Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

Phosphorylation of translational initiation factor 3 (eIF-3) by cyclic AMP-regulated protein kinase.

Translational initiation factor 3 (eIF-3) is phosphorylated by the cyclic AMP-regulated protein kinases from rabbit reticulocytes. eIF-3 is a large molecular weight complex which facilitates binding of the ternary complex containing met tRNA_f, GTP and initiation factor 2 to 40S ribosomal subunits. A single polypeptide with a molecular weight of 130,000 is modified. The phosphorylation is dependent upon the presence of cyclic AMP and is inhibited by the inhibitor protein diagnostic for cyclic AMP-regulated protein kinase. Assuming a molecular weight of 700,000 for eIF-3, one mole of phosphate is incorporated per mole of eIF-3. Thus the phosphorylation of two interacting components of the protein synthesizing system, 40S ribosomal subunits and eIF-3, is controlled by cyclic AMP.     

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.     

Differential activation of two protease-activated protein kinases from reticulocytes by a Ca2+-stimulated protease and identification of phosphorylated translational components

Two protein kinases have been partially purified from rabbit reticulocytes and shown to be activated by limited proteolysis with trypsin [S.M. Tahara and J.A. Traugh (1981) J. Biol. Chem. 256, 11558-11564; P.T. Tuazon, W.C. Merrick, and J.A. Traugh (1980) J. Biol. Chem. 255, 10954-10958]. Reticulocyte lysate was examined for protease activities which might be involved in activation of the protein kinases in vivo. Two neutral proteases, differentially activated by Fe2+ and Ca2+, were identified and partially purified. The Ca2+-stimulated protease specifically activated protease-activated kinase II; no effect was observed on protease-activated kinase I. The Fe2+-stimulated protease was not active on either protein kinase. The protease-activated kinases were examined using initiation factors (eIF) and 40-S ribosomal subunits as substrate. Protease-activated kinase I phosphorylated one subunit of eIF-3 (Mr 130000), eIF-4B and 40-S ribosomal protein S10. Protease-activated kinase II modified the beta subunit of eIF-2 (Mr 53000) and 40-S ribosomal protein S6. The substrate specificities are unique when compared with other cAMP-dependent and cAMP-independent protein kinases from reticulocytes.     

Protein kinase activity and ribosome phosphorylation in ethionine-treated rats.

The regulation of protein synthesis at the level of the ribosome was investigated using the model system of ethionine-induced inhibition of protein synthesis. The phosphorylation of ribosomal protein S6 was examined in vivo during ethionine intoxication and during the adenine-induced reversal of ethionine intoxication. The extent of phosphorylation of S6 correlated well with protein synthetic activity observed after ethionine, and ethionine followed by adenine treatments. No clear correlation was observed in the ethionine system between cyclic adenosine 3':5'-monophosphate concentration or the activity of ribosomal protein kinase and the phosphorylation of ribosomal protein S6. A role for a cyclic adenosine 3':5'-monophosphate-dependent ribosomal phosphoprotein phosphatase is postulated.     

Inhibition of rabbit reticulocyte protein kinases by hemin.

Hemin, at concentrations optimal for globin synthesis, produces inhibition of a specific cytoplasmic protein kinase and a ribosome-associated protein kinase isolated from rabbit reticulocytes. Both enzymes are cyclic AMP-dependent. This inhibition is noncompetitive and greater when ribosomal proteins are used as the phosphate acceptors. The inhibition of these protein kinases by hemin and its partial reversal by globin suggests that hemin regulates protein kinase activity in reticulocytes.     

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.     

Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes.

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).     

Cyclic nucleotide-independent protein kinases from rabbit reticulocytes. Purification and characterization of protease-activated kinase II

A cyclic nucleotide-independent protein kinase, protease-activated kinase II, which incorporates up to four phosphates into 40 S ribosomal protein S6, has been purified from the postribosomal supernatant of rabbit reticulocytes. Protease-activated kinase II was purified as an inactive proenzyme by chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-150, and hydroxylapatite. The enzyme was activated in vitro by limited digestion with trypsin or chymotrypsin. No other mode of activation for protease-activated kinase II in vitro was identified. The proenzyme had a molecular weight of 80,000 as measured by gel filtration; following tryptic digestion, the molecular weight of the activated protein kinase was 45,000-55,000. Protease-activated kinase II required Mg2+ for activity but was inhibited by other divalent cations, monovalent cations, and fluoride ion. ATP was the phosphoryl donor in the phosphorylation reaction; GTP had no effect. In vitro, multiple phosphorylation of S6 was observed with some phosphate incorporated into S10. Phosphorylation of S6 by protease-activated kinase II has been shown to be stimulated in serum-starved 3T3-L1 cells by insulin (Perisic, O., and Traugh, J. A. (1983) J. Biol. Chem. 258, 9589-9592) and in reticulocytes by altering the pH of the incubation medium (Perisic, O., and Traugh, J. A. (1983) J. Biol. Chem. 258, 13998-14002.     

Differential Responses of the Phosphorylation of Ribosomal Protein S6 to Nerve Growth Factor and Epidermal Growth Factor in PC 12 Cells

Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor-sensitive S6 kinase and of the epidermal growth factor-sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP-dependent kinase, kinase C, or the calcium/calmodulin-dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor-treated cells were also seen on phosphorylation of S6 by cyclic AMP-dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP-dependent kinase in vitro, the action of the nerve growth factor-sensitive S6 kinase was increased about twofold.     

Cyclic nucleotide-independent protein kinases from ribosomes and phosphorylation of a single 40S ribosomal subunit protein in zoospores of Blastocladiella emersonii

Cyclic nucleotide-independent protein kinase (EC 2.7.1.37) activity was found in the nuclear cap organelle, within which ribosomes of zoospores of Blastocladiella emersonii are sequestered. Two protein kinase activities were resolved from the high-salt wash fraction of zoospore ribosomes by selective adsorption to DEAE-cellulose. Both enzymes phosphorylated in vitro a 32,000 Mr protein of the 40S ribosomal subunit. Phosphorylation of this ribosomal protein, which exhibits electrophoretic properties similar to those of mammalian ribosomal protein S6, was also observed in vivo in 32P-labeled zoospores.     

A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.

A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.     

Isolation of ribosomal subparticles from human placenta containing intact rRNA and determination of the functional activity of the 80S ribosome

The method for isolation of human placenta ribosomal subunits containing intact rRNA has been determined. The method uses fresh unfrozen placenta. Activity of 80S ribosomes obtained via reassociation of 40S and 60S subunits in non-enzymatic poly(U)-mediated Phe-tRNAPhe binding, was near 75% (maximal [14C]Phe-tRNA(Phe) binding was 1.5 mol Phe-tRNA(Phe) per mol of 80S ribosomes). Activity of 80S ribosomes with damaged rRNA isolated from frozen placenta was 2 times lower (the maximum level of poly(U)-dependent Phe-tRNA(Phe) binding was 0.7 mol per mol of ribosomes). The activity 80S ribosomes in poly(U)-mediated synthesis of polyphenylalanine was determined by using fractionated ("ribosomeless") protein synthesising system from rabbit reticulocytes. In this system up to the 50 mol of Phe residues per mol of 80S ribosomes are incorporated in acid insoluble fraction in 1 hour, at 37 degrees C. The obtained level of [14C]phenylalanine incorporation is three times as much as the amount of Phe residues observed for the ribosomal subunits, isolated from frozen placenta.     

Eukaryotic ribosomal proteins stimulate Escherichia coli stringent factor to synthesize guanosine 5''-diphosphate, 3''-diphosphate (ppGpp) and guanosine 5''-triphosphate, 3''-diphosphate (ppGpp).

Summary When supplemented with Escherichia coli stringgent factor, 80S ribosomes from various sources failed to support guanosine tetra- and pentaphosphate ((p)ppGpp) synthesis. In contrast, ribosomal proteins from 80S, 60S or 40S particles (mouse embryos, rabbit reticulocytes) crossreacted with the E. coli stringent factor. Significant stimulation of (p)ppGpp synthesis was achieved proteins/ml. These observations may provide additional criteria to detect homologies between eukaryotic and prokaryotic ribosomal proteins.     

Further evidence for the participation of proteins S 3, S 14 and S 19 in tRNA binding to E. coli 30 S subunits

Previous studies have shown that iodination of 30 S subunits causes inactivation for both enzymatic fMet-tRNA and non-enzymatic phe-tRNA binding activities. This inactivation was shown to be due to the modification of three to five ribosomal proteins [1]. In this report the role of these proteins in tRNA binding activity has been further studied. Purified ribosomal proteins, isolated from modified subunits, are re-assembled into otherwise unmodified 30 S ribosomes and assayed for tRNA binding capacity. The presence of modified S 3, S 14 and S 19 (S 15) in the reconstituted particle results in substantial reduction of both fMet-tRNA and phe-tRNA binding activities. This reduction in tRNA binding activity does not appear to be due to an assembly defect.     

Ca2+-independent activation of protease-activated kinase II by phospholipids/diolein and comparison with the Ca2+/phospholipid-dependent protein kinase

The proenzyme form of protease-activated kinase (PAK) II from reticulocytes has been shown to be activated in vitro by limited proteolysis and characterized using 40 S ribosomal subunits as substrate (T.H. Lubben and J.A. Traugh (1983) J. Biol. Chem. 258, 13992-13997). In these studies, we have shown that PAK II can be activated in a Ca2+-independent manner with phospholipids/diolein using histone 1, eukaryotic initiation factor 2, and 40 S ribosomal subunits as substrates. The addition of Ca2+ results in a diminution of PAK II activity. The Ca2+/phospholipid-dependent protein kinase (protein kinase C) is present in reticulocytes and is separated from PAK II during purification by chromatography on ADP-agarose. PAK II activated by limited proteolysis has the same substrate specificity as PAK II activated by phospholipids/diolein as shown by two-dimensional finger-printing of tryptic phosphopeptides of histone 1 and ribosomal protein S6, indicating proteolysis did not alter the specificity of the enzyme. Lipid vesicles decrease the Km of PAK II for histone 1 by 10-fold, while no effect is observed on the Km or the Vmax of PAK II for ATP. These results are strikingly different from the kinetics reported for protein kinase C, where the activators increase the Vmax for ATP. The two enzymes have similar, if not identical, substrate specificity with histone 1, as determined by phosphopeptide mapping, but at least 8-fold more protein kinase C than PAK II is required to incorporate a comparable amount of phosphate into S6 and it is not possible to incorporate stoichiometric amounts of phosphate into S6 with protein kinase C. The two protein kinases also differentially phosphorylate other substrates. The data support the hypothesis that PAK II and protein kinase C are closely related, but unique enzymes.     

Demonstration of RNA N-glycosidase activity of a Vero toxin (VT2 variant) produced by Escherichia coli O91:H21 from a patient with the hemolytic uremic syndrome

A new Vero toxin purified from Escherichia coli O91:H21 isolated from a patient with the hemolytic uremic syndrome (VT2vh) was shown to inhibit elongation factor 1-dependent aminoacyl-tRNA binding to ribosomes, resulting in inhibition of protein synthesis in rabbit reticulocytes. VT2vh, like Shiga toxin, VT1 and VT2, showed RNA N-glycosidase activity and cleaved the N-glycosidic bond of the adenosine residue at position 4324 in 28S ribosomal RNA.     

A membrane-bound protein kinase from rabbit reticulocytes is an active form of multipotential S6 kinase

An active ribosomal protein S6 kinase has been highly purified from the membranes of rabbit reticulocytes by chromatography of the Triton X-100 extract on DEAE-cellulose, SP-Sepharose Fast Flow, and by FPLC on Mono Q and Superose-12. The S6 kinase elutes around 40 000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit molecular weight of 40–43 kDa as determined by protein kinase activity following denaturation/renaturation in SDS-polyacrylamide gels containing S6 peptide. It also phosphorylates translational initiation factors eIF-2 and eIF-4F, glycogen synthase, histone 1, histone 2B, myelin basic protein, but not prolactin, skeletal myosin light chain, histone 4, tubulin, and casein. Apparent K_m values have been determined to be 15 μM for ATP, 1.2 μM for S6 and 10 μM for S6 peptide. Two-dimensional tryptic phosphopeptide mapping shows the same sites on S6 are phosphorylated as those identified previously with proteolytically activated multipotential S6 kinase from rabbit reticulocytes, previously denoted as protease activated kinase II. Examination of relative rates of phosphorylation and kinetic constants of synthetic peptides based on previously identified phosphorylation sites, indicates a minimum substrate recognition sequence to be arginine at the n − 3 position. Based on these characteristics, including molecular weight and an expanded substrate specificity, the membrane S6 kinase can be distinguished from the p90 (Type I) and p70 (Type II) S6 kinases, and from protein kinase C and the catalytic subunit of cAMP-dependent protein kinase.     

Partial purification and characterization of reticulocyte phosphatase with activity for phosphorylated peptide initiation factor 2

An enzyme fraction containing phosphatase activity for phosphorylated eukaryotic peptide initiation factor 2 (eIF-2) has been isolated from rabbit reticulocytes and partially characterized. The enzyme efficiently catalyzes release of phosphate from the small subunit of eIF-2 (eIF-2 alpha) that has been phosphorylated by the hemin-controlled repressor. It is shown to restore activity of this phosphorylated eIF-2 for binding of methionyl-tRNAf to 40 S ribosomal subunits in a partial reaction of peptide initiation. The enzyme fraction also has phosphatase activity for eIF-2 phosphorylated in its largest subunit and for the 100,000-dalton peptide associated with the eIF-2 alpha kinase activity of the hemin-controlled repressor. The phosphoprotein phosphatase has been isolated by a procedure involving precipitation with ethanol at room temperature and has an apparent molecular weight in the order of 76,000. Its phosphatase activity for eIF-2 alpha is stimulated about 3-fold by optimal concentrations of Mn2+, but is not stimulated by Ca2+ or Mg2+. The enzyme is strongly inhibited by Fe2+ and by purine nucleoside diphosphates.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.