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Results for “Ribosomal Protein S6”

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At least 19 recordsLinked to original sources
PubMed1991

Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6.

Ribosomal protein S6 is phosphorylated in response to mitogens by activation of one or more protein kinase cascades. Phosphorylation of S6 in vivo is catalyzed by (at least) two distinct mitogen-activated S6 kinase families distinguishable by size, the 70 kDa and 90 kDa S6 kinases. Both S6 kinases are activated by serine/threonine phosphorylation. Members of each family have been cloned. The 90 kDa S6 kinases are activated more rapidly than the 70 kDa S6 kinase, and may have other intracellular targets. The 70 kDa S6 kinase is relatively specific for 40 S ribosomal subunits. No kinase capable of activating the 70 kDa S6 kinase has been identified. Members of the 90 kDa S6 kinases are activated in vitro by 42 kDa and 44 kDa MAP kinases, which are in turn activated by mitogen-dependent activators. The pathways for mitogen-stimulated S6 phosphorylation are discussed.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of cloned cDNAs that code for human ribosomal protein S6.

Ribosomal protein (rp) S6 is the major substrate of protein kinases in eukaryotic ribosomes. To facilitate the identification of cloned cDNAs for human rpS6, we used published amino acid (aa) sequence data for rat liver rpS6 and yeast (Saccharomyces carlsbergensis) rpS10 to design mixed oligodeoxynucleotide probes. Screening of several human cDNA libraries with these probes permitted the isolation of plasmids which encompass the entire coding sequence of rpS6 (249 aa residues), 27 bp of the 5'-untranslated leader and all 39 bp of the 3'-untranslated region. A comparison of the predicted human rpS6 amino acid sequence and the yeast rpS10 amino acid sequence shows highly conserved areas separated by regions of divergence.

Amino Acid Sequence↗
PubMed1988

Primary structure of mammalian ribosomal protein S6.

Ribosomal protein S6 was isolated from rat liver ribosomes by reversed-phase high-performance liquid chromatography (HPLC) and subjected to cyanogen bromide and proteolytic cleavages. The cleavage fragments were resolved by HPLC and sequenced by automated Edman degradation. The overall amino acid sequence of S6 (249 residues) was determined by alignment of the overlapping sequences of selected cyanogen bromide, chymotryptic, tryptic, and clostripain cleavage fragments. The only protein found to exhibit close homology with the S6 sequence is yeast ribosomal protein S10 (61% sequence identity). Previously, characterized phosphopeptide derivatives of S6 containing phosphorylation sites for adenosine 3',5'-cyclic phosphate dependent and protease-activated protein kinases originate from the carboxy-terminal region of S6 encompassing residues 233-249.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of a novel protein phosphatase highly specific for ribosomal protein S6.

Ribosomal protein S6 is the principal phosphoprotein of the eucaryotic ribosome that becomes multiply phosphorylated on serine residues in response to a wide variety of mitogenic stimuli. In this paper the principal protein phosphatases able to dephosphorylate S6 were characterized in Xenopus laevis ovary and eggs. Two enzymes termed peak I and peak II were found to account for most S6 phosphatase activity in both oocytes and eggs. The peak I enzyme had an apparent Mr of 200,000 on gel filtration, dephosphorylated the beta subunit of phosphorylase kinase and phosphorylase a, and was inhibited by inhibitor 1 and inhibitor 2, suggesting it was similar to protein phosphatase 1. The peak II enzyme was purified over 12,000-fold and had an apparent Mr = 55,000 on glycerol gradient centrifugation. This phosphatase could dephosphorylate all sites in S6 but was unable to dephosphorylate phosphorylase a or phosphorylase kinase. However, it was inhibited by nanomolar concentrations of inhibitor 1 and inhibitor 2. These results indicate the peak II enzyme represents a new class of highly specific protein phosphatase and suggest that inhibition of dephosphorylation in cellular extracts by inhibitor 1 and inhibitor 2 is not a sufficient criterion for implicating protein phosphatase 1 in a cellular process.

Animals↗
PubMed1987

Interleukin 2 and diacylglycerol stimulate phosphorylation of 40 S ribosomal S6 protein. Correlation with increased protein synthesis and S6 kinase activation.

Interleukin 2 (IL-2) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), a direct activator of protein kinase C, induce phosphorylation of the ribosomal S6 protein in a murine IL-2-dependent lymphocyte clone. The phosphorylation of S6 protein was correlated with increased protein synthesis in this cell line. Using cell-free assay systems, two unique kinases capable of phosphorylating the S6 protein were identified, namely, a calcium/phospholipid-dependent phosphotransferase, protein kinase C, and a second phospholipid-independent kinase detected in crude cytosolic fractions. Peptide mapping of the S6 protein demonstrated that the degree of S6 phosphorylation stimulated by IL-2 and OAG was similar to that achieved using the second (calcium/phospholipid-independent) kinase but not to the level of phosphorylation achieved with protein kinase C. The kinase responsible for phosphorylating S6 was soluble in stimulated cells and was induced in a time-dependent manner by either IL-2 or diacylglycerol treatment of intact cells. These data support the notion that, although protein kinase C is activated by IL-2 or OAG, subsequent events such as S6 phosphorylation may be the result of the activation of secondary phosphotransferase systems regulated by protein kinase C.

Animals↗
PubMed1984

Phosphorylation of ribosomal protein S6 and a peptide analogue of S6 by a protease-activated kinase isolated from rat liver.

A trypsin-activated protein kinase has been isolated from rat liver using a peptide analogue of ribosomal protein S6 as a substrate in kinase assays. The structure of the peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, was based on a region of S6 containing both an insulin- and cyclic AMP-regulated phosphorylation site. The trypsin-activated protein kinase phosphorylated a corresponding site in the peptide analogue and ribosomal protein S6 that was distinct from the preferred site for cyclic AMP-dependent protein kinase. Ribosomal S6 contained at least one other major site for the trypsin-activated protein kinase.

Amino Acid Sequence↗
PubMed2004

Continuous ethanol exposure inhibits agonist-stimulated phosphorylation of p70S6 kinase and ribosomal S6 protein in cultured rat astrocytes.

The effects of ethanol exposure on agonist-stimulated phosphorylation of p70S6 kinase and ribosomal S6 protein were determined in confluent astrocyte monolayers. Basal phosphorylation of p70S6 kinase and S6 protein was either unaffected or reduced, respectively, after exposure to 50 mM ethanol for 4 days. The abilities of norepinephrine, carbachol and epidermal growth factor to phosphorylate these proteins were significantly decreased after ethanol exposure. In contrast, ethanol exposure had no effect on the protein expression of either p70S6 kinase or S6 protein. Our data suggest that continuous ethanol exposure results in a generalized decrease in agonist-activation of the p70S6 pathway.

Animals↗
PubMed1986

An insulin-stimulated (ribosomal S6) protein kinase from soluble extracts of H4 hepatoma cells.

Insulin stimulates the phosphorylation of the 40 S ribosomal subunit protein, S6, in intact 32P-labeled H4IIE-C3 cells, a rat hepatoma line. Cell-free cytosolic extracts from H4 cells exhibit a 5- to 10-fold increase in S6 protein kinase activity (measured by transfer of 32P to exogenous 40 S rat liver ribosomal subunits) when prepared from cells exposed to insulin prior to homogenization. Stimulation of S6 phosphorylation in intact cells and activation of S6 protein kinase in cell-free extracts are both detectable within 2 min after insulin, and are maximally stimulated by 10 min. Half-maximal stimulation is observed at 10(-11) M insulin. The stimulated S6 kinase activity requires ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to be present during the kinase assay for full expression. Despite the presence of a 5- to 10-fold increase in S6 protein kinase activity, the extracts from insulin-treated cells exhibit no stimulated kinase activity toward casein, histone, or ATP-citrate lyase assayed under the conditions employed for S6. Thus, insulin mediates the rapid activation of protein kinase specific for ribosomal protein S6 by an as yet unidentified mechanism.

Animals↗
PubMed1977

Ethionine and the phosphorylation of ribosomal protein S6.

Ribosome phosphorylation was studied by monitoring the phosphorylation state of small subunit protein S6 as visualized on two-dimensional electrophoretograms of ribosomal proteins isolated from rat liver. No phosphorylation of S6 was observed under conditions of ethionine-induced inhibition of protein synthesis. Moderate phosphorylation, detected as the appearance of S6 and four or five phosphorylated derivatives, was observed in saline-treated animals. Reversal of ethionine-induced inhibition of protein synthesis by treatment with adenine led to extensive phosphorylation of S6. A model for protein synthesis which includes requisite phosphorylation of ribosomes during initiation is proposed. Cyclic adenosine 3':5'-monophosphate concentration was significantly elevated in liver of both ethionine- and ethionine plus adenine-treated rats, relative to that of saline-treated animals.

Adenine↗
PubMed1998

Cloning and structural analysis of the gene encoding the ribosomal protein S6 from the parasite Leishmania infantum.

We have cloned the S6 ribosomal protein encoding gene from a Leishmania infantum cDNA library. This parasite protozoon, responsible for leishmaniasis in Europe, is able to undergo developmental changes in vitro and results a good model to study cell differentiation processes. The LiS6 protein sequence indicates its pertinence to the S6 protein family, related to the early mechanisms of cell division, differentiation and activation, and shows an intermediate position between the yeasts and higher eukaryotes. Thus, LiS6 protein has the same amino acid length as that of the higher eukaryotes and certain common features such nucleus entrance sequences and several kinase phosphorylation sites. However, the key functional protein kinase C phosphorylation sites are at different locations and present several threonine instead of the usual serine residues. The gene structural analysis suggest the presence of three different encoding genes that do not present remarkable changes along the different phases of the parasite.

Amino Acid Sequence↗
PubMed2000

Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6.

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.

Animals↗
PubMed1989

Activation of a ribosomal S6 protein kinase in rapidly emerging diethylnitrosamine-induced gamma-glutamyltranspeptidase-positive hyperplastic liver lesions of the rat.

The gamma-glutamyl transpeptidase (GGT)-positive hyperplastic liver lesions which developed in the Fisher 344 rat 7 and 60 days following a single carcinogenic dose of diethylnitrosamine (DENA, 200 mg/kg body weight), short-term dietary exposure to 0.02% 2-acetylaminofluorene (AAF) to suppress the growth of normal hepatocytes, and partial hepatectomy to actuate rapid growth of DENA altered hepatocytes not suppressed by AFF, showed an increased activity of a kinase which specifically phosphorylates the ribosomal S6 protein in vitro. Sham-operated animals showed, on the contrary, no GGT-positive cells and low S6 kinase activity, under the same conditions. After partial hepatectomy, activation of S6 kinase and elevated levels of phosphorylated S6 protein in vitro were detected in the early phases of "normal" hepatocyte proliferation, during liver regeneration, in DENA-treated, GGT-negative preparations, when the "selection" agent AAF was omitted from the diet. The observed activation of S6 kinase in GGT-positive hepatocytes and/or liver nodules could represent an early manifestation of the enhanced proliferation of altered hepatocytes during tumor induction and/or promotion under these conditions.

Animals↗
PubMed1990

Identification of phosphoprotein NP33 as a nucleus-associated ribosomal S6 protein and its phosphorylation in hematopoietic cells.

Exposure of HL-60 promyelocytes to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased incorporation of 32P into a Mr approximately 33,000 protein (NP33) found in the nuclear matrices prepared by treating cells with Triton X-100, nucleases, and 2 M NaCl (D. E. Macfarlane, J. Biol. Chem., 261: 6947-6953, 1986). We now report that 12-O-tetradecanoylphorbol-13-acetate causes phosphorylation of NP33 in U937, K562, HEL, Molt-3, and Raji cell lines, all of which are rapidly proliferating cells of hematopoietic origin. 12-O-Tetradecanoylphorbol-13-acetate caused a lesser degree of NP33 phosphorylation in peripheral blood lymphocytes and monocytes and none in granulocytes or platelets. The incorporation of 32P into NP33 was complete in about 10 min, and it was prevented or reversed by staurosporin, indicating that NP33 is continuously phosphorylated and dephosphorylated. NP33 was purified to homogeneity from Triton X-100-washed nuclei or whole cells by extraction with H2SO4, acetone precipitation, and preparative two-dimensional gel electrophoresis. The amino-terminal amino acid sequence of NP33 appears to be the same as that of ribosomal S6 protein. NP33 appears to be S6 protein copurifying with the nuclear matrix.

Alkaloids↗
PubMed1995

Use of alternative polyadenylation sites by the chicken S6 ribosomal protein gene.

Two chicken ribosomal protein S6 cDNAs were isolated from a liver cDNA library and their DNA sequence determined. Each of the cDNAs was found to use a different polyadenylation signal and to be polyadenylated at a different site. This is the first observation of alternative Poly(A) site usage by a ribosmal protein gene. Northern blot analysis revealed that the chicken rpS6 transcripts can be alternatively polyadenylated in brain tissue. Southern blot analysis indicates that there is only one rpS6 gene in the chicken genome. The long open reading frame of the chicken rpS6 gene specifies 249 amino acids which show high homology with rpS6 homologues from other species. The four serine residues in rpS6 which are major sites of phosphorylation in mammals are conserved in chicken.

Amino Acid Sequence↗
PubMed2002

Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM).

Although the cellular functions of TSC2 and its protein product, tuberin, are not known, somatic mutations in the TSC2 tumor suppressor gene are associated with tumor development in lymphangioleiomyomatosis (LAM). We found that ribosomal protein S6 (S6), which exerts translational control of protein synthesis and is required for cell growth, is hyperphosphorylated in the smooth muscle-like cell lesions of LAM patients compared with smooth muscle cells from normal human blood vessels and trachea. Smooth muscle (SM) cells derived from these lesions (LAMD-SM) also exhibited S6 hyperphosphorylation, constitutive activation of p70 S6 kinase (p70S6K), and increased basal DNA synthesis. In parallel, TSC2-/- smooth muscle cells (ELT3) and TSC2-/- epithelial cells (ERC15) also exhibited hyperphosphorylation of S6, constitutive activation of p70S6K, and increased basal DNA synthesis. Re-introduction of wild type tuberin into LAMD-SM, ELT3, and ERC15 cells abolished phosphorylation of S6 and significantly inhibited p70S6K activity and DNA synthesis. Rapamycin, an immunosuppressant, inhibited hyperphosphorylation of S6, p70S6K activation, and DNA synthesis in LAMD-SM cells. Interestingly, the basal levels of phosphatidylinositol 3-kinase, Akt/protein kinase B, and p42/p44 MAPK activation were unchanged in LAMD-SM and ELT3 cells relative to levels in normal human tracheal and vascular SM. These data demonstrate that tuberin negatively regulates the activity of S6 and p70S6K specifically, and suggest a potential mechanism for abnormal cell growth in LAM.

DNA↗
PubMed2004

Functional characterization of a maize ribosomal S6 protein kinase (ZmS6K), a plant ortholog of metazoan p70(S6K).

Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.

Animals↗
PubMed1987

Comparative studies on phosphorylation of synthetic peptide analogue of ribosomal protein S6 and 40-S ribosomal subunits between Ca2+/phospholipid-dependent protein kinase and its protease-activated form.

Ca2+/phospholipid-dependent protein kinase (protein kinase C) and trypsin-activated protein kinase C (protein kinase M) phosphorylated the synthetic peptide R1-A13 (Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala-Ser-Thr-Ser-Lys-Ala) which contains both cAMP- and insulin-regulated phosphorylation sites in rat liver ribosomal protein S6 [Wettenhall, R. E. H. & Morgan, F. J. (1984) J. Biol. Chem. 259, 2084-2091]. Both enzymes showed essentially the same kinetic properties; V and apparent Km were determined to be 0.16 mumol min-1 mg-1 and 30 microM, respectively. At first, tryptic phosphopeptides were prepared at the early stage of phosphorylation and purified by high-performance liquid chromatography (HPLC). Through these analyses, four radioactive peptides were isolated. When protein kinase C was employed, phosphorylation was observed on all four peptides in a Ca2+/phospholipid-dependent manner. Irrespective of the protein kinase employed, phosphate incorporation into these peptides increased linearly with time; the peptide concentration did not affect the ratio of phosphate distribution into these four peptides. Analysis of amino acid composition and phosphoamino acid of radioactive peptides obtained after extensive phosphorylation showed that phosphates were incorporated into Ser-4, Ser-5, Ser-9 and Ser-11. The latter three serine residues were major phosphorylated sites. When rat liver 40-S ribosomal subunits were employed as substrate for protein kinases C and M, a radioactive protein with Mr,app = 31,000, which corresponded to S6 protein, was detected on an autoradiogram of a sodium dodecyl sulfate/polyacrylamide slab gel. The rate of phosphorylation with protein kinase M was twice as fast as that with protein kinase C. The elution profile of radioactive tryptic peptides in HPLC suggest that phosphorylation occurred on the sites in S6 protein corresponding to Ser-5, Ser-9 and Ser-11 as major sites and Ser-4 as the minor one. These results indicate that protein kinase C has an ability to recognize at least four sites derived from hormone-dependent phosphorylation sites in ribosomal protein S6 irrespective of the mode of activation of this enzyme.

Amino Acid Sequence↗
PubMed2005

Inactivation of S6 ribosomal protein gene in T lymphocytes activates a p53-dependent checkpoint response.

Ribosome biogenesis has been associated with regulation of cell growth and cell division, but the molecular mechanisms that integrate the effect of ribosome biogenesis on these processes in mammalian cells remain unknown. To study the effect of impaired ribosome functions in vivo, we conditionally deleted one or two alleles of the 40S ribosomal protein S6 gene in T cells in the mouse. While complete deletion of S6 abrogated T-cell development, hemizygous expression did not have any effect on T-cell maturation in the thymus, but inhibited the accumulation of T cells in the spleen and lymph nodes, as a result of their decreased survival in the peripheral lymphoid organs. Additionally, TCR-mediated stimulation of S6-heterozygous T cells induced a normal increase in their size, but cell cycle progression was impaired. Genetic inactivation of p53 tumor suppressor rescued development of S6-homozygous null thymocytes and proliferative defect of S6-heterozygous T cells. These results demonstrate the existence of a p53-dependent checkpoint mechanism that senses changes in the fidelity of the translational machinery to prevent aberrant cell division or eliminate defective T cells in vivo. Failure to activate this checkpoint response could potentially lead to a development of pathological processes such as tumors and autoimmune diseases.

Animals↗