Conserved signaling pathways that activate the mitogen-activated protein kinases (MAPKs) are involved in relaying extracellular stimulations to intracellular responses. The MAPKs coordinately regulate cell proliferation, differentiation, motility, and survival, which are functions also known to be mediated by members of a growing family of MAPK-activated protein kinases (MKs; formerly known as MAPKAP kinases). The MKs are related serine/threonine kinases that respond to mitogenic and stress stimuli through proline-directed phosphorylation and activation of the kinase domain by extracellular signal-regulated kinases 1 and 2 and p38 MAPKs. There are currently 11 vertebrate MKs in five subfamilies based on primary sequence homology: the ribosomal S6 kinases, the mitogen- and stress-activated kinases, the MAPK-interacting kinases, MAPK-activated protein kinases 2 and 3, and MK5. In the last 5 years, several MK substrates have been identified, which has helped tremendously to identify the biological role of the members of this family. Together with data from the study of MK-knockout mice, the identities of the MK substrates indicate that they play important roles in diverse biological processes, including mRNA translation, cell proliferation and survival, and the nuclear genomic response to mitogens and cellular stresses. In this article, we review the existing data on the MKs and discuss their physiological functions based on recent discoveries

SUMMARY The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries.

We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.

The mammalian translational initiation machinery is a tightly controlled system that is composed of eukaryotic initiation factors, and which controls the recruitment of ribosomes to mediate cap-dependent translation. Accordingly, the mTORC1 complex functionally controls this cap-dependent translation machinery through the phosphorylation of its downstream substrates 4E-BPs and S6Ks. It is generally accepted that rapamycin, a specific inhibitor of mTORC1, is a potent translational repressor. Here we report the unexpected discovery that rapamycin's ability to regulate cap-dependent translation varies significantly among cell types. We show that this effect is mechanistically caused by rapamycin's differential effect on 4E-BP1 versus S6Ks. While rapamycin potently inhibits S6K activity throughout the duration of treatment, 4E-BP1 recovers in phosphorylation within 6 h despite initial inhibition (1-3 h). This reemerged 4E-BP1 phosphorylation is rapamycinresistant but still requires mTOR, Raptor, and mTORC1's activity. Therefore, these results explain how cap-dependent translation can be maintained in the presence of rapamycin. In addition, we have also defined the condition by which rapamycin can control cap-dependent translation in various cell types. Finally, we show that mTOR catalytic inhibitors are effective inhibitors of the rapamycin-resistant phenotype.cap-dependent translation ͉ mTORC1 ͉ rapamycin resistance T he mammalian translational initiation machinery governs the recruitment of ribosomes to mRNA to commence the production of protein synthesis. This machinery consists of various eukaryotic initiation factors (eIFs) that tightly regulate protein synthesis based on environmental cues. Importantly, initiation is an important step for cellular control because it is the ratelimiting step of translation (1).Two predominant pathways translate mammalian mRNA through cap-dependent and independent mechanisms. The capping of the 5Ј end of mRNA by m 7 GTP allows the recruitment of the eIF4F complex, eIF3, and the 40S ribosomal subunit to the 5Ј mRNA cap. Capindependent translation is mediated by an internal RNA structure called internal ribosome entry site (IRES), which recruits the ribosome independent of both the cap and the entire eIF4F complex (2).The initiation of cap-dependent translation is tightly regulated by extracellular conditions including glucose, nutrient, and growth factor levels. These factors control cap-dependent translation by regulating the evolutionarily conserved mTORC1 (mTOR, Raptor, mLST8) pathway (3). Activation of mTORC1 positively stimulates mRNA translation via its downstream substrates S6Ks and 4E-BP1/ eIF4E (4-7). Phosphorylation of 4E-BP1 by mTORC1 results in its dissociation from eIF4E, promoting assembly of the eIF4F complex. It is thought that S6K1 can phosphorylate translational regulators such as eIF4B and PDCD4 to enhance the translational efficiency of mRNAs with highly structured 5Ј UTRs (8-10).Therefore, growth factors positively regulate cap-dependent translation via mTORC...

Converging signals from the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) pathways are well established to modulate translation initiation. Less is known regarding the molecular basis of protein synthesis regulated by other inputs, such as agonists of the Ras/extracellular signal-regulated kinase (ERK) signaling cascade. Ribosomal protein (rp) S6 is a component of the 40S ribosomal subunit that becomes phosphorylated at several serine residues upon mitogen stimulation, but the exact molecular mechanisms regulating its phosphorylation and the function of phosphorylated rpS6 is poorly understood. Here, we provide evidence that activation of the p90 ribosomal S6 kinases (RSKs) by serum, growth factors, tumor promoting phorbol esters, and oncogenic Ras is required for rpS6 phosphorylation downstream of the Ras/ERK signaling cascade. We demonstrate that while ribosomal S6 kinase 1 (S6K1) phosphorylates rpS6 at all sites, RSK exclusively phosphorylates rpS6 at Ser 235/236 in vitro and in vivo using an mTORindependent mechanism. Mutation of rpS6 at Ser 235/236 reveals that phosphorylation of these sites promotes its recruitment to the 7-methylguanosine cap complex, suggesting that Ras/ERK signaling regulates assembly of the translation preinitiation complex. These data demonstrate that RSK provides an mTORindependent pathway linking the Ras/ERK signaling cascade to the translational machinery.In eukaryotic cells, the main rate-limiting step of translation is initiation, which is controlled by an array of proteins that respond to signaling cascades activated by extracellular signals (reviewed in Refs. 1-3). The mammalian target of rapamycin, mTOR, 4 is a conserved serine/threonine kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate mammalian cell growth (reviewed in Refs. 4, 5-8).Under conditions of nutrient and energy sufficiency and insulin or mitogen stimulation, mTOR stimulates two important translational regulators, the ribosomal S6 kinases (S6K1 and S6K2) and the eukaryotic initiation factor 4E (eIF4E). eIF4E is crucial for ribosome recruitment as it binds to the 7-methylguanosine cap structure (m7GpppN, where N is any nucleotide) at the 5Ј-end of nearly all transcribed mRNAs to initiate cap-dependent translation (reviewed in Ref. 7). When mTOR is active, eIF4E nucleates the assembly of the translation preinitiation complex through recruitment of numerous initiation factors, resulting in association of the ribosomal subunits to the mRNA. S6K1 and S6K2 are serine/threonine kinases directly stimulated by mTOR which in turn, phosphorylate substrates involved in cell and body size (5, 6). S6K1 phosphorylates several substrates located in the cytoplasm and the nucleus, including the ribosomal protein (rp) S6 (reviewed in Ref. 9).Ribosomal protein S6 is one of 33 proteins that comprise the 40 S ribosomal subunit and represents the most extensively studied substrate of S6K1 (10). Because the initial discovery that liver-derived rpS6 was phosphoryla...

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Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. In this study, we show that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 ribosomal S6 kinase 1 (S6K1). The mitogen-activated protein kinase (MAPK)-activated kinase, p90 ribosomal S6 kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin͞hamartin complex, resulting in increased mTOR signaling to S6K1. Together, our data unveil a regulatory mechanism by which the Ras͞MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function.

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