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E Erikson

Publications and source records attributed to E Erikson.

At least 37 records · Page 2Linked to original sources
PubMed1989

Biochemical characterization of the p34cdc2 protein kinase component of purified maturation-promoting factor from Xenopus eggs.

Genetic studies in the fission yeast Schizosaccharomyces pombe and biochemical data in oocytes and eggs of Xenopus laevis have implicated the product of the cdc2+ gene as critical for the G2 to M transition in the cell cycle. The product of the cdc2+ gene is a 34-kDa serine/threonine protein kinase, designated p34cdc2, that is a component of purified maturation-promoting factor (MPF) and also of purified mammalian growth-associated histone H1 kinase. The biochemical properties of p34cdc2 H1 kinase activity in the MPF complex were studied. Phosphorylation of the p45cyclin component in the MPF complex by p34cdc2 exhibited kinetics consistent with an intramolecular reaction. On glycerol gradient centrifugation, MPF kinase against several substrates sedimented with an apparent Mr = 45,000-55,000. p34cdc2 was found to utilize ATP, GTP, and adenosine 5'-O-(3-thiotriphosphate) with apparent Km values of 75, 700, and 250 microM, respectively. The kinase activity was inhibited by beta-glycerophosphate, NaF, and zinc, whereas p-nitrophenyl phosphate was slightly stimulatory. The relative rates of phosphorylation of various substrates by MPF and growth-associated H1 kinase were similar. These findings should prove useful in further work on the regulation of MPF kinase activity and characterization of its substrates.

Animals↗
PubMed1989

In vivo phosphorylation and activation of ribosomal protein S6 kinases during Xenopus oocyte maturation.

Previous studies have shown that increased ribosomal protein S6 kinase activity in unfertilized Xenopus eggs can be resolved by DEAE-Sephacel chromatography into two peaks, designated S6 kinase I and S6 kinase II. We show here that antibody against bacterially expressed S6 kinase II cross-reacts with S6 kinase I. Both S6 kinases undergo marked phosphorylation when they are activated during oocyte maturation, and both become deactivated and dephosphorylated upon activation of eggs. Immunoblotting of extracts of oocytes reveals that all S6 kinase molecules undergo a decrease and increase in electrophoretic mobility upon activation and deactivation, respectively. The increase in electrophoretic mobility can be produced in vitro by incubation of activated S6 kinase with purified phosphatases. Phosphoamino acid analysis of S6 kinase II labeled in vivo during maturation reveals both phosphoserine and phosphothreonine, and phosphopeptide maps suggest that several kinases may phosphorylate and activate S6 kinase II in vivo. These results demonstrate that, during oocyte maturation and early development, S6 kinase activation and deactivation are regulated by phosphorylation and dephosphorylation, suggesting a probable mechanism for S6 kinase regulation in other mitogenically stimulated cells.

Amino Acids↗
PubMed1988

Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II.

Ribosomal protein S6 is a component of the eukaryotic 40S ribosomal subunit that becomes phosphorylated on multiple serine residues in response to a variety of mitogens, including insulin, growth factors, and transforming proteins of many oncogenic viruses. Recently, an activated S6 kinase (S6 K II) has been purified to homogeneity from Xenopus eggs, and characterized immunologically and at the molecular level. Purified S6 K II can be deactivated in vitro by incubation with either protein phosphatase 1 or protein phosphatase 2A. Reactivation and phosphorylation of S6 K II occurs in vitro with an insulin-stimulated microtubule-associated protein-2 (MAP-2) protein kinase which is itself a phosphoprotein that can be deactivated by protein phosphatase 2A. These studies suggest that a step in insulin signalling involves sequential activation by phosphorylation of at least two serine/threonine protein kinases.

Animals↗
PubMed1988

A Xenopus ribosomal protein S6 kinase has two apparent kinase domains that are each similar to distinct protein kinases.

We report the molecular cloning of cDNAs for S6 kinase II (S6KII) mRNAs present in Xenopus ovarian tissue. Two cDNAs were isolated by hybridization to oligonucleotide probes designed to encode tryptic peptides isolated from S6KII. The two cDNAs show 91% sequence similarity to each other. These two cDNAs predict proteins of 733 (S6KII alpha) and 629 (S6KII beta) amino acids that show 95% sequence similarity over the 629 amino acids where they are colinear. Amino acids 44-733 of S6KII alpha were expressed in Escherichia coli and the recombinant protein was used to raise antiserum in rabbits. This antiserum reacted with authentic S6KII prepared from Xenopus eggs. This interaction was specifically blocked by the recombinant protein from E. coli. The sequences of S6KII alpha and -beta predict four tryptic peptides whose sequences are identical to four peptides isolated from a tryptic digest of S6KII. The S6KII proteins have a very unusual structure when compared with previously studied protein kinases. They contain two apparent kinase domains, each similar to distinct protein kinases. The amino-terminal 366 amino acids show high sequence similarity to the regions of protein kinase C, the catalytic subunit of cAMP-dependent protein kinase, and cGMP-dependent protein kinase that contain the sites for ATP binding and are believed to be the catalytic centers for phosphotransferase activity. The remainder of the S6 kinase molecule shows high sequence similarity to the ATP-binding and presumed catalytic domain of the catalytic subunit of phosphorylase b kinase.

Amino Acid Sequence↗
PubMed1988

Molecular analyses of gene products associated with the response of cells to mitogenic stimulation.

We have completed the molecular analysis of a ribosomal protein S6 kinase identified in unfertilized Xenopus eggs. Although some of our results support the notion that an antigenically related enzyme is present in animal cells, we are uncertain if such an enzyme accounts for all of the mitogen-stimulated phosphorylation of 40S subunits. Other experiments have led to the identification of mRNAs that are rapidly expressed in mitogen-stimulated cells. The relevance of such genes and their products is in doubt, however, until a function can be demonstrated for each of them. Preliminary experiments with the pCEF-4 gene product described here have failed to show that it acts as a mitogen for cells in culture, for example. Thus, although studies such as these may lead to the identification of novel genes, we may need to search elsewhere for a physiologically significant function.

Amino Acid Sequence↗
PubMed1988

Substrate specificity of ribosomal protein S6 kinase II from Xenopus eggs.

The substrate specificity of ribosomal protein S6 kinase II (S6 K II) from Xenopus eggs was evaluated using several protein substrates and a synthetic peptide corresponding to two phosphorylation sites in ribosomal protein S6. Previous studies had shown that S6 K II is unable to phosphorylate histones, casein, or phosvitin, proteins commonly used as substrates for protein kinases. In the present study S6 K II was found to phosphorylate with a significant stoichiometry rabbit skeletal muscle glycogen synthase, cardiac and skeletal muscle troponin I, and lamin C. In addition, the S6 peptide was phosphorylated by S6 K II to the same extent as observed with the catalytic subunit of cAMP-dependent protein kinase. Studies with oocytes undergoing progesterone-induced meiotic maturation and with activated or fertilized eggs revealed identical oscillations in both S6 and lamin C kinase activity. These results indicate that S6 K II does not have an absolute specificity for S6 in vitro. Therefore, since this enzyme is regulated during the cell cycle, it may phosphorylate several other proteins of interest during mitogenic stimulation.

Animals↗
PubMed1987

Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

Ribosomal protein S6 becomes highly phosphorylated during progesterone- or insulin-induced maturation of Xenopus laevis oocytes. We have previously purified an Mr 92,000 protein as one of the major S6 kinases from Xenopus unfertilized eggs. In this paper we confirm by renaturation of activity from a sodium dodecyl sulfate-polyacrylamide gel that this protein is an S6 kinase. This enzyme, termed S6 kinase II (S6 K II), was used for the preparation of polyclonal antiserum. Immunocomplexes formed with the antiserum and purified S6 K II were able to express kinase activity with the same substrate specificity as that of the purified enzyme, including autophosphorylation of S6 K II itself. The antiserum did not react with S6 kinase I, another major S6 kinase present in Xenopus eggs, which is chromatographically distinct from S6 K II. The administration of progesterone to oocytes resulted in a 20- to 25-fold increase in S6 kinase activity in extracts of these cells. Immunocomplex kinase assays done on extracts revealed that anti-S6 K II serum reacted with S6 kinase from progesterone-treated oocytes. This antiserum also reacted with the activated S6 kinase from insulin-stimulated oocytes. In addition, anti-S6 K II serum reacted with activated S6 kinase from chicken embryo fibroblasts stimulated with serum or transformed by Rous sarcoma virus. These results indicate that S6 K II or an antigenically related S6 kinase(s) is subject to regulation by mitogenic stimuli in various cell types.

Animals↗
PubMed1986

Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6.

In Xenopus oocytes ribosomal protein S6 becomes phosphorylated on serine residues in response to hormones or growth factors and following microinjection of the tyrosine-specific protein kinases associated with Rous sarcoma virus or Abelson murine leukemia virus. To begin characterization of the enzymes responsible for S6 phosphorylation in this system, we have undertaken the purification of S6 protein kinases from unfertilized Xenopus eggs. DEAE-Sephacel chromatography of crude extracts revealed two peaks of S6 kinase activity, and the peak eluting at 160 mM NaCl was chosen for further purification. Successive chromatography on Mono S, Sephacryl S-200, Mono Q, and heparin-Sepharose resulted in purification of the enzyme to a single protein migrating at Mr = 92,000 on polyacrylamide gels. The final preparation was purified about 500-fold from the DEAE-Sephacel peak with a recovery of 10%. Apparent Km values of the enzyme for ATP and 40 S subunits were 28 and 5 microM, respectively, and the specific activity with 330 microM ATP and 5.6 microM 40 S subunits was 300 nmol/min/mg. The enzyme was inhibited by beta-glycerophosphate, sodium fluoride, potassium phosphate, ADP, heparin, quercetin, and spermine. The availability of a purified S6 protein kinase should facilitate elucidation of the molecular mechanism of S6 phosphorylation during growth stimulation.

Adenosine Triphosphate↗
PubMed1986

Activation of a ribosomal protein S6 protein kinase in Xenopus oocytes by insulin and insulin-receptor kinase.

Previous studies in this laboratory have shown that insulin treatment of Xenopus oocytes leads to an increase in phosphorylation of ribosomal protein S6. To investigate the mechanism of this increase, S6 kinase activity was measured in lysates of oocytes exposed to insulin. Insulin caused a rapid 4- to 6-fold increase in S6 kinase activity, which was maximal by 20 min and which could be reversed by removal of insulin prior to homogenization. Dose-response curves showed a detectable increase in specific activity at 1 nM insulin with a maximal effect at 100 nM. Treatment of oocytes with puromycin did not prevent this increase in S6 kinase activity, suggesting activation rather than synthesis of the enzyme. DEAE-Sephacel chromatography of extracts from insulin-treated oocytes revealed two peaks of S6 kinase activity, and the specific activity of the peak eluting at 300 nM NaCl was increased 3-fold in oocytes treated with insulin. The same peak of S6 kinase activity was increased 40% within 10 min in oocytes injected with highly purified insulin-receptor kinase. These results indicate that the insulin-dependent increase in S6 phosphorylation is due, at least in part, to activation of an S6 protein kinase, and this activation may result from the action of the insulin receptor at an intracellular location.

Animals↗
PubMed1985

Inter- and intramolecular interactions of highly purified Rous sarcoma virus-transforming protein, pp60v-src.

We have purified intact pp60v-src, the product of the Rous sarcoma virus src gene, over 2400-fold, based on the phosphorylation of tumor-bearing rabbit IgG. The purification procedure involved detergent extraction of the particulate fraction of the cells and sequential chromatography on hydroxylapatite, butyl agarose, DEAE-Sephacel, ADP-agarose, and Sephacryl S-200. Analysis of the preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-stained band with an apparent molecular weight of 60,000. Our results show that the activities of this preparation were qualitatively similar to those described previously for partially purified pp60v-src. Upon analysis by two-dimensional gel electrophoresis, the purified pp60v-src yielded one major species which migrated to the same position as the least acidic of the three major species detectable in cellular lysates, suggesting that the pp60v-src had been dephosphorylated during the purification procedure. We found that pp60v-src was very prone to aggregation; to maintain it as a monomer both Nonidet P-40 and KCl were required. Under conditions which maintained pp60v-src as a monomer, the rate of autophosphorylation was independent of its concentration and thus proceeded via an intramolecular process. Preincubation of pp60v-src with ATP or GTP as well as nonphosphorylating analogs of ATP or GTP preserved its phosphorylating activity toward alpha-casein whereas its activity was reduced 80% upon preincubation in the absence of nucleotides. We suggest that protection with nucleotides rather than autophosphorylation accounts for the apparent increase in the activity of pp60v-src after incubation of the enzyme with ATP.

Adenosine Triphosphate↗
PubMed1985

Phosphorylation of ribosomal protein S6 on serine after microinjection of the Abelson murine leukemia virus tyrosine-specific protein kinase into Xenopus oocytes.

Phosphorylation of ribosomal protein S6 in NIH 3T3 fibroblasts is dependent on the presence of serum, but after transformation of these cells by Abelson murine leukemia virus (Ab-MuLV), S6 remained highly phosphorylated on serine residues either in the absence or the presence of serum. To investigate whether S6 phosphorylation in this system was a consequence of the action of the Ab-MuLV tyrosine-specific protein kinase, purified Ab-MuLV kinase made in Escherichia coli was microinjected into Xenopus oocytes and was observed to cause a 7- to 15-fold increase in the phosphorylation of S6 on serine residues. Two-dimensional phosphopeptide maps of S6 phosphorylated in Ab-MuLV-transformed NIH cells in the absence of serum were identical to those of S6 isolated from normal cells grown in the presence of serum. In addition, S6 from oocytes injected with Ab-MuLV kinase yielded an S6 phosphopeptide map indistinguishable from that of serum-stimulated NIH 3T3 cells, whereas S6 from control oocytes lacked several phosphopeptides. Ab-MuLV kinase did not phosphorylate S6 directly in vitro, and microinjection of a mutant Ab-MuLV protein lacking kinase activity had no effect. These results indicate that the Ab-MuLV kinase interacts with a cellular pathway to enhance S6 phosphorylation by directly or indirectly activating an S6 protein kinase and/or inactivating an S6 protein phosphatase.

Abelson murine leukemia virus↗
PubMed1985

A protein kinase from Xenopus eggs specific for ribosomal protein S6.

A protein kinase specific for ribosomal protein S6 has been purified from eggs of Xenopus laevis. As visualized on a silver-stained polyacrylamide gel, the major protein in the preparation migrated with a Mr of 90,000. Incubation of the enzyme preparation with [gamma-32P]ATP led to phosphorylation of this protein on serine residues. Upon glycerol gradient centrifugation, the S6 kinase activity and the Mr 90,000 protein both sedimented with a Mr of 50,000-55,000. Two-dimensional gel electrophoresis demonstrated that up to 4-5 phosphate groups per S6 molecule could be incorporated with this enzyme in vitro, and two-dimensional peptide mapping demonstrated that the phosphopeptides from S6 labeled in vitro with the enzyme comigrated with those from highly phosphorylated S6 labeled in vivo in response to progesterone treatment. The purified S6 protein kinase did not phosphorylate at a significant rate ribosomal protein S10, histone H1, histone H4, mixed histones, casein, or phosvitin, indicating a high degree of substrate specificity. These results indicate that activation of a single S6 protein kinase may be sufficient to account for increased S6 phosphorylation after a growth stimulus.

Animals↗
PubMed1984

Biochemical characterization of a 34-kilodalton normal cellular substrate of pp60v-src and an associated 6-kilodalton protein.

Transformation of fibroblasts by several retroviruses that produce transforming gene products associated with protein kinase activity results in the phosphorylation of a normal cellular protein with an Mr of 34,000 (the 34K protein). Evidence is presented here that, as extracted from chicken embryo fibroblasts, this protein exists in two forms that differ both in their elution from hydroxylapatite and in their native molecular weight. The form that eluted from hydroxylapatite at 210 to 295 mM potassium phosphate displayed a native molecular weight of 30,000 to 40,000, whereas the form that eluted at 320 to 440 mM displayed a native molecular weight of 60,000 to 70,000. The latter form copurified with a low-molecular-weight protein with an approximate Mr of 6,000 (6K). Both forms of 34K were completely separable from malate dehydrogenase activity. Phosphorylated 34K, isolated from Rous sarcoma virus-transformed cells, was also present in two forms; hence, in the cell neither form serves as a preferential substrate for pp60v-src. We found that the expression of 34K differed greatly in various avian tissues. In particular, it was present in the highest concentration in cultured fibroblasts and in very low concentration in brain tissue. Its expression in this tissue seems to be controlled at the level of transcription, since 34K mRNA in brain tissue was barely detectable. The expression of 6K was similar to that of 34K.

Animals↗
PubMed1983

Antigenic and structural studies on the transforming proteins of Rous sarcoma virus and Yamaguchi 73 avian sarcoma virus.

A widely cross reactive antiserum raised against denatured pp60v-src, the transforming protein encoded by Rous sarcoma virus, was used to test antigenic relationships with the transforming gene products encoded by other avian sarcoma viruses. The results showed that P90gag-yes, the transforming protein of a representative of Class III avian sarcoma viruses, is antigenically related to pp60v-src. Tryptic phosphopeptide analysis of P90gag-yes revealed two phosphotyrosine-containing peptides and one phosphoserine-containing peptide. One of the phosphotyrosine-containing peptides comigrated with the phosphotyrosine-containing tryptic peptide from pp60v-src.

Antigens, Viral↗
PubMed1983

The effect of quercetin on the phosphorylation activity of the Rous sarcoma virus transforming gene product in vitro and in vivo.

The phosphotransferase activity of the Rous sarcoma virus src gene product, pp60src, was inhibited both in vitro and in vivo by the bioflavonoid quercetin. The Ki for the inhibitory effect was in the range of 6-11 microM under conditions in vitro. The inhibitory effect of quercetin was competitive towards the nucleotides ATP and GTP as substrates for pp60src and was non-competitive towards alpha-casein as the protein substrate of this kinase activity. In contrast, studies in vitro of the phosphotransferase activity of the catalytic subunit of the cAMP-dependent protein kinase showed that this flavonoid did not inhibit the phosphorylation of physiological substrates of this enzyme. In cultured cells the half-maximal inhibition of tyrosine phosphorylation of pp60src as well as the phosphorylation of the Mr = 34000 protein, a physiological substrate of pp60src, was in the range 0.06-0.08 mM.

Animals↗
PubMed1983

Characterization of the Rous sarcoma virus transforming gene product.

This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus transforming gene product, pp60src, using ATP or GTP as phosphate donors. The Km values for the phosphorylation of pp60src by ATP and GTP were similar (10-36 and 25-36 microM, respectively) and the Vmax values were different (5-7 and 1.5-1.7 nmol min-1 mg-1 of pp60src, respectively). The radiolabeling of pp60src by [gamma-32P] ATP was inhibited by ADP and dATP at 20-fold higher concentrations by 75 and 83%, respectively. Other nucleotides served as weaker inhibitors under the same conditions. The radiolabeling of pp60src by [gamma-32P]GTP had lower specificity for this nucleotide, since ATP, dATP, ADP, dGTP, GDP, and TTP had at least a 50% inhibitory effect. The phosphorylated products of approximate Mr = 60,000 that were produced with ATP or GTP were shown to be the same protein molecule since they both could be immunoprecipitated with antibody raised against p60src produced by bacterial recombinants. Structural analysis revealed that the use of GTP resulted in phosphorylation of a tyrosine residue on the COOH-terminal region of pp60src, apparently the same site which contains the tyrosine phosphorylated in infected cells. In contrast, the use of ATP resulted in phosphorylation of several additional tyrosine residues on the NH2-terminal region of the molecule. In thermolability studies, the t1/2 values for the phosphorylation of pp66src in preparations from wild type virus-infected chicken cells were 5.1 min for both ATP and GTP, whereas the t1/2 values for the phosphorylation of pp60src in preparations from temperature-sensitive transformation mutant-infected cells were 1.1 min for both phosphate donors. Similar observations were found with alpha-casein as substrate.

Adenosine Triphosphate↗
PubMed1983

Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity.

This communication provides biochemical, immunological, and genetic evidence that pp60src, the Rous sarcoma virus transforming gene product, is associated with glycerol kinase activity. Our investigations demonstrated that the compound phosphorylated by pp60src or by glycerol kinase (EC 2.7.1.30) from Candida mycoderma share the same electrophoretic and chromatographic mobilities. The glycerol kinase and protein kinase activities of pp60src were inhibited similarly by preincubation with immune IgG. Both activities were reduced 6-9-fold in pp60src preparations derived by immunoaffinity chromatography from cells which were infected with NY68, a temperature-sensitive transformation mutant of Rous sarcoma virus. The thermolability at 41 degrees C of the glycerol kinase activity of pp60src from the mutant virus-infected cells was greater (t/2 = 1.3 min) than the same activity in pp60src preparations from wild type virus-infected cells (t/2 = 4.8 min).

Animals↗