RESUMO
In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) catalytic subunit with multiple regulatory roles thought to be specified by association with different cyclin partners (Pcls). Pcl10p is one of four Pcls with little sequence similarity to cyclins involved in cell cycle control. It has been implicated in specifying the phosphorylation of glycogen synthase (Gsy2p). We report that recombinant Pho85p and Pcl10p produced in Escherichia coli reconstitute an active Gsy2p kinase in vitro. Gsy2p phosphorylation required Pcl10p, occurred at physiologically relevant sites, and resulted in inactivation of Gsy2p. The activity of the reconstituted enzyme was even greater than Pho85p-Pcl10p isolated from yeast, and we conclude that, unlike many Cdks, Pho85p does not require phosphorylation for activity. Pcl10p formed complexes with Gsy2p, as judged by (i) gel filtration of recombinant Pcl10p and Gsy2p, (ii) coimmunoprecipitation from yeast cell lysates, and (iii) enzyme kinetic behavior consistent with Pcl10p binding the substrate. Synthetic peptides modeled on the sequences of known Pho85p sites were poor substrates with high K(m) values, and we propose that Pcl10p-Gsy2p interaction is important for substrate selection. Gel filtration of yeast cell lysates demonstrated that most Pho85p was present as a monomer, although a portion coeluted in high-molecular-weight fractions with Pcl10p and Gsy2p. Overexpression of Pcl10p sequestered most of the Pho85p into association with Pcl10p. We suggest a model for Pho85p function in the cell whereby cyclins like Pcl10p recruit Pho85p from a pool of monomers, both activating the kinase and targeting it to substrate.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas Fúngicas/metabolismo , Glicogênio Sintase/metabolismo , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ativação Enzimática , Escherichia coli/genética , Proteínas Fúngicas/genética , Espectrometria de Massas , Modelos Biológicos , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Recombinantes/metabolismoRESUMO
The ABRF amino acid analysis study evaluated the general utility of amino acid analysis (AAA) for identification of proteins after denaturing gel electrophoresis and electroblotting to polyvinylidene difluoride (PVDF) membrane.Thirty-eight participating laboratories analyzed a known control (ovalbumin, 5 microg applied to the gel) and either lysozyme or bovine serum albumin as unknown samples (1-, 5-, and 10-microg amounts applied to the gel). Analyses of the unknowns yielded average compositional errors of approximately 30%, 19%, and 18%, respectively, from the low, intermediate, and higher sample amounts; the ovalbumin control exhibited an approximately 17% average error. Compositional data were submitted to the ExPASy and PROPSEARCH Internet sites for protein identification.Without search parameter adjustments or restrictions, both computer programs provided identification of about 20%, 66%, and 74% of the data from the 1-, 5-, and 10-microg gel samples, respectively. Deleting problematic data (Gly, Met, and Pro) did not always facilitate protein identification. Incorporating control results into the ExPASy search increased identifications 2% to 10%, and restricting search parameters by species, isoelectric pH, and molecular weight increased identifications by more than 80%. Average amounts analyzed for correct identifications were approximately 0.4 microg, 1.8 microg, and 2.9 microg for the 1-, 5-, and 10-microg gel samples, respectively.The results support the efficacy of AAA in the low microgram and nanogram range for the identification of PVDF-immobilized proteins from two-dimensional gels.
RESUMO
The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.
Assuntos
Precursores Enzimáticos/química , Fosfotirosina/química , Proteínas Tirosina Quinases/química , Tirosina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/química , Camundongos , Dados de Sequência Molecular , Fosfolipase C gama , Proteínas Quinases/química , Proteínas Recombinantes/química , Transdução de Sinais , Quinase Syk , Fosfolipases Tipo C/química , Domínios de Homologia de srcRESUMO
The biogenesis of glycogen involves a specific initiation event mediated by the initiator protein, glycogenin, which undergoes self-glucosylation to generate an oligosaccharide primer from which the glycogen molecule grows. Rabbit muscle glycogenin was expressed at high levels in Escherichia coli and purified close to homogeneity in a procedure that involved binding to a UDP-agarose affinity column. The resulting protein had subunit molecular weight of 38,000 as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Analysis of peptide fragments by mass spectroscopy indicated that the recombinant glycogenin was already glucosylated at Tyr-194 and contained from 1 to 8 glucose residues attached. The enzyme was active as a glucosyl transferase and could incorporate a further approximately 5 mol of glucose/mol. The apparent Km for the glucosyl donor UDP-glucose was 4.5 microM, and the pH optimum was pH 8. Of a number of nucleotides and related compounds surveyed, UDP and UTP were the most effective inhibitors. There was also a correlation between inhibition and the presence of a pyrophosphate group. Of several oligosaccharides of glucose, only maltose caused significant inhibition. The glucosylation reaction was first order with respect to glycogenin suggesting that it was intramolecular. The efficacy of the purified glycogenin as a substrate for the elongation reaction catalyzed by glycogen synthase was significantly enhanced if glycogenin was first allowed to undergo self-glucosylation. The length of the priming oligosaccharide is thus important for glycogen synthase action. A mutant of glycogenin, in which Tyr-194 was changed to Phe, behaved identically to the wild-type through purification and in particular bound to the UDP-agarose affinity matrix. Despite these indications of the protein's overall structural integrity, it was unable to self-glucosylate. This result indicates that Tyr-194 is necessary for glycogenin function and is consistent with Tyr-194 being the sole site of glucosylation.
Assuntos
Glicoproteínas/metabolismo , Proteínas Musculares/metabolismo , Músculos/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Escherichia coli , Glucose/metabolismo , Glucosiltransferases , Glicogênio/metabolismo , Glicogênio Sintase/metabolismo , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Dados de Sequência Molecular , Proteínas Musculares/genética , Proteínas Musculares/isolamento & purificação , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Coelhos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Sequence information was obtained from low picomole amounts of nonspecific cross-reacting antigen (NCA) 160 (M(r) 160,000), a granulocyte membrane glycoprotein. Following affinity purification and SDS-polyacrylamide gel electrophoresis, the protein was electrotransferred to nitrocellulose, digested with trypsin, and the peptides were isolated using capillary reversed-phase liquid chromatography. Analysis of these peptides by Edman microsequencing and mass spectrometry established that NCA-160 was identical to biliary glycoprotein I, a protein that we previously cloned from a human colon library (1). NCA-160 from human granulocytes is a CD15-positive glycoprotein belonging to the carcinoembryonic antigen family and possesses putative transmembrane and cytoplasmic domains. Previous efforts to characterize this antigen at the protein level were hampered by a blocked NH2 terminus. In this study, we confirmed 20% of the deduced amino acid sequence starting with approximately 50 pmol of sample. Carbohydrate structural data is also presented on a single N-linked oligosaccharide moiety located in the A' domain. The capillary high performance liquid chromatography techniques used here, as well as mass spectrometry, were essential for high sensitivity analysis of the blotted, digested glycoprotein.
Assuntos
Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Glicoproteínas/química , Glicoproteínas de Membrana/química , Neutrófilos/química , Sequência de Aminoácidos , Sequência de Carboidratos , Moléculas de Adesão Celular , Reações Cruzadas , Humanos , Antígenos CD15 , Espectrometria de Massas , Dados de Sequência MolecularRESUMO
We have reported previously that phosphofructokinase from the liver fluke Fasciola hepatica is activated by phosphorylation with cAMP-dependent protein kinase, and that this event appears to be important in vivo for regulation of PFK (E. S. Kamemoto, L. Lan, and T. E. Mansour (1989) Arch. Biochem. Biophys. 271, 553-559). Here, we report the amino acid sequence of the single tryptic phosphopeptide generated after phosphorylation of the purified enzyme with cAMP-dependent protein kinase and [gamma 32P]ATP. Through a combination of Edman microsequence analysis, fast atom bombardment mass spectroscopy, and phosphoamino acid analysis, the sequence of the phosphorylated peptide was determined to be: R-S-T(P)-M-M-I-P-G-M-E-G-K. This sequence is not homologous to any previously determined phosphofructokinase phosphopeptides. Regulatory differences between the mammalian and parasite enzymes are discussed with particular emphasis on regulation by protein phosphorylation.
Assuntos
Fasciola hepatica/enzimologia , Fosfofrutoquinase-1/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Fosfofrutoquinase-1/química , Fosfofrutoquinase-1/isolamento & purificação , Fosforilação , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Especificidade por Substrato , TripsinaRESUMO
Phosphofructokinase (PFK) from sheep heart was shown to be phosphorylated by Ca2+/calmodulin protein kinase (CaM-kinase) as well as by cyclic AMP-dependent protein kinase (PKA). HPLC analysis of phosphorylated PFK indicated that phosphorylation by CaM-kinase occurs at least at two sites that are distinct from those recognized by PKA. Phosphorylation by either CaM-kinase of PKA resulted in an increase in sensitivity to ATP inhibition and a small but consistent decrease in Ki for ATP. Phosphorylation by either protein kinase caused a slight increase in the Km of PFK for fructose-6-P. Protein kinase C failed to phosphorylate PFK. Combinations of PKA, CaM-kinase and protein kinase C did not alter the stoichiometry of phosphorylation and did not change the effect on enzyme activity.
Assuntos
Miocárdio/enzimologia , Fosfofrutoquinase-1/metabolismo , Proteínas Quinases/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Cromatografia Líquida de Alta Pressão , Cinética , Fosfofrutoquinase-1/isolamento & purificação , Fosfopeptídeos/isolamento & purificação , Fosforilação , OvinosRESUMO
Rabbit skeletal muscle glycogen synthase was inhibited by pyridoxal 5'-phosphate and irreversibly inactivated after sodium borohydride reduction of the enzyme-pyridoxal-P complex. The irreversible inactivation by pyridoxal-P was opposed by the presence of the substrate UDP-glucose. With [3H]pyridoxal-P, covalent incorporation of 3H label into the enzyme could be monitored. UDP-glucose protected against 3H incorporation, whereas glucose-6-P was ineffective. Peptide mapping of tryptic digests indicated that two distinct peptides were specifically modified by pyridoxal-P. One of these peptides contained the NH2-terminal sequence of the glycogen synthase subunit. Chymotrypsin cleavage of this peptide resulted in a single-labeled fragment with the sequence: Glu-Val-Ala-Asn-(Pyridoxal-P-Lys)-Val-Gly-Gly-Ile-Tyr. This sequence is identical to that previously reported (Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260. 6670-6676) for a peptide specifically modified by a substrate analogue and inferred to form part of the active site of the enzyme. Sequence analysis revealed that the modified lysine was located at residue 38 from the NH2 terminus of the rabbit muscle glycogen synthase subunit. An analogous tryptic peptide obtained from the rabbit liver isozyme displayed a high degree of sequence homology in the vicinity of the modified lysine. We propose that the extreme NH2 terminus of the glycogen synthase subunit forms part of the catalytic site, in close proximity to one of the phosphorylated regions of the enzyme (site 2, serine 7). In addition, the work extends the known NH2-terminal amino acid sequences of both the liver and muscle glycogen synthase isozymes.
Assuntos
Glicogênio Sintase/metabolismo , Isoenzimas/metabolismo , Lisina , Músculos/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Boroidretos/farmacologia , Glicogênio Sintase/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Cinética , Fígado/enzimologia , Dados de Sequência Molecular , Especificidade de Órgãos , Fragmentos de Peptídeos/análise , Fosfato de Piridoxal/farmacologia , Coelhos , TripsinaRESUMO
Glycogen synthase from rabbit skeletal muscle was found to be phosphorylated by a protein-tyrosine kinase, p40, purified from bovine thymus. The phosphorylation, to a stoichiometry of 0.4-0.5 mol/mol subunit, was specific for a single tyrosine residue in the sequence EEDGERYDEDEE. This acidic sequence has considerable similarity to the site recognized by p40 in erythrocyte band 3 protein. In the analysis of the phosphorylated peptide, it was noted that the sequence -RY(P)- impeded cleavage by either trypsin or automatic Edman degradation.
Assuntos
Glicogênio Sintase/metabolismo , Proteínas Tirosina Quinases/fisiologia , Timo/enzimologia , Sequência de Aminoácidos , Animais , Bovinos , Glicogênio Sintase/isolamento & purificação , Cinética , Dados de Sequência Molecular , Fosfopeptídeos/isolamento & purificação , Fosforilação , CoelhosRESUMO
The mechanism for synergistic phosphorylation by glycogen synthase kinase 3 (GSK-3) and casein kinase II was studied using a synthetic peptide which contains the sequence of a potentially important proline/serine-rich regulatory region of rabbit muscle glycogen synthase. The peptide, Ac-PRPAS(3a)VPPS(3b)PSLS(3c)RHSS(4)PHQS(5) EDEEEP-amide, has five known phosphorylation sites of the native enzyme designated sites 3a, 3b, 3c, 4, and 5, which are spaced every fourth residue. The peptide was phosphorylated specifically at site 5 by casein kinase II with an apparent Km of 23 microM, but it was not phosphorylated by GSK-3. However, after initial phosphorylation of site 5 by casein kinase II, the peptide became an effective substrate for GSK-3 with an apparent Km of 2 microM. GSK-3 introduced up to four phosphates and appeared to catalyze the sequential modification of sites 4, 3c, 3b, and 3a, respectively. The results can be explained if GSK-3 recognizes the sequence -SXXXS(P). Phosphorylation of site 5 by casein kinase II creates this recognition site. Thereafter, each successive phosphorylation introduced by GSK-3 generates a new recognition site. The results provide a molecular basis to explain the synergistic action of casein kinase II and GSK-3 that is also observed with native glycogen synthase. In addition, this investigation emphasizes how protein recognition sites in some cellular targets may have to be formed post-translationally.
Assuntos
Proteínas Quinases/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Caseína Quinases , Quinases da Glicogênio Sintase , Focalização Isoelétrica , Cinética , Músculos/enzimologia , Peptídeos/síntese química , Fosfopeptídeos/análise , Fosforilação , Coelhos , Especificidade por Substrato , TripsinaRESUMO
The histidine residue at the amino terminus of lysine-12 protected glucagon was replaced by its D-isomer by an established semisynthetic strategy to extend a stepwise series of replacements at this position. The product was examined for its secondary structure and its function. Circular dichroism spectra obtained at concentrations from 0.25 to 1.09 mg/ml at pH 10.2 in 0.2 M phosphate buffer were similar to those obtained with native hormone. Competitive binding assays and adenylate cyclase activation assays with partially purified rat liver plasma membranes show this D-His1 analog of glucagon to be a full agonist, causing the same maximum activation of adenylate cyclase as native hormone; but both binding and activation assays show the binding affinity to be diminished about 10-fold. The data suggest that the adjustment of the bonding of the imidazole group to the receptor to bring about transduction results in constraints on the conformation along the peptide sequence which interfere with the peptide adopting the same binding conformation achieved by the native hormone.
Assuntos
Glucagon/análogos & derivados , Adenilil Ciclases/metabolismo , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Dicroísmo Circular , Glucagon/síntese química , Glucagon/metabolismo , Glucagon/farmacologia , Fígado/metabolismo , Conformação Proteica , Ratos , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Glucagon , Relação Estrutura-AtividadeRESUMO
The ability of catfish glucagon and glucagon-like peptide to bind and activate mammalian glucagon receptors was investigated. Neither catfish peptide binds to glucagon receptors of rat liver, hypothalamus or pituitary. Neither stimulates adenylate cyclase activity in liver membranes. Catfish glucagon fails to activate adenylate cyclase in hypothalamic or pituitary membranes in contrast to mammalian glucagon. However, catfish glucagon-like peptide does stimulate hypothalamic and pituitary adenylate cyclase (EC50 approximately 1 pM) possibly through mammalian glucagon-like peptide receptors.