK kinase activation of MAPK.

Mitogen-activated protein kinase (MAPK) 1 cascades control the activity of three sets of effector protein kinases (extracellular signal-regulated protein kinases (ERKs), Jun kinases (JNKs), and p38s). The central element in each MAPK pathway is a module of three protein kinases, MAPKK kinase, MAPKK, and MAPK (1). The three sets of effector MAPK differ in type of activating stimulus: JNKs and p38/HOG-1 primarily respond to stress (e.g. heat shock), and ERKs primarily respond to mitogens. However, a stimulus can activate more than one class of MAPK; the contribution of each pathway is cell typedependent, and MAPK pathways can both synergize and antagonize. This is caused in part by regulatory proteins influencing signaling by a range of mechanisms including scaffolding (e.g. JIP-1, STE5), regulating localization (e.g. Ksr), or recruitment to targets (e.g. 14-3-3 proteins) (2)(3)(4). Here we describe a novel family of MAPK control proteins, homologues of fly tribbles.
Drosophila tribbles was shown to regulate String activity and hence mitosis during ventral furrow formation (5)(6)(7)(8). A canine Trb-2-like protein has been described in the literature as a transiently expressed, mitogen induced, and highly labile cytoplasmic phosphoprotein, but its biological function was not characterized (9,10). Rat Trb was shown to be rapidly upregulated during neuronal cell apoptosis (11). Recently Trb-3 has been reported to regulate Akt activation in liver by insulin (12) and regulate ATF4 activity (13,14). We show here that Trbs bind to MAPKK and regulate MAPK activation suggesting that Trb function may be broader than reported previously.
Cell Cultures and Transfections-HeLa (ECACC, 85060701) and NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and penicillin-streptomycin. Raw cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and penicillin-streptomycin. Cells (1.5 ϫ 10 4 per well) were seeded into 96-well tissue culture plates 24 h prior to transfection. Transfections were performed using SuperFect (Qiagen) according to the manufacturer's advice; each well received 500 ng of inducible reporter construct (pIL-8 luc, pAP-1 luc, pNFB luc, or pLHRE-TK luc), 100 ng of pTK-RLuc (Promega) for normalization of transfection efficiency, and 50 ng of htrb-1 or htrb-3 expression vectors under investigation unless stated otherwise in the appropriate figure legend. 500 ng of pFR luc, 100 ng of pTK-RLuc, and 10 ng of pFA-CHOP or pFA2-Elk-1, 25 ng pMEK-1, or pMEK-3 plasmids were transfected to specifically activate p38 or ERK and to study the effect of htrb-3 on the activation. Sufficient pCDNA3.1 (Invitrogen) ("empty vector") was added to keep the total DNA dose constant at 700 ng/well. 2 h after transfection, cells were washed, and 100 l of fresh medium was added. Triplicate wells were transfected for each treatment. Stimulations were performed for 4 h (unless indicated otherwise) 24 h later. 2 nM IL-1␤ or 10 ng/ml tumor necrosis factor ␣, 0.5 g/ml human growth hormone, 50 ng/ml PMA, or 10 nM of the other cytokines listed on Supplementary Table I was used  (unless stated otherwise on the figure). Agonists were prepared and added as 10ϫ stocks in 11 l of phosphate-buffered saline. Reporter levels were measured following 4 h of stimulation using the dualluciferase system (Promega) as recommended by the manufacturer.
Cytokines-IL-␤ was a kind gift from the Immunex Corporation. The other human cytokine preparations were kindly provided by Dr clonal antibodies were purchased from Sigma. Protein concentrations of cell lysates were determined, and an equal amount of total protein was loaded in each lane. Kinase assays were performed by using the appropriate kits from New England Biolabs.

RESULTS
Human tribbles Identification and Sequence-Using a transcription expression screen for gene products mediating inflammatory cytokine signaling (19 -21), we identified a human tribbles homologue (htrb-1) which regulated the human IL-8 promoter in HeLa cells. The library clone encoded a portion of the 3Ј-untranslated region extending to the poly(A) tail (the library was oligo(dT)-primed) transcribed in the expression vector (pCDM8) in the sense orientation. PCR experiments using coding region primers to detect endogenous trb-1 message but not the truncated noncoding libraryderived transcript showed that transfection of the library clone into HeLa cells caused a Ͼ10-fold increase in the level of endogenous trb-1 message, which correlated with inhibition of IL-8 promoter activity. 2 Two other human tribbles homologues, htrb-2 and htrb-3 (41 and 51% identical to Htrb-1, respectively), were identified by data base searching; sequences have been submitted to GenBank TM (accession numbers AF250310 and AF250311).
A family comprised of three trbs is found in mammals, and orthologs have been identified in both vertebrates and invertebrates. 3 All three Trb proteins share a central Trb domain. In addition, each has N-terminal (70 -100 residues) and C-terminal (ϳ25 residues) domains, which are neither closely related to any other sequences nor closely related to each other. The Trb domain is homologous to protein serine-threonine kinases but lacks the active site lysine and is predicted to be kinase dead as shown for canine Trb-2 and Tribbles (5,9).
Quantitative real time-PCR experiments showed that trb-1 mRNA was expressed in most human tissues with the highest levels in skeletal muscle, thyroid gland, pancreas, peripheral blood leukocytes, and bone marrow ( Supplementary Fig. 1). trb-2 levels were highest in peripheral blood leukocytes, and trb-3 levels were highest in pancreas peripheral blood leukocytes and bone marrow; in addition HeLa cells were found to express trbs 1-3. (Supplementary Fig. 1).
Trbs Regulate AP-1-When overexpressed, Htrb-1 repressed basal activity of the NFB and AP-1-regulated IL-8 promoter in HeLa cells in culture but not its activation by IL-1 and tumor necrosis factor ␣ (Fig. 1A). The NFB sites mediate cytokine activation, whereas the AP-1 sites control basal activity (22). Our results therefore suggest that Trb-1 acts selectively on pathways leading to AP-1. Consistent with this, testing specific NFB or AP-1 reporters activated by MEKK-1 co-transfection showed that Htrb-1 inhibited AP-1 activity but not NFB (Fig.  1, B and C) nor a signal transducers and activators of transcription-responsive LHRE promoter (Fig. 1D). Specificity was also analyzed by testing a panel of human cytokines in the presence or absence of Htrb-3; AP-1 activation was inhibited (Fig. 1H), whereas NFB activation was not (Supplementary  Table I). Thus, whether activated by overexpression of upstream components or by physiological agonists, pathways leading to AP-1 activation were specifically inhibited by Trb overexpression.
We have shown above that up-regulation of endogenous Trb-1 and ectopic overexpression of Trb-1 and Trb-3 inhibited AP-1 activity in HeLa cells. Next we examined the effects of suppressing endogenous Trb expression. An antisense construct encoding the htrb-3 5Ј-untranslated region and N-terminal domain was co-transfected with AP-1 or NFB reporters using either MEKK-1 or NFB inducing kinase as activators (Fig. 1, E-F). Trb-3 mRNA expression detected by quantitative real time-PCR using primers spanning the central Trb domain was reduced by Ͼ70% (Fig. 1G), whereas Trb-1 levels were only slightly altered by transfecting a high dose of AS-Trb-3 con- struct, suggesting a Trb-3-specific action. As observed with overexpression, AP-1 but not NF-B activity was inhibited (Fig.  1, E and F).
Trbs Regulate ERKs-Because Trbs are mitogen-induced and regulate String/cdc25, we examined the effect of Trb levels on the control of the Ras/MEK/ERK module. V12Ras-driven AP-1 activation was blocked by overexpression of both Trb-1 and Trb-3 ( Fig. 2A). Also, HeLa cells were stimulated with PMA, and phospho-ERK levels were determined by Western blotting; basal levels were increased by Htrb-3, and both the extent and rate of phosphorylation were enhanced by Htrb-1 or Htrb-3 (Fig. 2B), correlating with enhanced ERK activity measured in an in vitro kinase assay (Fig. 2D). Although expression levels of Trb-1 and Trb-3 were comparable (see Fig. 4), ERK activation was enhanced only using low doses of Trb-3 (Fig.  2C), and ERK potentiation was enhanced in the presence of all doses of Trb-1 within the range tested (Fig. 2C), suggesting that the system is more sensitive toward Trb-3 levels. Comparison between constitutive (transfection, Fig. 2A) and transient (PMA treatment, Fig. 2, B-D) stimuli showed that Trbs are capable of either up-or down-regulating MAPK activity.
MAPKK Interacts with Trbs-Overexpression of htrb-1 inhibited MEKK-1 mediated AP-1 but not NFB activity (Fig. 1, B and C); a similar effect was seen when an AP-1 reporter was stimulated by PMA (Fig. 3A). To characterize the possible site of Htrb action, we tested whether increasing the dose of PMA (Fig. 3A) or MEKK-1 (Fig. 3B) could override the effect of Htrb-3. This was not the case, suggesting that htrb-3 controls a rate-limiting step downstream of MEKK-1. MEKK-1 phosphorylates MKK7 and/or MKK4, and activation of these kinases leads (via JNKs) to phosphorylation of transcription factors including c-Jun and CREB2. Western blotting showed that both the kinetics and the extent (as judged by gel mobility) of PMA-induced jun phosphorylation were altered by Trb-3 (Fig.  3C). Thus the locus of action of trbs in the JNK pathway is at or downstream of MAPKKs and at or upstream of c-jun. p38 MAPK was not activated by PMA in this system. However, overexpression of Trb proteins inhibited basal p38 activity as detected in an in vitro kinase assay (Fig. 3D).
These data suggest that Trbs act at the level of MAPKK/ MAPK and hence control MAPK phosphorylation/activity. To identify the molecular target(s) of Trbs, we used transfection and co-immunoprecipitation experiments with Myc-tagged Trbs and FLAG-tagged MAPKs, MAPKKs, and MAPKK kinases. We found that MEK-1 interacts with both Trb-1 and Trb-3, MKK7 specifically interacts with Trb-3, and MKK4 specifically interacts with Trb-1 (Fig. 4B); however, no interactions were detected between Trb and MEKK-1, MLK-3, ERK-2, JNK-1, or p38 (data not shown). These findings were confirmed in yeast 2 hybrid assays; interactions were detected between both Trbs and MEK-1, between Trb-3 and MKK7, but not between Trb and JNK (data not shown). These data are consistent with the results of the functional assays (Figs. 2 and 3), suggesting that trbs control MAPK activation by binding to MAPKKs (Fig. 5C).
Dose responses for all Trb/MAPKK combinations (Fig. 4A) showed that Htrb-3 levels were increased by MKK7 co-transfection (Fig. 4A, fourth row) and MEK-1 (second row) but not MKK4 (third row), which does not bind to Trb-3. Htrb-1 levels were enhanced by MKK4 (fifth row) but not MKK7 (sixth row), which does not bind to trb-1; thus Trb-MAPKK interactions stabilize Trbs. In contrast, no effect was seen on MAPKK levels, as illustrated by Fig. 4A (first row). These findings are consistent with the functional, immunoprecipitation, and yeast 2 hybrid data, and suggest that Trb protein levels may be regulated by MAPKK.
Trbs Regulate the Relative Activation of MAPK-Because Trbs interact with more than one MAPKK and appear to have differential effects on ERK, JNK, and p38 (Figs. 2B and 3, C and D) activation, we examined whether Trbs differentially regulate MAPK activation. Because Htrb-3 interacts with both MEK-1 and MKK7, and MKK7 activates JNK in HeLa cells, we examined Htrb-3 using MAPK pathway-specific reporter assays to distinguish between ERK, JNK, and p38. At low doses, Htrb-3 enhanced JNK and ERK activation (with a more pronounced effect on ERKs) but inhibited p38 (Fig. 5A). At higher doses the activation of all three pathways was inhibited relative to control. Thus there appears to be a different optimal set point for each pathway (Fig. 5A), suggesting that Htrbs may regulate relative activation of the three classes of MAPK. DISCUSSION We show that Trbs appear necessary for MAP kinase pathway function and are inhibitory at high levels. Several mechanisms can account for these findings. For example, Trbs might be scaffolds, both over-and underexpression of which has been shown to inhibit MAPK signaling (23)(24)(25)(26).
Our data show that MAPKKs not only bind Trbs but also appear to stabilize them. Further, Trb proteins have been reported to be rapidly mitogen induced and to have short halflives (9, 10), and we have found that Trb mRNAs are also rapidly induced by mitogens and have short half-lives. 3 Taken together, these data suggest that Trb/MAPK interactions are dynamic and regulated by external signals. In addition, MAPK cascades have been suggested to incorporate a positive feedback loop leading to nonlinear behavior (24). Modeling using ordinary differential equations shows that with these elements in place, Trb proteins could act either as activators or as inhibitors of MAPK activity, depending on the ratio of Trb to MAPKK levels in the cell. 4 Similar observations have been made by us and others for the NFB (27,28) and the cell cycle control systems (29). We also note that these systems are capable of producing much more complex behaviors than the simple nonmonotonic dose responses we report here, for example the oscillations observed in cells from IB knock-out mice and reproduced in simple mathematical models (28).
Although the inhibition at higher levels might be argued to be an artifact of Trb overexpression, Trb-3 has been shown to 4 S. K. Dower and E. Kiss-Toth, unpublished data. be an Akt inhibitor, UAS-driven Trb overexpression causes G 2 mitotic arrest in imaginal disk cells, and the original trb clone we detected in our screen was active by virtue of its capacity to modulate endogenous Trb expression. Fly tribbles regulates cell cycle progression, blocking String action by promoting its degradation. Thus trb(Ϫ) flies show defective gastrulation because of elevated zygotic String activity driving premature mitosis (5,7,8). However, these experiments did not identify a molecular mechanism of tribbles action. A large body of literature shows MAPK pathways to be regulators of String/cdc25 (30 -35). We speculate that tribbles may also regulate MAPKK activity.
There are apparent discrepancies between our results and those reported for Drosophila tribbles. First we observed no effect on cell cycle progression in HeLa cells caused by either overexpression or repression (with antisense) of either Trb-1 or Trb-3. 3 This may be because HeLa cells express all three Trbs, and the two tested here are partially functionally redundant. We also found that both overexpression and repression of expression of Trbs inhibited AP-1 activity and that whereas Trb-3 overexpression inhibited the activity of all three MAPK cascades relative to control, at intermediate levels a modest enhancement of JNK and ERK activity was observed. However, overexpression of String and/or tribbles in Drosophila appears to have opposite effects on mitosis in cystocytes during oogenesis and in the ventral region during embryogenesis, an effect suggested to be caused by changes in the duration of G 2 resulting from perturbations in the relative levels of String and Trb (8). Further, it has been reported that tribbles action in Drosophila is cell type-specific (7). We speculate that this may well apply for the mammalian homologues as well. In line with this model, we have found that MEKK1-mediated AP-1 activation is blocked by various tribbles in a cell type-dependent manner (Fig. 5B).
A recent report shows that Trb-3 interacts with Akt and that elevated Trb levels block Akt activation in the liver (12). Our current understanding of Akt signaling cascades places this protein kinase at the level of MAPKKs in the "signaling hierarchy" (Fig. 5C), suggesting that the observations of Du et al. (12) are consistent with a broader picture of Trb function as regulators of intermediate steps in these protein kinase cascades. In conclusion, the data in this report suggest that mammalian Trbs bind to and regulate MAPKK and may thus control the relative activation of MAPK by incoming signals.