John Blenis

ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions

SUMMARY 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.

Molecular mechanisms of mTOR-mediated translational control

The process of translation requires substantial cellular resources. Cells have therefore evolved complex mechanisms to control overall protein synthesis as well as the translation of specific mRNAs that are crucial for cell growth and proliferation. At the heart of this process is the mammalian target of rapamycin (mTOR) signalling pathway, which senses and responds to nutrient availability, energy sufficiency, stress, hormones and mitogens to modulate protein synthesis. Here, we highlight recent findings on the regulators and effectors of mTOR and discuss specific cases that serve as paradigms for the different modes of mTOR regulation and its control of translation.

Identification of the Tuberous Sclerosis Complex-2 Tumor Suppressor Gene Product Tuberin as a Target of the Phosphoinositide 3-Kinase/Akt Pathway

The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. Here, we show that an approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin, as a potential target of Akt/PKB. We demonstrate that, upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. We also show that S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and that T1462 is constitutively phosphorylated in PTEN(-/-) tumor-derived cell lines. Finally, we find that a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity.

Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression.

Cell growth (an increase in cell mass and size through macromolecular biosynthesis) and cell cycle progression are generally tightly coupled, allowing cells to proliferate continuously while maintaining their size. The target of rapamycin (TOR) is an evolutionarily conserved kinase that integrates signals from nutrients (amino acids and energy) and growth factors (in higher eukaryotes) to regulate cell growth and cell cycle progression coordinately. In mammals, TOR is best known to regulate translation through the ribosomal protein S6 kinases (S6Ks) and the eukaryotic translation initiation factor 4E-binding proteins. Consistent with the contribution of translation to growth, TOR regulates cell, organ, and organismal size. The identification of the tumor suppressor proteins tuberous sclerosis1 and 2 (TSC1 and 2) and Ras-homolog enriched in brain (Rheb) has biochemically linked the TOR and phosphatidylinositol 3-kinase (PI3K) pathways, providing a mechanism for the crosstalk that occurs between these pathways. TOR is emerging as a novel antitumor target, since the TOR inhibitor rapamycin appears to be effective against tumors resulting from aberrantly high PI3K signaling. Not only may inhibition of TOR be effective in cancer treatment, but rapamycin is an FDA-approved immunosuppressive and cardiology drug. We review here what is known (and not known) about the function of TOR in cellular and animal physiology.

Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.

Nuclear localization and regulation of erk- and rsk-encoded protein kinases.

We demonstrate that members of the erk-encoded family of mitogen-activated protein (MAP) kinases (pp44/42mapk/erk) and members of the rsk-encoded protein kinases (RSKs or pp90rsk) are present in the cytoplasm and nucleus of HeLa cells. Addition of growth factors to serum-deprived cells results in increased tyrosine and threonine phosphorylation and in the activation of cytosolic and nuclear MAP kinases. Activated MAP kinases then phosphorylate (serine/threonine) and activate RSKs. Concurrently, a fraction of the activated MAP kinases and RSKs enter the nucleus. In addition, a distinct growth-regulated RSK-kinase activity (an enzyme[s] that phosphorylates recombinant RSK in vitro and that may be another member of the erk-encoded family of MAP kinases) was found associated with a postnuclear membrane fraction. Regulation of nuclear MAP kinase and RSK activities by growth factors and phorbol ester is coordinate with immediate-early gene expression. Indeed, in vitro, MAP kinase and/or RSK phosphorylates histone H3 and the recombinant c-Fos and c-Jun polypeptides, transcription factors phosphorylated in a variety of cells in response to growth stimuli. These in vitro studies raise the possibility that the MAP kinase/RSK signal transduction pathway represents a protein-Tyr/Ser/Thr phosphorylation cascade with the spatial distribution and temporal regulation that can account for the rapid transmission of growth-regulating information from the membrane, through the cytoplasm, and to the nucleus.

Rapamycin-FKBP Specifically Blocks Growth-Dependent Activation of and Signaling by the 70 kd S6 Protein Kinases

The macrolide rapamycin blocks cell cycle progression in yeast and various animal cells by an unknown mechanism. We demonstrate that rapamycin blocks the phosphorylation and activation of the 70 kd S6 protein kinases (pp70S6K) in a variety of animal cells. The structurally related drug FK506 had no effect on pp70S6K activation but at high concentrations reversed the rapamycin-induced block, confirming the requirement for the rapamycin and FK506 receptor, FKBP. Rapamycin also interfered with signaling by these S6 kinases, blocking serum-stimulated S6 phosphorylation and delaying entry of Swiss 3T3 cells into S phase. Neither rapamycin nor FK506 blocked activation of a distinct family of S6 kinases (RSKs) or the MAP kinases. These studies identify a rapamycin-sensitive signaling pathway, argue for a ubiquitous role for FKBPs in signal transduction, indicate that FK506-FKBP-calcineurin complexes do not interfere with pp70S6K signaling, and show that in fibroblasts pp70S6K, not RSK, is the physiological S6 kinase.

Tuberous Sclerosis Complex Gene Products, Tuberin and Hamartin, Control mTOR Signaling by Acting as a GTPase-Activating Protein Complex toward Rheb

BACKGROUND Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear. RESULTS We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. CONCLUSIONS 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.

Molecular interpretation of ERK signal duration by immediate early gene products

The duration of intracellular signalling is associated with distinct biological responses, but how cells interpret differences in signal duration are unknown. We show that the immediate early gene product c-Fos functions as a sensor for ERK1 (extracellular-signal-regulated kinase 1) and ERK2 signal duration. When ERK activation is transient, its activity declines before the c-Fos protein accumulates, and under these conditions c-Fos is unstable. However, when ERK signalling is sustained, c-Fos is phosphorylated by still-active ERK and RSK (90K-ribosomal S6 kinase). Carboxy-terminal phosphorylation stabilizes c-Fos and primes additional phosphorylation by exposing a docking site for ERK, termed the FXFP (DEF) domain. Mutating the DEF domain disrupts the c-Fos sensor and c-Fos-mediated signalling. Other immediate early gene products that control cell cycle progression, neuronal differentiation and circadium rhythms also contain putative DEF domains, indicating that multiple sensors exist for sustained ERK signalling. Together, our data identify a general mechanism by which cells can interpret differences in ERK activation kinetics.

mTOR and S6K1 Mediate Assembly of the Translation Preinitiation Complex through Dynamic Protein Interchange and Ordered Phosphorylation Events

In response to nutrients, energy sufficiency, hormones, and mitogenic agents, S6K1 phosphorylates several targets linked to translation. However, the molecular mechanisms whereby S6K1 is activated, encounters substrate, and contributes to translation initiation are poorly understood. We show that mTOR and S6K1 maneuver on and off the eukaryotic initiation factor 3 (eIF3) translation initiation complex in a signal-dependent, choreographed fashion. When inactive, S6K1 associates with the eIF3 complex, while the S6K1 activator mTOR/raptor does not. Cell stimulation promotes mTOR/raptor binding to the eIF3 complex and phosphorylation of S6K1 at its hydrophobic motif. Phosphorylation results in S6K1 dissociation, activation, and subsequent phosphorylation of its translational targets, including eIF4B, which is then recruited into the complex in a phosphorylation-dependent manner. Thus, the eIF3 preinitiation complex acts as a scaffold to coordinate a dynamic sequence of events in response to stimuli that promote efficient protein synthesis.