DOCSLIB.ORG

Tyrosine Phosphorylation (Rous Sarcoma Virus/P60"/Avian Sarcoma Viruses Y73 and PRCII/Abelson Virus) TILO PATSCHINSKY*, TONY HUNTER*, FRED S

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Tyrosine Phosphorylation (Rous Sarcoma Virus/P60 Proc. Natl Acad. Sci. USA Vol. 79, pp. 973-977, February 1982 Biochemistry Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation (Rous sarcoma virus/p60"/avian sarcoma viruses Y73 and PRCII/Abelson virus) TILO PATSCHINSKY*, TONY HUNTER*, FRED S. ESCHt, JONATHAN A. -COOPER*, AND BARTHOLOMEW M. SEFTON* *Tumor Virology Laboratory and tLaboratories of Endocrinology, The Salk Institute, P.O. Box 85800, San Diego, California 92138 Communicated by Robert W. Holley, October 13, 1981 ABSTRACT We have identified the single phosphorylated ty- MATERIALS AND METHODS rosine in p60, the transforming protein of Rous sarcoma virus, as part of the sequence Cells and Viruses. Preparation and infection of chicken fi- broblasts has been described (16). The Schmidt-Ruppin strain NH2-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-COOH. of RSV of subgroup A (SR.RSV-A) originated in the laboratory Therefore,. this is a sequence that is recognized efficiently by a of H. Hanafusa, The Rockefeller University. Avian sarcoma vi- tyrosine protein kinase in vivo. Phosphorylation oftyrosine in cel- rus Y73 was obtained from K. Toyoshima, the University of lular proteins appears to play a role in malignant transformation Tokyo, and the avian sarcoma virus PRCII was a gift from J. by four classes of genetically distinct RNA tumor viruses. Phos- Neil, the University of Southern California. The ANN-i line of phorylated tyrosines in several other proteins resemble the ty- NIH/3T3 cells nonproductively infected with Abelson virus (17) rosine in p60 in that they are located 7 residues to the COOH- was the source ofp120. Mink lung cells nonproductively trans- terminal side of a basic amino acid and either 4 residues to the formed by the Snyder-Theilin strain offeline sarcoma virus (ST- COOH-terminal side of, or in close proximity to, a glutamic acid FeSV) (18) were provided by M. Barbacid, the National Cancer residue. Therefore, it is possible that these features play a role in Institute. the selection of sites ofphosphorylation by some tyrosine protein Radioactive Labeling. Labeling ofinfected chicken cells and kinases. However, several clear exceptions to this rule exist. ofthe ANN-1 cells with [32P]orthophosphate (ICN, carrier-free) Rous sarcoma virus (RSV) transforms cells to a malignant state was performed as described (19). RSV-infected chicken cells, through the expression of a single gene, src (reviewed in ref. growing on 100-mm plastic Petri dishes, were labeled with in- 1). This transforming gene encodes a single 60,000-dalton phos- dividual radioactive amino acids overnight at 41°C in Dul- phoprotein, p6Orc (2). p6Osrc is aprotein kinase with the unusual becco's modified Eagle's medium containing only 5% of the specificity ofphosphorylating tyrosine residues in its target pro- normal concentration of the labeled amino acid and supple- teins (3-6). This unscheduled phosphorylation of tyrosine al- mented with 4% complete calf serum. Each sample was from most certainly plays a role in cellular transformation by this virus a single culture. Labeling was in 5 ml ofmedium containing one (7) and probably also by three other groups of RNA tumor vi- of the following: 2 mCi of [35S]cysteine (470 Ci/mmol; 1 Ci = ruses genetically distinct from RSV (8-10). Tyrosine protein 3.7 x 10'° becquerels); 10 mCi of [3H]isoleucine (84 Ci/ kinases may also play an important role in normal metabolism mmol); 3 mCi of [3S]methionine (1000 Ci/mmol); 5 mCi of because they are implicated in the cellular response to epider- [3H]tryptophan (26 Ci/mmol); 5 mCi of [3H]tyrosine (53 Ci/ mal growth factor (11). mmol). [3S]Cysteine and [3H]tyrosine were from New England An important question is how tyrosine protein kinases select Nuclear; all others were from Amersham/Searle. and recognize their substrates. In the case of the well-charac- Cell Lysis and Immunoprecipitation. The transforming pro- terized protein kinases that phosphorylate serine and threo- teins of RSV, Y73 virus, PRCII virus, ST-FeSV, and Abelson nine, the amino acid sequence of the substrate plays a crucial virus were isolated by immunoprecipitation from transformed role in selection ofthe phosphorylation site. The cAMP-depen- cells as dent protein kinase has a strong preference for serines that are described (4, 16, 19-21). located 2-5 residues to the COOH-terminal side ofone or two Gel Electrophoresis, Elution of Proteins, and Tryptic basic amino acids, most commonly arginine (12, 13). In contrast, Digestion. Immunoprecipitated proteins were purified on the casein protein kinases as a class show a strong preference preparative NaDodSO4polyacrylamide gels (22) and localized for sites in the vicinity ofacidic residues (14, 15). In casein itself, by autoradiography. The position of 3H-labeled proteins was all but one ofthe sites ofphosphorylation are located 2 residues identified by 32p_ or 3S- labeled markers that were run in par- to the NH2-terminal side of an acidic amino acid (14). allel. All other procedures were as described (22). Because of these precedents, we have characterized the se- Peptide Mapping. Two-dimensional mapping was done by quence of amino acids at sites oftyrosine phosphorylation. We using electrophoresis at pH 8.9 in the first dimension and as- deduced the complete amino acid sequence surrounding the cending chromatography in the second, as described (4). 32P- single phosphorylated tyrosine in p6(5rc and analyzed indirectly Labeled peptides were detected by using preflashed film and the sequence at sites of tyrosine phosphorylation in a number an intensifying screen. 3H- and 3S-labeled peptides were visu- of other proteins. alized by fluorography. Analysis ofmixtures of 32P-labeled and The publication costs ofthis article were defrayed in part by page charge Abbreviations: RSV, Rous sarcoma virus; SR-RSV-A, Schmidt-Ruppin payment. This article must therefore be hereby marked "advertise- RSV ofsubgroup A; Tyr(P), phosphotyrosine; ST-FeSV, Snyder-Theilin ment" in accordance with 18 U. S.- C. §1734 solely to indicate this fact. feline sarcoma virus. 973 Downloaded by guest on September 25, 2021 974 Biochemistry: Patschinsky et al. Proc. Natl. Acad. Sci.. USA 79 (1982) 'H- or 'S-labeled peptides involved the use ofjust enough 32P- sequence of the COOH-terminal half of p6Osrc contained iso- labeled material to give an image after 3-6 days ofexposure with leucine and tyrosine and was devoid of methionine, cysteine, an intensifying screen. The thin-layer plates were then dipped glycine, tryptophan, and histidine-the tryptic peptide con- in molten 2-methylnaphthalene containing 0.4% diphenyloxa- taining residues 413-422. This tryptic peptide contained only zole and re-exposed without an intensifying screen. a single tyrosine, tyrosine-419-(Fig. 2). Staphylococcus aureusV8 Protease Digestion. Whole tryptic This conclusion was also supported by automated Edman digests or isolated tryptic peptides were digested with 5-10 Ug degradation of peptide P3. Phenylthiohydantoin-Tyr(P) was re- of S. aureus protease (Miles) at pH 4.0 or pH 7.8 as described leased at the seventh cycle ofdegradation ofthe tryptic peptide (19). and at the first cycle ofdegradation ofthe product ofsecondary Amino Acid Sequence Analysis. Automated Edman degra- digestion ofthe peptide with S. aureus protease (Table 1). These dation was performed as described (23). The residues from each results are consistent with the positions ofboth arginine-412 and step were dried and assayed directly by using Cerenkov radia- glutamic acid-418 (Fig. 2). It should be noted that the predicted tion. In some cases, the anilinothiazolinone derivatives were sequence ofpeptide 3contains two glutamic acids, at positions converted into phenylthiohydantoin derivatives and separated 415 and 418. However, complete digestion of peptide P with by electrophoresis at pH 3.5 in the presence of phenylthiohy- S. aureus protease yielded only one phosphorylated prod- dantoin-phosphotyrosine [Tyr(P)] marker to verify that the re- uct-that resulting from cleavage at glutamic acid-418. Smart leased radioactivity was indeed associated with tyrosine. et aL (30) also concluded that tyrosine-419 is phosphorylated in In Vitro Kinase Assay. This assay was performed as described p6srC (20) except that the buffer used contained 0.01 M sodium 1,4- The approach we used to identify tyrosine-419 as the site of piperazinebis(ethanesulfonic acid) at pH 7.0 and 0.01 M MnCl2. tyrosine phosphorylation in p60rc is indirect and requires that Isolation of p36 and p50. p50 was isolated from preparative the deduced sequence of the protein is correct. However, ad- gels containing immunoprecipitated p6osrc as described (4). p36 ditional evidence substantiates this conclusion. A synthetic pep- was purified by column chromatography and gel electrophoresis tide, identical in sequence to the deduced sequence ofpeptide as described (24). 3,can be phosphorylated on tyrosine in vitro by a protein kinase Analysis of the Synthetic Peptide. A peptide with the se- present in immunoprecipitates containing either p60Orc or the quence Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg was syn- putative transforming protein of Y73 virus (unpublished data). thesized and purified (unpublished data). The peptide was This phosphorylated synthetic peptide comigrated with biosyn- phosphorylated in vitro by incubation with immunoprecipitates thetically labeled peptide , from p60srC when analyzed by pep- containing p90 of Y73 virus as described above. The phosphor- tide mapping (Fig. 1). Additionally,
Load more

Recommended publications
  • The Cross-Talk Between Methylation and Phosphorylation in Lymphoid-Specific Helicase Drives Cancer Stem-Like Properties
    Signal Transduction and Targeted Therapy www.nature.com/sigtrans ARTICLE OPEN The cross-talk between methylation and phosphorylation in lymphoid-specific helicase drives cancer stem-like properties Na Liu1,2,3, Rui Yang1,2, Ying Shi1,2, Ling Chen1,2, Yating Liu1,2, Zuli Wang1,2, Shouping Liu1,2, Lianlian Ouyang4, Haiyan Wang1,2, Weiwei Lai1,2, Chao Mao1,2, Min Wang1,2, Yan Cheng5, Shuang Liu4, Xiang Wang6, Hu Zhou7, Ya Cao1,2, Desheng Xiao1 and Yongguang Tao1,2,6 Posttranslational modifications (PTMs) of proteins, including chromatin modifiers, play crucial roles in the dynamic alteration of various protein properties and functions including stem-cell properties. However, the roles of Lymphoid-specific helicase (LSH), a DNA methylation modifier, in modulating stem-like properties in cancer are still not clearly clarified. Therefore, exploring PTMs modulation of LSH activity will be of great significance to further understand the function and activity of LSH. Here, we demonstrate that LSH is capable to undergo PTMs, including methylation and phosphorylation. The arginine methyltransferase PRMT5 can methylate LSH at R309 residue, meanwhile, LSH could as well be phosphorylated by MAPK1 kinase at S503 residue. We further show that the accumulation of phosphorylation of LSH at S503 site exhibits downregulation of LSH methylation at R309 residue, which eventually promoting stem-like properties in lung cancer. Whereas, phosphorylation-deficient LSH S503A mutant promotes the accumulation of LSH methylation at R309 residue and attenuates stem-like properties, indicating the critical roles of LSH PTMs in modulating stem-like properties. Thus, our study highlights the importance of the crosstalk between LSH PTMs in determining its activity and function in lung cancer stem-cell maintenance.
    [Show full text]
  • Mendelian Randomization Study on Amino Acid Metabolism Suggests Tyrosine As Causal Trait for Type 2 Diabetes
    nutrients Article Mendelian Randomization Study on Amino Acid Metabolism Suggests Tyrosine as Causal Trait for Type 2 Diabetes Susanne Jäger 1,2,* , Rafael Cuadrat 1,2, Clemens Wittenbecher 1,2,3, Anna Floegel 4, Per Hoffmann 5,6, Cornelia Prehn 7 , Jerzy Adamski 2,7,8,9 , Tobias Pischon 10,11,12 and Matthias B. Schulze 1,2,13 1 Department of Molecular Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, 14558 Nuthetal, Germany; [email protected] (R.C.); [email protected] (C.W.); [email protected] (M.B.S.) 2 German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany; [email protected] 3 Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA 4 Leibniz Institute for Prevention Research and Epidemiology-BIPS, 28359 Bremen, Germany; fl[email protected] 5 Human Genomics Research Group, Department of Biomedicine, University of Basel, 4031 Basel, Switzerland; per.hoff[email protected] 6 Institute of Human Genetics, Division of Genomics, Life & Brain Research Centre, University Hospital of Bonn, 53105 Bonn, Germany 7 Research Unit Molecular Endocrinology and Metabolism, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany; [email protected] 8 Chair of Experimental Genetics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, 85354 Freising-Weihenstephan, Germany 9 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive,
    [Show full text]
  • H-Tyrosine-Glycine-Phenylalanine-Glycine-Glycine-OH for the Treatment of Chronic Idiopathic Myelofibrosis
    European Medicines Agency Pre-authorisation Evaluation of Medicines for Human Use Document Date: London, 5 October 2009 Doc.Ref.: EMEA/COMP/1579/2003 Rev.1 Please note that this product was withdrawn from the Community Register of designated Orphan Medicinal Products in March 2009 on request of the Sponsor. Committee for Orphan Medicinal Products Public summary of positive opinion for orphan designation of H-Tyrosine-Glycine-Phenylalanine-Glycine-Glycine-OH for the treatment of chronic idiopathic myelofibrosis On 20 October 2003, orphan designation (EU/3/03/167) was granted by the European Commission to Abiogen Pharma S.p.A, Italy, for H-Tyrosine-Glycine-Phenylalanine-Glycine-Glycine-OH for the treatment of chronic idiopathic myelofibrosis. What is chronic idiopathic myelofibrosis? Chronic idiopathic myelofibrosis is a disease in which cancer cells are found in the blood and in the bone marrow. The bone marrow is the spongy tissue inside the large bones in the body. Normally the bone marrow makes cells called “blasts” that mature into several different types of blood cells that have specific functions in the body. These include red cells, white cells and platelets. Red blood cells carry oxygen and other materials to all tissues of the body. White blood cells fight infection. Platelets make the blood clot. When myelofibrosis develops, the bone marrow produces large number of abnormal blood cells. In chronic idiopathic myelofibrosis the abnormal population of cells produce substances that alter the growth media of bone marrow and makes bone marrow very dense and rigid. As the abnormal bone marrow environment is no more adequate for the cells, some migrate to other sites where proliferation and maturation take place.
    [Show full text]
  • Phosphorylation of Chicken Protein Tyrosine Phosphatase 1 by Casein Kinase II in Vitro
    EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 29, No 4, 229-233, December 1997 Phosphorylation of chicken protein tyrosine phosphatase 1 by casein kinase II in vitro Eun Joo Jung,1 Kee Ryeon Kang1 and Introduction Yoon-Se Kang1,2 The phosphorylation of protein tyrosine residues is an early event in signal transduction initiated by binding of 1 Department of Biochemistry and Gyeongsang Institute of Cancer growth factors and hormones to their cognate receptors Research, College of Medicine, Gyeongsang National University, and it leads to regulation of cellular activities which include Chinju 660-280, Korea proliferation, differentiation, and also malignant transfor- 2 Corresponding author mation of cells (Hunter, 1989; Ullirich and Schlessinger, Accepted 17 November 1997 1990; Cantley et al., 1991). Under normal conditions, the level of tyrosine phosphorylation within a cell is determined by a balance between the actions of protein tyrosine Abbreviations: CPTP, chicken protein tyrosine phosphatase; HPTP1B, human placenta kinases (PTKs) and protein tyrosine phosphatases (PTPs) protein tyrosine phosphatase 1B; CKII, casein kinase II; MAP kinase, mitogen-activated (Hunter, 1989; Fischer et al., 1991; Trowbridge, 1991). protein kinase; GST, glutathione S-transferase; pNPP, p-nitrophenyl phosphate; EGF, PTPs do not simply reverse the action of tyrosine kinases, epidermal growth factor but rather, PTP itself may play a central role in cellular regulation. PTPs are generally classified as transmem- brane (receptor-type) and nontransmembrane (nonrecep- tor-type) enzymes based on the presence or absence of extracellular and transmembrane portions of their predicted sequence (Fischer et al., 1991). Because the activity of Abstract tyrosine kinase can be controlled by phosphorylation, it has been postulated that PTP activity may be regulated The phosphorylation and dephosphorylation of by phosphorylation as well.
    [Show full text]
  • Mechanism of Insulin Receptor Kinase Inhibition in Non-Insulin- Dependent Diabetes Mellitus Patients
    Mechanism of insulin receptor kinase inhibition in non-insulin- dependent diabetes mellitus patients. Phosphorylation of serine 1327 or threonine 1348 is unaltered. M Kellerer, … , K Siddle, H U Häring J Clin Invest. 1995;96(1):6-11. https://doi.org/10.1172/JCI118073. Research Article The tyrosine kinase activity of insulin receptor isolated from the skeletal muscle of NIDDM patients has previously been found to be decreased compared with the activity of receptor from nondiabetic subjects but the mechanism underlying this defect is unknown. Phosphorylation of receptor serine/threonine residues has been proposed to exert an inhibitory influence on receptor tyrosine kinase activity and Ser 1327 and Thr 1348 have been identified as specific sites of phosphorylation in the insulin receptor COOH terminal domain. To address the potential negative regulatory role of phosphorylation of these residues in vivo, we assessed the extent of phosphorylation of each site in insulin receptor isolated from the skeletal muscle of 12 NIDDM patients and 13 nondiabetic, control subjects. Phosphorylation of Ser 1327 and Thr 1348 was determined using antibodies that specifically recognize insulin receptor phosphorylated at these sites. In addition, a phosphotyrosine-specific antibody was used to monitor receptor tyrosine phosphorylation. The extent of insulin-induced tyrosine autophosphorylation was decreased in receptor isolated from diabetic versus nondiabetic muscle, thus confirming earlier reports. In contrast, there was no significant difference in the extent of phosphorylation of either Ser 1327 or Thr 1348 in receptor isolated from diabetic or nondiabetic muscle as assessed by immunoprecipitation (Ser 1327: 5.6 +/- 1.6% diabetics vs. 4.7 +/- 2.0% control; Thr 1348: 3.8 +/- 1.0% diabetics vs.
    [Show full text]
  • Citric Acid Cycle
    CHEM464 / Medh, J.D. The Citric Acid Cycle Citric Acid Cycle: Central Role in Catabolism • Stage II of catabolism involves the conversion of carbohydrates, fats and aminoacids into acetylCoA • In aerobic organisms, citric acid cycle makes up the final stage of catabolism when acetyl CoA is completely oxidized to CO2. • Also called Krebs cycle or tricarboxylic acid (TCA) cycle. • It is a central integrative pathway that harvests chemical energy from biological fuel in the form of electrons in NADH and FADH2 (oxidation is loss of electrons). • NADH and FADH2 transfer electrons via the electron transport chain to final electron acceptor, O2, to form H2O. Entry of Pyruvate into the TCA cycle • Pyruvate is formed in the cytosol as a product of glycolysis • For entry into the TCA cycle, it has to be converted to Acetyl CoA. • Oxidation of pyruvate to acetyl CoA is catalyzed by the pyruvate dehydrogenase complex in the mitochondria • Mitochondria consist of inner and outer membranes and the matrix • Enzymes of the PDH complex and the TCA cycle (except succinate dehydrogenase) are in the matrix • Pyruvate translocase is an antiporter present in the inner mitochondrial membrane that allows entry of a molecule of pyruvate in exchange for a hydroxide ion. 1 CHEM464 / Medh, J.D. The Citric Acid Cycle The Pyruvate Dehydrogenase (PDH) complex • The PDH complex consists of 3 enzymes. They are: pyruvate dehydrogenase (E1), Dihydrolipoyl transacetylase (E2) and dihydrolipoyl dehydrogenase (E3). • It has 5 cofactors: CoASH, NAD+, lipoamide, TPP and FAD. CoASH and NAD+ participate stoichiometrically in the reaction, the other 3 cofactors have catalytic functions.
    [Show full text]
  • Protein Kinases Phosphorylation/Dephosphorylation Protein Phosphorylation Is One of the Most Important Mechanisms of Cellular Re
    Protein Kinases Phosphorylation/dephosphorylation Protein phosphorylation is one of the most important mechanisms of cellular responses to growth, stress metabolic and hormonal environmental changes. Most mammalian protein kinases have highly a homologous 30 to 32 kDa catalytic domain. • Most common method of reversible modification - activation and localization • Up to 1/3 of cellular proteins can be phosphorylated • Leads to a very fast response to cellular stress, hormonal changes, learning processes, transcription regulation .... • Different than allosteric or Michealis Menten regulation Protein Kinome To date – 518 human kinases known • 50 kinase families between yeast, invertebrate and mammaliane kinomes • 518 human PKs, most (478) belong to single super family whose catalytic domain are homologous. • Kinase dendrogram displays relative similarities based on catalytic domains. • AGC (PKA, PKG, PKC) • CAMK (Casein kinase 1) • CMGC (CDC, MAPK, GSK3, CLK) • STE (Sterile 7, 11 & 20 kinases) • TK (Tryosine kinases memb and cyto) • TKL (Tyrosine kinase-like) • Phosphorylation stabilized thermodynamically - only half available energy used in adding phosphoryl to protein - change in free energy forces phosphorylation reaction in one direction • Phosphatases reverse direction • The rate of reaction of most phosphatases are 1000 times faster • Phosphorylation occurs on Ser/The or Tyr • What differences occur due to the addition of a phosphoryl group? • Regulation of protein phosphorylation varies depending on protein - some turned on or off
    [Show full text]
  • Amino Acids Amino Acids
    Amino Acids Amino Acids What Are Amino Acids? Essential Amino Acids Non Essential Amino Acids Amino acids are the building blocks of proteins; proteins are made of amino acids. Isoleucine Arginine (conditional) When you ingest a protein your body breaks it down into the individual aminos, Leucine Glutamine (conditional) reorders them, re-folds them, and turns them into whatever is needed by the body at Lysine Tyrosine (conditional) that time. From only 20 amino acids, the body is able to make thousands of unique proteins with different functions. Methionine Cysteine (conditional) Phenylalanine Glycine (conditional) Threonine Proline (conditional) Did You Know? Tryptophan Serine (conditional) Valine Ornithine (conditional) There are 20 different types of amino acids that can be combined to make a protein. Each protein consists of 50 to 2,000 amino acids that are connected together in a specific Histidine* Alanine sequence. The sequence of the amino acids determines each protein’s unique structure Asparagine and its specific function in the body. Asparate Popular Amino Acid Supplements How Do They Benefit Our Health? Acetyl L- Carnitine: As part of its role in supporting L-Lysine: L-Lysine, an essential amino acid, is mental function, Acetyl L-Carnitine may help needed to support proper growth and bone Proteins (amino acids) are needed by your body to maintain muscles, bones, blood, as support memory, attention span and mental development. It can also support immune function. well as create enzymes, neurotransmitters and antibodies, as well as transport and performance. store molecules. N-Acetyl Cysteine: N-Acetyl Cysteine (NAC) is a L-Arginine: L-Arginine is a nonessential amino acid form of the amino acid cysteine.
    [Show full text]
  • Interpretive Guide for Amino Acids
    Interpretive Guide for Amino Acids Intervention Options LOW HIGH Essential Amino Acids Arginine (Arg) Arg Mn Histidine (His) Folate, His Isoleucine (Ile) * B6, Check for insulin insensitivity Leucine (Leu) * B6, Check for insulin insensitivity Lysine (Lys) Carnitine Vitamin C, Niacin, B6, Iron, a-KG Methionine(Met) * B6, á-KG, Mg, SAM Phenylalanine (Phe) * Iron,VitaminC,Niacin,LowPhediet Threonine(Thr) * B6, Zn Tryptophan(Trp) Trpor5-HTP Niacin, B6 Valine (Val) * B6, Check for insulin insensitivity Essential Amino Acid Derivatives Neuroendocrine Metabolism y-Aminobutyric Acid (GABA) a-KG, B6 Glycine (Gly) Gly Folate, B6,B2,B5 Serine (Ser) B6, Mn, Folate * Taurine (Tau) Tau, B6 Vit. E, Vit. C, B-Carotene, CoQ10, Lipoate Tyrosine(Tyr) Iron,Tyr,VitaminC,Niacin Cu, Iron, Vitamin C, B6 Ammonia/Energy Metabolism a-Aminoadipic Acid B6, a-KG Asparagine (Asn) Mg Aspartic Acid (Asp) a-KG, B6 Mg, Zn Citrulline (Cit) Mg, Aspartic acid Glutamic Acid (Glu) B6, a-KG Niacin, B6 Glutamine (Gln) a-KG, B6 Ornithine (Orn) Arg Mg, a-KG, B6 Sulfur Metabolism Cystine (Cys) NAC B2 Cystathionine B6 Homocystine (HCys) B6, Folate, B12, Betaine Additional Metabolites a-Amino-N-Butyric Acid a-KG, B6 B6, a-KG Alanine (Ala) * B6 Anserine Zn n-Alanine Lactobacillus and Bifidobacteria, B6 n-Aminoisobutyric Acid B6 Carnosine Zn Ethanolamine Mg Hydroxylysine (HLys) Vitamin C, Iron, a-KG Hydroxyproline (HPro) Vitamin C, Iron, a-KG 1-Methylhistidine Vitamin E, B12, Folate 3-Methylhistidine BCAAs, Vit. E, Vit. C, n-Carotene, CoQ10, Lipoate Phosphoethanolamine (PE) SAM, B12, Folate, Betaine Phosphoserine Mg Proline (Pro) a-KG Vitamin C, Niacin Sarcosine B2 * Use balanced or custom mixtures of essential amino acids Nordic Laboratiroes∙ Nygade 6, 3.sal ∙ 1164 Copenhagen K ∙ DenmarkTel: +45 33 75 1000 ∙ e-mail: [email protected] In association with ©Metametrix, Inc.
    [Show full text]
  • Solutions to 7.012 Problem Set 1
    MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel Solutions to 7.012 Problem Set 1 Question 1 Bob, a student taking 7.012, looks at a long-standing puddle outside his dorm window. Curious as to what was growing in the cloudy water, he takes a sample to his TA, Brad Student. He wanted to know whether the organisms in the sample were prokaryotic or eukaryotic. a) Give an example of a prokaryotic and a eukaryotic organism. Prokaryotic: Eukaryotic: All bacteria Yeast, fungi, any animial or plant b) Using a light microscope, how could he tell the difference between a prokaryotic organism and a eukaryotic one? The resolution of the light microscope would allow you to see if the cell had a true nucleus or organelles. A cell with a true nucleus and organelles would be eukaryotic. You could also determine size, but that may not be sufficient to establish whether a cell is prokaryotic or eukaryotic. c) What additional differences exist between prokaryotic and eukaryotic organisms? Any answer from above also fine here. In addition, prokaryotic and eukaryotic organisms differ at the DNA level. Eukaryotes have more complex genomes than prokaryotes do. Question 2 A new startup company hires you to help with their product development. Your task is to find a protein that interacts with a polysaccharide. a) You find a large protein that has a single binding site for the polysaccharide cellulose. Which amino acids might you expect to find in the binding pocket of the protein? What is the strongest type of interaction possible between these amino acids and the cellulose? Cellulose is a polymer of glucose and as such has many free hydroxyl groups.
    [Show full text]
  • Phosphorylation of Protein 4.1 on Tyrosine-418 Modulates Its Function
    Proc. Nati. Acad. Sci. USA Vol. 88, pp. 5222-5226, June 1991 Biochemistry Phosphorylation of protein 4.1 on tyrosine-418 modulates its function in vitro (epidermal growth factor receptor/spectrin/actin assembly) GOSUKONDA SUBRAHMANYAM*, PAUL J. BERTICStt, AND RICHARD A. ANDERSON**§ Department of *Pharmacology and tPhysiological Chemistry, and the tCell and Molecular Biology Program, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706 Communicated by Vincent T. Marchesi, March IS, 1991 ABSTRACT Protein 4.1 was initially characterized as a actin/protein 4.1 complex (12). However, phosphorylation of protein that regulates cytoskeletal assembly in erythrocytes. protein 4.1 by protein kinase C also reduces protein 4.1 However, recent studies have shown that protein 4.1 is ubiq- binding to band 3 but not to glycophorin, while phosphory- uitous in mammalian cells. Here, we show that protein 4.1 is lation by cAMP-dependent kinase does not affect membrane phosphorylated on tyrosine by the epidermal growth factor interactions (12). These results suggest that protein 4.1 is receptor (EGFR) tyrosine kinase. The phosphorylation site has phosphorylated at multiple sites by different protein kinases, been localized to the 8-kDa domain, which has one tyrosine, and each phosphorylation event selectively modulates pro- tyrosine-418. The 8-kDa region is required for the assembly of tein 4.1 function. The phosphorylation sites by cAMP- the spectrin/actin complex, and phosphorylation by EGFR dependent protein kinase and protein kinase C are in the reduced the ability ofprotein 4.1 to promote the assembly ofthe 8-kDa and 16-kDa domains (10).
    [Show full text]
  • Structural Basis of O-Glcnac Recognition by Mammalian 14-3-3 Proteins
    Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins Clifford A. Tolemana,1, Maria A. Schumachera,1, Seok-Ho Yub, Wenjie Zenga, Nathan J. Coxa, Timothy J. Smitha, Erik J. Soderblomc, Amberlyn M. Wandsb, Jennifer J. Kohlerb, and Michael Boycea,2 aDepartment of Biochemistry, Duke University School of Medicine, Durham, NC 27710; bDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390; and cDuke Proteomics and Metabolomics Core Facility, Center for Genomic and Computational Biology, Duke University, Durham, NC 27710 Edited by Carolyn R. Bertozzi, Stanford University, Stanford, CA, and approved April 23, 2018 (received for review December 24, 2017) O-GlcNAc is an intracellular posttranslational modification that gov- Results erns myriad cell biological processes and is dysregulated in human We developed a biochemical approach to test the hypothesis that diseases. Despite this broad pathophysiological significance, the O-GlcNAc is specifically recognized by mammalian reader pro- biochemical effects of most O-GlcNAcylation events remain unchar- teins. First, we derived a consensus O-GlcNAcylated peptide acterized. One prevalent hypothesis is that O-GlcNAc moieties may sequence by aligning 802 mapped Ser-O-GlcNAc sites (34–36) be recognized by “reader” proteins to effect downstream signaling. (Fig. 1A)(www.phosphosite.org). We noted that a Pro-Val-Ser However, no general O-GlcNAc readers have been identified, leav- tripeptide observed previously in smaller datasets (37, 38) also ing a considerable gap in the field. To elucidate O-GlcNAc signaling emerged in our sequence, suggesting that this motif may be mechanisms, we devised a biochemical screen for candidate O-GlcNAc important for O-GlcNAc modification and/or recognition.
    [Show full text]