ABSTRACT
The bacterial macrolide rapamycin is an efficacious anticancer agent against solid tumors. In a hypoxic environment, the increase in mass of solid tumors is dependent on the recruitment of mitogens and nutrients. When nutrient concentrations change, particularly those of essential amino acids, the mammalian Target of Rapamycin (mTOR) functions in regulatory pathways that control ribosome biogenesis and cell growth. In bacteria, ribosome biogenesis is independently regulated by amino acids and adenosine triphosphate (ATP). Here we demonstrate that the mTOR pathway is influenced by the intracellular concentration of ATP, independent of the abundance of amino acids, and that mTOR itself is an ATP sensor.
Subject(s)
Adenosine Triphosphate/metabolism , Protein Kinases/metabolism , Adaptor Proteins, Signal Transducing , Amino Acids/metabolism , Androstadienes/pharmacology , Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Line , Deoxyglucose/pharmacology , Enzyme Activation , Homeostasis , Humans , Insulin/pharmacology , Kinetics , Phosphoproteins/metabolism , Phosphorylation , RNA, Transfer, Amino Acyl/metabolism , Recombinant Fusion Proteins/metabolism , Ribosomal Protein S6 Kinases/antagonists & inhibitors , Ribosomal Protein S6 Kinases/metabolism , Ribosomes/metabolism , Rotenone/pharmacology , Signal Transduction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases , WortmanninABSTRACT
Because ribosome biogenesis plays an essential role in cell proliferation, control mechanisms may have evolved to recognize lesions in this critical anabolic process. To test this possibility, we conditionally deleted the gene encoding 40S ribosomal protein S6 in the liver of adult mice. Unexpectedly, livers from fasted animals deficient in S6 grew in response to nutrients even though biogenesis of 40S ribosomes was abolished. However, liver cells failed to proliferate or induce cyclin E expression after partial hepatectomy, despite formation of active cyclin D-CDK4 complexes. These results imply that abrogation of 40S ribosome biogenesis may induce a checkpoint control that prevents cell cycle progression.
Subject(s)
Cell Division , Liver/cytology , Liver/physiology , Protein Biosynthesis , Proto-Oncogene Proteins , Ribosomal Proteins/physiology , Animals , Cyclin D1/biosynthesis , Cyclin D1/metabolism , Cyclin E/genetics , Cyclin E/metabolism , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Cyclin-Dependent Kinases/metabolism , DNA/biosynthesis , Food Deprivation , G1 Phase , Gene Deletion , Gene Targeting , Hepatectomy , Interferon-alpha/pharmacology , Liver/metabolism , Liver Regeneration , Mice , Mice, Inbred Strains , Phosphorylation , Polyribosomes/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA, Ribosomal/metabolism , Ribosomal Protein S6 , Ribosomal Proteins/genetics , Ribosomes/metabolism , S PhaseABSTRACT
The proliferation of most normal cells depends on the co-operation of several growth factors and hormones, each with a specific role, but the key events involved in the action of each necessary stimulant remain largely uncharacterized. In the present study, the pathways involved in the mechanism(s) of co-operation have been investigated in primary cultures of dog thyroid epithelial cells. In this physiologically relevant system, thyroid stimulating hormone (TSH) acting through cAMP, epidermal growth factor (EGF) and phorbol esters (such as PMA) induce DNA synthesis. Their effect requires stimulation of the insulin-like growth factor-1 (IGF-1) receptor by either IGF-1 or insulin, which are not themselves mitogenic agents. In contrast, hepatocyte growth factor (HGF) is itself fully mitogenic. The results of the study demonstrate that cAMP, EGF, HGF and PMA stimulate p70 ribosomal S6 kinase (p70 S6 kinase). However, insulin/IGF-1 also stimulate p70 S6 kinase. Thus stimulation of p70 S6 kinase might be necessary, but is certainly not sufficient, for the induction of DNA synthesis and is not specific for any stimulated pathway. In contrast, phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB) activation by insulin and HGF is strong and sustained, whereas it is weak and transient with EGF and absent in the presence of TSH or PMA. These findings suggest that: (i) stimulation of PI 3-kinases and/or PKB is not involved in the cAMP-dependent pathways leading to thyrocyte proliferation, or in the action of PMA, (ii) the stimulation of the PI 3-kinase/PKB pathway may account for the permissive action of insulin/IGF-1 in the proliferation of these cells, and (iii) the stimulation of this pathway by HGF may explain why this agent does not require insulin or IGF-1 for its mitogenic action.
Subject(s)
Cell Division/drug effects , Colforsin/pharmacology , Cyclic AMP/physiology , Epithelial Cells/cytology , Epithelial Cells/physiology , Growth Substances/pharmacology , Insulin/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Ribosomal Protein S6 Kinases/metabolism , Thyroid Gland/cytology , Thyroid Gland/physiology , Androstadienes/pharmacology , Animals , Cells, Cultured , Dogs , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/pharmacology , Epithelial Cells/drug effects , Hepatocyte Growth Factor/pharmacology , Kinetics , Phosphatidylinositols/metabolism , Proto-Oncogene Proteins c-akt , Sirolimus/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Thyrotropin/pharmacology , WortmanninABSTRACT
Insulin controls glucose homeostasis by regulating glucose use in peripheral tissues, and its own production and secretion in pancreatic beta cells. These responses are largely mediated downstream of the insulin receptor substrates, IRS-1 and IRS-2 (refs 4-8), through distinct signalling pathways. Although a number of effectors of these pathways have been identified, their roles in mediating glucose homeostasis are poorly defined. Here we show that mice deficient for S6 kinase 1, an effector of the phosphatidylinositide-3-OH kinase signalling pathway, are hypoinsulinaemic and glucose intolerant. Whereas insulin resistance is not observed in isolated muscle, such mice exhibit a sharp reduction in glucose-induced insulin secretion and in pancreatic insulin content. This is not due to a lesion in glucose sensing or insulin production, but to a reduction in pancreatic endocrine mass, which is accounted for by a selective decrease in beta-cell size. The observed phenotype closely parallels those of preclinical type 2 diabetes mellitus, in which malnutrition-induced hypoinsulinaemia predisposes individuals to glucose intolerance.
Subject(s)
Glucose Intolerance , Insulin/blood , Islets of Langerhans/ultrastructure , Ribosomal Protein S6 Kinases/metabolism , Animals , Blood Glucose/metabolism , Cell Size , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Fasting , Female , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/metabolism , Male , Mice , Mice, Inbred C57BL , Ribosomal Protein S6 Kinases/deficiencyABSTRACT
Cell proliferation requires cell growth; that is, cells only divide after they reach a critical size. However, the mechanisms by which cells grow and maintain their appropriate size have remained elusive. Drosophila deficient in the S6 kinase gene (dS6K) exhibited an extreme delay in development and a severe reduction in body size. These flies had smaller cells rather than fewer cells. The effect was cell-autonomous, displayed throughout larval development, and distinct from that of ribosomal protein mutants (Minutes). Thus, the dS6K gene product regulates cell size in a cell-autonomous manner without impinging on cell number.
Subject(s)
Drosophila melanogaster/enzymology , Drosophila melanogaster/growth & development , Ribosomal Protein S6 Kinases/metabolism , Wings, Animal/cytology , Animals , Base Sequence , Body Constitution , Cell Count , Cell Division , Cell Size , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Epithelial Cells/cytology , Female , Genes, Insect , Larva/cytology , Larva/growth & development , Male , Metamorphosis, Biological , Molecular Sequence Data , Mutation , Ribosomal Protein S6 Kinases/genetics , Wings, Animal/growth & developmentABSTRACT
In mammalian cells, p70(S6K) plays a key role in translational control of cell proliferation in response to growth factors. Because of the reliance on translational control in early vertebrate development, we cloned a Xenopus homolog of p70(S6K) and investigated the activity profile of p70(S6K) during Xenopus oocyte maturation and early embryogenesis. p70(S6K) activity is high in resting oocytes and decreases to background levels upon stimulation of maturation with progesterone. During embryonic development, three peaks of activity were observed: immediately after fertilization, shortly before the midblastula transition, and during gastrulation. Rapamycin, an inhibitor of p70(S6K) activation, caused oocytes to undergo germinal vesicle breakdown earlier than control oocytes, and sensitivity to progesterone was increased. Injection of a rapamycin-insensitive, constitutively active mutant of p70(S6K) reversed the effects of rapamycin. However, increases in S6 phosphorylation were not significantly affected by rapamycin during maturation. mos mRNA, which does not contain a 5'-terminal oligopyrimidine tract (5'-TOP), was translated earlier, and a larger amount of Mos protein was produced in rapamycin-treated oocytes. In fertilized eggs rapamycin treatment increased the translation of the Cdc25A phosphatase, which lacks a 5'-TOP. Translation assays in vivo using both DNA and RNA reporter constructs with the 5'-TOP from elongation factor 2 showed decreased translational activity with rapamycin, whereas constructs without a 5'-TOP or with an internal ribosome entry site were translated more efficiently upon rapamycin treatment. These results suggest that changes in p70(S6K) activity during oocyte maturation and early embryogenesis selectively alter the translational capacity available for mRNAs lacking a 5'-TOP region.
Subject(s)
Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Oocytes/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , Ribosomal Protein S6 Kinases/metabolism , Amino Acid Sequence , Animals , Blastocyst/metabolism , Cloning, Molecular , Down-Regulation , Female , Gastrula/metabolism , Molecular Sequence Data , Oogenesis/drug effects , Ribosomal Protein S6 Kinases/genetics , Sirolimus/pharmacology , Up-Regulation , Xenopus laevis/genetics , Xenopus laevis/metabolismABSTRACT
Recent studies have shown that the p70(s6k)/p85(s6k) signaling pathway plays a critical role in cell growth by modulating the translation of a family of mRNAs termed 5'TOPs, which encode components of the protein synthetic apparatus. Here we demonstrate that homozygous disruption of the p70(s6k)/p85(s6k) gene does not affect viability or fertility of mice, but that it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70(s6k)/p85(s6k)-deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum-induced S6 phosphorylation and translational up-regulation of 5'TOP mRNAs were equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70(s6k)/p85(s6k)-deficient and wild-type mice. A search of public databases identified a novel p70(s6k)/p85(s6k) homolog which contains the same regulatory motifs and phosphorylation sites known to control kinase activity. This newly identified gene product, termed S6K2, is ubiquitously expressed and displays both mitogen-dependent and rapamycin-sensitive S6 kinase activity. More striking, in p70(s6k)/p85(s6k)-deficient mice, the S6K2 gene is up-regulated in all tissues examined, especially in thymus, a main target of rapamycin action. The finding of a new S6 kinase gene, which can partly compensate for p70(s6k)/p85(s6k) function, underscores the importance of S6K function in cell growth.
Subject(s)
Ribosomal Protein S6 Kinases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers , DNA, Complementary , Embryo, Mammalian/cytology , Embryonic and Fetal Development/genetics , Fibroblasts/cytology , Mice , Mice, Mutant Strains , Molecular Sequence Data , Phenotype , Phosphorylation , Ribosomal Protein S6 Kinases/chemistry , Ribosomal Protein S6 Kinases/metabolism , Sequence Homology, Amino AcidABSTRACT
Here we have employed p70(s6k) truncation and point mutants to elucidate the role played by the carboxyl-terminal autoinhibitory domain S/TP phosphorylation sites in kinase activation. Earlier studies showed that truncation of the p70(s6k) amino terminus severely impaired kinase activation but that this effect was reversed by deleting the carboxyl terminus, which in parallel led to deregulation of Thr229 phosphorylation in the activation loop (Dennis, P. B., Pullen, N., Kozma, S. C., and Thomas, G. (1996) Mol. Cell. Biol. 16, 6242-6251). In this study, substitution of acidic residues for the four autoinhibitory domain S/TP sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr229 phosphorylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr389 phosphorylation, because substitution of an acidic residue at this position in the p70(s6k) variant containing the S/TP mutations leads to a large increase in basal Thr229 phosphorylation and kinase activity. In contrast, an alanine substitution at Thr389 blocks both responses. Consistent with these data, we show that a mutant harboring the acidic S/TP and Thr389 substitutions is an excellent in vitro substrate for the newly identified Thr229 kinase, phosphoinositide-dependent kinase-1 (Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S., Hemmings, B. A., and Thomas, G. (1998) Science 279, 707-710), whereas phosphoinositide-dependent kinase-1 poorly utilizes the two p70(s6k) variants that have only one set of mutations. These findings indicate that phosphorylation of the S/TP sites, in cooperation with Thr389 phosphorylation, controls Thr229 phosphorylation through an intrasteric mechanism.
Subject(s)
Ribosomal Protein S6 Kinases/chemistry , 3-Phosphoinositide-Dependent Protein Kinases , Cell Line , Enzyme Activation/physiology , Humans , Kidney/embryology , Mutagenesis/genetics , Phosphopeptides/analysis , Phosphorylation , Phosphothreonine/analysis , Protein Serine-Threonine Kinases/metabolism , Ribosomal Protein S6 Kinases/genetics , Sequence Deletion/genetics , Threonine/genetics , Threonine/metabolismABSTRACT
In mammalian cells, mitogen-induced phosphorylation of ribosomal protein S6 by p70s6k has been implicated in the selective translational upregulation of 5'TOP mRNAs. We demonstrate here that the homologous Arabidopsis thaliana protein, AtS6k2, ectopically expressed in human 293 cells or isolated from plant cells, phosphorylates specifically mammalian and plant S6 at 25 degrees C but not at 37 degrees C. When Arabidopsis suspension culture cells are shifted from 25 to 37 degrees C, the kinase becomes rapidly inactivated, consistent with the observation that heat shock abrogates S6 phosphorylation in plants. Treatment with potato acid phosphatase reduced the specific activity of immunoprecipitated AtS6k2 threefold, an effect which was blocked in the presence of 4-nitrophenyl phosphate. In quiescent mammalian cells, AtS6k2 is activated by serum stimulation, a response which is abolished by the fungal metabolite wortmannin but is resistant to rapamycin. Treatment of mammalian cells with rapamycin abolishes in vivo S6 phosphorylation by p70s6k; however, ectopic expression of AtS6k2 rescues the rapamycin block. Collectively, the data demonstrate that AtS6k2 is the functional plant homolog of mammalian p70s6k and identify a new signalling pathway in plants.
Subject(s)
Arabidopsis/enzymology , Ribosomal Protein S6 Kinases/metabolism , Amino Acid Sequence , Antibodies/metabolism , Arabidopsis/genetics , Cell Line , Cells, Cultured , DNA, Plant/chemistry , DNA, Plant/isolation & purification , Hot Temperature , Humans , Kidney/embryology , Molecular Sequence Data , Ribosomal Protein S6 Kinases/genetics , Ribosomal Protein S6 Kinases/immunology , Signal TransductionABSTRACT
Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB was demonstrated, as PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activated p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocked insulin-induced activation of p70s6k.
Subject(s)
Protein Serine-Threonine Kinases/metabolism , Ribosomal Protein S6 Kinases/metabolism , 3-Phosphoinositide-Dependent Protein Kinases , Amino Acid Sequence , Androstadienes/pharmacology , Animals , Binding Sites , Calcium-Calmodulin-Dependent Protein Kinase Type 4 , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Catalysis , Cell Line , Enzyme Activation , Insulin/pharmacology , Insulin Antagonists/pharmacology , Molecular Sequence Data , Phosphorylation , Phosphothreonine/metabolism , Polyenes/pharmacology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Recombinant Proteins/metabolism , Sirolimus , WortmanninABSTRACT
The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.
Subject(s)
Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Binding Sites , Catalysis , Cells, Cultured , Consensus Sequence , Enzyme Activation , Humans , Molecular Sequence Data , Peptide Mapping , Phosphorylation , Protein Kinase C/chemistry , Ribosomal Protein S6 KinasesABSTRACT
Mitogen-induced activation of p70(s6k) is associated with the phosphorylation of specific sites which are negatively affected by the immunosuppressant rapamycin, the fungal metabolite wortmannin, and the methylxanthine SQ20006. Recent reports have focused on the role of the amino terminus of the p85(s6k) isoform in mediating kinase activity, with the observation that amino-terminal truncation mutants are activated in the presence of rapamycin while retaining their sensitivity to wortmannin. Here we show that the effects of previously described amino- and carboxy-terminal truncations on kinase activity are ultimately reflected in the phosphorylation state of the enzyme. Mutation of the principal rapamycin-targeted phosphorylation site, T-389, to an acidic residue generates a form of the kinase which is as resistant to wortmannin or SQ20006 as it is to rapamycin, consistent with the previous observation that T-389 was a common target of all three inhibitors. Truncation of the first 54 residues of the amino terminus blocks the serum-induced phosphorylation of three rapamycin-sensitive sites, T-229 in the activation loop and T-389 and S-404 in the linker region. This correlates with a severe reduction in the ability of the kinase to be activated by serum. However, loss of mitogen activation conferred by the removal of the amino terminus is reversed by additional truncation of the carboxy-terminal domain, with the resulting mutant demonstrating phosphorylation of the remaining two rapamycin-sensitive sites, T-229 and T-389. In this double-truncation mutant, phosphorylation of T-229 occurs in the basal state, whereas mitogen stimulation is required to induce acute upregulation of T-389 phosphorylation. The phosphorylation of both sites proceeds unimpaired in the presence of rapamycin, indicating that the kinases responsible for the phosphorylation of these sites are not inhibited by the macrolide. In contrast, activation of the double-truncation mutant is blocked in the presence of wortmannin or SQ20006, and these agents completely block the phosphorylation of T-389 while having only a marginal effect on T-229 phosphorylation. When the T-389 site is mutated to an acidic residue in the double-truncation background, the activation of the resulting mutant is insensitive to the wortmannin and SQ20006 block, but interestingly, the mutant is activated to a significantly greater level than a control in the presence of rapamycin. These data are consistent with the hypothesis that T-389 is the principal regulatory phosphorylation site, which, in combination with hyperphosphorylation of the autoinhibitory domain S/TP sites, is acutely regulated by external effectors, whereas T-229 phosphorylation is regulated primarily by internal mechanisms.
Subject(s)
Polyenes/pharmacology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Tyrosine , Amino Acid Sequence , Androstadienes/pharmacology , Cell Line , Humans , Immunosuppressive Agents/pharmacology , Kidney , Mitogen-Activated Protein Kinase Kinases , Molecular Sequence Data , Mutagenesis, Site-Directed , Nicotinic Acids/pharmacology , Peptide Mapping , Phosphates/metabolism , Phosphodiesterase Inhibitors/pharmacology , Phosphopeptides/chemistry , Phosphopeptides/isolation & purification , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribosomal Protein S6 Kinases , Sequence Deletion , Sirolimus , Transfection , WortmanninABSTRACT
The protein p70s6k/p85s6k lies on a mitogen-stimulated signaling pathway and plays a key role in G1 progression of the cell cycle. Activation of this enzyme is mediated by a complex set of phosphorylation events, which has largely contributed to the difficulty in identifying the upstream kinases that mediate p70s6k activation. Genetics has proved a powerful complementary approach for such problems, providing an alternative means to identify components of signaling cascades and their functional end targets. As a first step toward implementing such an approach, we have cloned cDNAs encoding the Drosophila melanogaster p70s6k homolog (Dp70s6k). Dp70s6k is encoded by a single gene, which generates three mRNA transcripts and exhibits an overall identity of 78% in the catalytic domain with its mammalian counterpart. Importantly, this high identity extends beyond the catalytic domain to the N terminus, linker region, and the autoinhibitory domain. Furthermore, all the critical phosphorylation sites required for mammalian p70s6k activation are conserved within these same domains of Dp70s6k. Chief amongst these conserved sites are those associated with the selective rapamycin-induced p70s6k dephosphorylation and inactivation. Consistent with this observation, analysis of total S6 kinase activity in fractionated Drosophila Schneider line 2 cell extracts reveals two peaks of activity, only one of which is rapamycin sensitive. By employing a monospecific polyclonal antibody generated against Dp70s6k, we show that the cloned DP70s6k cDNA has identity with only the rapamycin sensitive peak, suggesting that this biological system would be useful in determining not only the mechanism of p70s6k activation, but also in elucidating the mechanism by which rapamycin acts to inhibit cell growth.
Subject(s)
Drosophila melanogaster/enzymology , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary/genetics , Gene Expression , Molecular Sequence Data , Phosphoproteins/metabolism , Polyenes/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/genetics , Ribosomal Protein S6 Kinases , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , SirolimusABSTRACT
The immunosuppressive agent rapamycin induces inactivation of p70s6k with no effect on other mitogen-activated kinases. Here we have employed a combination of techniques, including mass spectrometry, to demonstrate that this effect is associated with selective dephosphorylation of three previously unidentified p70s6k phosphorylation sites: T229, T389 and S404. T229 resides at a conserved position in the catalytic domain, whose phosphorylation is essential for the activation of other mitogen-induced kinases. However, the principal target of rapamycin-induced p70s6k inactivation is T389, which is located in an unusual hydrophobic sequence outside the catalytic domain. Mutation of T389 to alanine ablates kinase activity, whereas mutation to glutamic acid confers constitutive kinase activity and rapamycin resistance. The importance of this site and its surrounding motif to kinase function is emphasized by its presence in a large number of protein kinases of the second messenger family and its conservation in putative p70s6k homologues from as distantly related organisms as yeast and plants.
Subject(s)
Antifungal Agents/pharmacology , Polyenes/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , 3T3 Cells , Amino Acid Sequence , Animals , Conserved Sequence , Enzyme Activation/drug effects , Humans , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Ribosomal Protein S6 Kinases , Sequence Analysis , SirolimusABSTRACT
The enzymes p70s6k and p85s6k are two isoforms of the same kinase and are important in mitogenesis. Both isoforms are activated by a complex phosphorylation event and lie on a common signalling pathway, distinct from that of the p42mapk/p44mapk kinases. Activation of p42mapk/p44mapk is triggered by sequential activation of the GDP-GTP exchange factor Sos, the GTP-binding protein p21ras, and protein kinases p74raf and p47mek (refs 7-10). As p21ras transformed cells have increased S6 phosphorylation, we tested whether the p70s6k/p85s6k signalling pathway bifurcates between p21ras and p42mapk/p44mapk. We found that mutants of p74raf and p21ras blocked activation of epitope-tagged p44mapk but not epitope-tagged p70s6k. Moreover, in cells expressing human platelet-derived growth factor receptors lacking the kinase-insert domain, the growth factor activates p21ras but not p70s6k/p85s6k. The critical autophosphorylation site for p70s6k/p85s6k activation within this domain is a tyrosine at residue 751. Our results show that the p70s6k/p85s6k signalling pathway is independent of p21ras, that it bifurcates from the p21ras pathway at the receptor, and that it is initiated by autophosphorylation at a specific site.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases , Proto-Oncogene Proteins c-raf , Proto-Oncogene Proteins p21(ras)/metabolism , 3T3 Cells , Animals , Cell Line , Enzyme Activation , Humans , Mice , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Mutation , Phosphatidylinositol 3-Kinases , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor) , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Receptors, Platelet-Derived Growth Factor/metabolism , Ribosomal Protein S6 Kinases , Signal Transduction , SwineABSTRACT
The p70s6k/p85s6k represent two isoforms of the same kinase which are derived by differential splicing from a common gene. The p85s6k isoform is identical to p70s6k except for a 23 amino acid extension at its N-terminus, which constitutively targets it to the nucleus. Both isoforms are activated by multisite phosphorylation in response to mitogens and reside on the same signaling pathway, a pathway which is distinct from that of p42mapk/p44mapk pathway. Inhibitory p70s6k/p85s6k antibodies or the immunosuppressant rapamycin selectively inhibit kinase activity and repress or abolish cell growth depending on the inhibitory agent employed and the cell type examined. Recent studies imply that these effects are exerted through inhibition of 40S ribosomal protein S6 phosphorylation, the kinase target, which in turn suppresses the translation of a family of transcripts essential for cell growth.
Subject(s)
Cell Division/physiology , Protein Serine-Threonine Kinases/physiology , Amino Acid Sequence , Animals , Enzyme Activation , Humans , Molecular Sequence Data , Phosphorylation , Polyenes/pharmacology , Protein Biosynthesis , Ribosomal Protein S6 Kinases , SirolimusABSTRACT
The immunosuppressant rapamycin blocks p70s6k/p85s6k activation and phosphorylation of 40S ribosomal protein S6 in Swiss 3T3 cells. The same net result is obtained when the macrolide is added 3 hr after serum stimulation. In stimulated cells p70s6k/p85s6k inactivation is achieved within minutes, whereas S6 dephosphorylation requires 1-2 hr, supporting the concept that S6 dephosphorylation results from kinase inactivation. In parallel, rapamycin treatment causes a small, but significant, reduction in the initiation rate of protein synthesis, as measured both by [35S]methionine incorporation into protein and by recruitment of 80S ribosomes into polysomes. More striking, analysis of individual mRNA transcripts revealed that rapamycin selectively suppresses the translation of a family of mRNAs that is characterized by a polypyrimidine tract immediately after their N7-methylguanosine cap, a motif that can act as a translational modulator. This family includes transcripts for ribosomal proteins, elongation factors of protein synthesis, and proteins of as-yet-unknown function. The results imply that (i) 40S ribosomes containing phosphorylated S6 may selectively recognize this motif or proteins which bind to it and (ii) rapamycin may inhibit cell growth by blocking S6 phosphorylation and, thus, translation of these mRNAs.
Subject(s)
Actins/biosynthesis , Immunosuppressive Agents/pharmacology , Peptide Elongation Factors/biosynthesis , Polyenes/pharmacology , Protein Biosynthesis/drug effects , RNA, Messenger/metabolism , 3T3 Cells , Animals , Methionine/metabolism , Mice , Molecular Sequence Data , Oligonucleotide Probes , Peptide Elongation Factor 1 , Phosphorylation , Polyribosomes/metabolism , Pyrimidines , RNA, Messenger/biosynthesis , RNA, Messenger/chemistry , Ribosomal Protein S6 , Ribosomal Proteins/drug effects , Ribosomal Proteins/metabolism , Sirolimus , Transcription, GeneticABSTRACT
The immunosuppressant rapamycin selectively abolishes phosphorylation and activation of p70s6k/p85s6k at concentrations that either block or suppress cell growth. The four sites of phosphorylation associated with p70s6k/p85s6k activation all display Ser/Thr-Pro motifs and are closely clustered within a putative autoinhibitory domain of the enzyme. To produce a constitutively active, rapamycin-resistant form of the kinase, these four sites were converted to either Asp or Glu. When overexpressed in human 293 cells, the activity of the mutant is similar to that of the parent enzyme, under conditions where the parent is phosphorylated and active. Unexpectedly, however, the mutant remains sensitive to rapamycin and is inactivated in vitro by protein phosphatase 2A. Peptide maps reveal that rapamycin abolishes the activity of the overexpressed p70s6k through the dephosphorylation of a novel set of sites distinct from those associated with mitogenic activation.
Subject(s)
Immunosuppressive Agents/pharmacology , Polyenes/pharmacology , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Binding Sites , Cell Division , Cells, Cultured , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Mapping , Phosphorylation , Ribosomal Protein S6 Kinases , SirolimusABSTRACT
Rat p70s6k and p85s6k have been expressed in baculovirus recombinants propagated in Sf9 insect cells. Surprisingly, both recombinant isoforms were active without coinfection of other kinases which lie upstream in the signaling pathway. Treatment of either recombinant form with phosphatase 2A leads to immediate inactivation in the absence of phosphatase inhibitors. Further studies show that the same four major Ser/Thr-Pro sites associated with p70s6k activation following mitogenic stimulation in vivo are also the four major sites phosphorylated in both the p70s6k and p85s6k during the infection process. It is proposed that the production of phosphorylated and activated recombinant p70s6k and p85s6k is due to activation of a host cell signaling pathway which is triggered by viral infection. In support of this hypothesis, wild-type virus-, but not mock-infected cells, exhibit the multiple phosphorylation of a ribosomal protein which migrates similar to ribosomal protein S6 on two-dimensional-polyacrylamide gels and extracts from these same cells contain elevated levels of S6 kinase activity.
Subject(s)
Baculoviridae/physiology , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Line , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Moths , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribosomal Protein S6 Kinases , Ribosomal Proteins/metabolism , Virus ReplicationABSTRACT
Employing oligonucleotide primers derived from the DNA sequence of rat p70s6k a homologous sequence was shown by polymerase chain reaction (PCR) to be present as a maternal transcript in stage IV-VI Xenopus laevis oocytes. The sequence covered 665 bp of p70s6k and was 97% identical at the amino acid level. When used to probe a Northern blot of the poly(A)+ mRNA from stage VI oocytes, this sequence recognized four transcripts of 1.9, 2.5, 3.2 and 5.2 kb. Specific rat p70s6k antibodies immunoprecipitated active S6 kinase from stage VI oocytes but not unfertilized eggs. The basal level of activity was 3- to 5-fold higher in primed versus non-primed oocytes indicating that p70s6k activation was an early event in maturation. Consistent with this observation, progesterone induced a 10-fold activation of the kinase in non-primed oocytes within 1 h post-induction at a time critical for early activation of protein synthesis. A much smaller, variable peak of activation was observed at 85% germinal vesicle breakdown (GVBD), which was dependent on the rate of maturation. Two members of the pp90rsk family, thought to be the sole S6 kinases present in X.laevis oocytes, exhibited distinct kinetics of activation. Finally, the S6 kinase activity present 1 h post-progesterone stimulation was purified and shown to have a Mr of 70K.