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 NH 2 -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) (2-4). 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 (5-8). 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. , FLAG-MEK-1 (16), , and LHRE-TK-luc (18) were described earlier. V12 Ras was a gift of J. Downward. pAP-1 luc, pNFB luc, pFR luc, pFA-CHOP, pFA2-Elk-1, pMEKK-1 pMEK-1, and pMEK-3 were part of the PathDetect system (Stratagene). Quantitative real time-PCR was performed to characterize the expression profile of human tribbles genes by using the Human Rapid-Scan panel (Origene). MRNA levels are expressed as relative units normalized for glyceraldehyde-3-phosphate dehydrogenase expression. MATERIALS AND METHODS PlasmidsCell 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 ...

Migration and proliferation of smooth muscle cells are key to a number of physiological and pathological processes, including wound healing and the narrowing of the vessel wall. Previous work has shown links between inflammatory stimuli and vascular smooth muscle cell proliferation and migration through mitogenactivated protein kinase (MAPK) activation, although the molecular mechanisms of this process are poorly understood. Here we report that tribbles-1, a recently described modulator of MAPK activation, controls vascular smooth muscle cell proliferation and chemotaxis via the Jun kinase pathway. Our findings demonstrate that this regulation takes place via direct interactions between tribbles-1 and MKK4/SEK1, a Jun activator kinase. The activity of this kinase is dependent on tribbles-1 levels, whereas the activation and the expression of MKK4/SEK1 are not. In addition, tribbles-1 expression is elevated in human atherosclerotic arteries when compared with non-atherosclerotic controls, suggesting that this protein may play a role in disease in vivo. In summary, the data presented here suggest an important regulatory role for trb-1 in vascular smooth muscle cell biology.

Hu protein R (HuR) binds to the AU-rich element (ARE) in the 3'UTR to stabilize TNF-α mRNA. Here, we identified chemical inhibitors of the interaction between HuR and the ARE of TNF-α mRNA using RNA electrophoretic mobility gel shift assay (EMSA) and filter binding assay. Of 179 chemicals screened, we identified three with a half-maximal inhibitory concentration (IC 50 ) below 10 μM. The IC 50 of quercetin, b-40, and b-41 were 1.4, 0.38, and 6.21 μM, respectively, for binding of HuR protein to TNF-α mRNA. Quercetin and b-40 did not inhibit binding of tristetraprolin to the ARE of TNF-α mRNA. When LPS-treated RAW264.7 cells were treated with quercetin and b-40, we observed decreased stability of TNF-α mRNA and decreased levels of secreted TNF-α. From these results, we could find inhibitors for the TNF-α mRNA stability, which might be used advantageously for both the study for post-transcriptional regulation and the discovery of new anti-inflammation drugs.

No abstract