DOCSLIB.ORG

Autophosphorylation in Vitro of Recombinant 42-Kilodalton Mitogen

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Autophosphorylation in Vitro of Recombinant 42-Kilodalton Mitogen Proc. Nadl. Acad. Sci. USA Vol. 88, pp. 9508-9512, November 1991 Biochemistry Autophosphorylation in vitro of recombinant 42-kilodalton mitogen-activated protein kinase on tyrosine (mitogen-activated protein kinase/cell cycle/peptide mapping/recombinant expression) JIE WU*, ANTHONY J. ROSSOMANDOt, JENG-HORNG HERt, ROBERT DEL VECCHIOt, MICHAEL J. WEBERt, AND THOMAS W. STURGILL*t Departments of tMicrobiology, and *Internal Medicine and Pharmacology, Health Sciences Center, University of Virginia, Charlottesville, VA 22908 Communicated by Oscar L. Miller, Jr., July 29, 1991 (receivedfor review June 21, 1991) ABSTRACT Mitogen-activated protein kinase (MAP ki- lation sites are locatedjust upstream ofthe Ala-Pro-Glu motif nase) is a serine/threonine protein kinase that becomes enzy- in a region where activating autophosphorylations occur in matically activated and phosphorylated on tyrosine and thre- many other kinases (5, 6, 14). onine following treatment of quiescent cells with a variety of To investigate the mechanism(s) for p42maPk activation, we stimulatory agonists. Phosphorylation on both tyrosine and have cloned a full-length cDNA encoding p42maPk (6) and threonine is necessary to maintain full activity, and these two expressed the enzyme in Escherichia coli in soluble form. We regulatory phosphorylations occur close to each other, sepa- now report that recombinant p42maPk is basally active and rated by a single glutamate. To study the mechanisms by which autophosphorylates on tyrosine. This autophosphorylation MAP kinase becomes phosphorylated and activated, we have can occur on the site of regulatory phosphorylation and is cloned a full-length cDNA encoding MAP kinase and have accompanied by a partial enzymatic activation of p42maPk. expressed the enzyme in Escherichia coli as a soluble nonfusion Thus, upstream components regulating MAP kinase activity protein. We find that the enzyme displays a basal, intramo- may conceivably do so by enhancing the rate of autophos- lecular autophosphorylation on tyrosine-185 that is accompa- phorylation on tyrosine. nied by activation of the enzyme's kinase activity towards an exogenous substrate. The tyrosine-phosphorylated protein dis- plays a small fraction of the activity seen with the fully MATERIALS AND METHODS activated, doubly phosphorylated enzyme isolated from mam- Materials. The bacterial expression vector pETila and the malian cells but is activated 10- to 20-fold relative to the E. coli strains HB101 and BL21(DE3) (15) were purchased unphosphorylated enzyme. These findings raise the possibility from Novagen (Madison, WI). Isopropyl f3-D-thiogalactopy- that regulation of MAP kinase activity in response to agonist ranoside (IPTG) and enzymes used in DNA manipulations stimulation could occur in part through the enhancement of were obtained from GIBCO/BRL. The sources for chroma- autophosphorylation on tyrosine. tography media and reagents for culturing E. coli were Pharmacia-LKB and Difco, respectively. Oligonucleotides Mitogen-activated protein kinase (MAP kinase) was orig- were purchased from Operon Technologies (Alameda, CA), inally identified as a serine/threonine protein kinase that and used without further purification. A synthetic peptide became phosphorylated on tyrosine and threonine and en- (Val-Ala-Asp-Pro-Asp-His-Asp-His-Thr-Gly-Phe-Leu-Thr- zymatically activated following insulin-stimulation of 3T3-L1 Glu-Tyr-Val-Ala-Thr-Arg) corresponding to the tryptic pep- cells (1, 2). This enzyme was subsequently found to be tide containing the regulatory phosphorylation sites of identical or closely related to pp42, a protein that becomes p42mapk was purchased from Synthecell (Rockville, MD). tyrosine-phosphorylated in cells treated with various mito- Antibody 1798, which recognizes p42maPk, was raised against gens (3). pp42/MAP kinase recently has been shown to be the carboxyl-terminal 10 amino acids of the predicted ERKi encoded by a member ofa gene family (4-8). Accordingly, we sequence (5) by the MAPS procedure (16). now refer to it as p2mapk to distinguish it from other members Construction of Plasmids for Expression of Unfused p42Pk ofthe family, while still retaining recognizable features ofthe in E. coli. Plasmid pETila was digested with Nde I, and the earlier nomenclature. In addition to being phosphorylated recessive 3' end was filled in with Klenow fragment. The and activated during the Go -* G, transition, p42maPk and/or DNA was then cut with BamHI, generating a 5639-base-pair members ofthis family become phosphorylated and activated (bp) fragment, which was isolated by gel electrophoresis. The during M phase in Xenopus oocytes and in various differen- blunt end ofthe purified fragment starts at the first nucleotide tiated, nonmitogenic cells following treatment with stimula- ofthe ATG start codon ofthe phage T7 4510 gene. The p42maPk tory agonists (9-11). Therefore, we suspect that this enzyme cDNA was excised from plasmid pA4 (6) by sequential plays a fundamental role in some process common to a treatment with Bgl I, mung bean nuclease, and BamHI. The number of regulatory events (for review, see ref. 11). -1.25-kilobase (kb) 42mapk cDNA fragment was ligated to Phosphorylation of$2maPk on both tyrosine and threonine the above 5639-bp fragment of pETila and transformed into is required for it to display full enzymatic activity (12). The E. coliHB1O1. Plasmids in which the second nucleotide ofthe sites of regulatory phosphorylation were identified by mass p42mIPk cDNA were ligated directly to the first nucleotide of spectrometry and found to be on a single peptide separated the phage T7 )10 gene on pETlia reconstitute a translational by only one amino acid, glutamic acid, in a sequence (Thr- start codon ATG and thus an unfused 42mapk coding se- Glu-Tyr-Val-Ala-Thr-Arg) that is absolutely conserved in the related protein kinases ERKi (extracellular signal-related Abbreviations: MAP kinase, mitogen-activated protein kinase; protein kinase 1), KSS1, and FUS3 (13). These phosphory- p42maPk, 42-kDa MAP kinase; IPTG, isopropyl f-D-thiogalactopy- ranoside; MBP, myelin basic protein; ERK1, extracellular signal- related protein kinase 1. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Box 419, De- payment. This article must therefore be hereby marked "advertisement" partment of Internal Medicine, University of Virginia Health Sci- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ences Center, Charlottesville, VA 22908. 9508 Downloaded by guest on September 26, 2021 Biochemistry: Wu et al. Proc. Natl. Acad. Sci. USA 88 (1991) 9509 quence under the control of T7 transcriptional and transla- BL21 MK K52R tional control signals. One such plasmid, pET-MK, was - + - + - + IPTG isolated and transformed into expression host E. coli BL21(DE3). The kinase-defective Lys-52 -- Arg mutant was generated using the Promega "Altered Sites" in vitro muta- 45 kDa - genesis kit. _ Expression of p42IPk in E. coli. A 10-ml overnight culture 40 ~p42'P42 mapk ofE. coli BL21(DE3)[pET-MK] was inoculated into 1 liter of Luria-Bertoni medium (10% tryptone/5% yeast extract/10%o NaCI) containing 0.1 mg of ampicillin per ml. The bacteria were grown at 370C with vigorous shaking to late logarithmic phase (ODwo = 0.8). IPTG was then added to a final con- 45 kDa - centration of 0.4 mM, and the cells were induced for 4-5 hr - p42 mapk before harvesting by centrifugation. For labeling of the phosphorylated p42maPk synthesized in E. coli, 0.2 mCi (1 Ci = 37 GBq) of 32p, per ml was added to the bacteria prior to the IPTG induction. The bacterial pellets were frozen at FIG. 1. Expression and tyrosine phosphorylation of p42mapk in -700C. bacteria. Wild-type p42mapk (lanes MK) and the kinase-defective Purification ofp421)Pk Produced in E. colU. Bacterial pellets Lys-52 -- Arg mutant (lanes K52R) were expressed in E. coli by were thawed, resuspended in 25 mM Tris-HCl, pH 7.5 induction with IPTG for 4 hr. The bacterial lysates were separated by (40C)/40 mM p-nitrophenyl phosphate/2 mM EGTA/2 mM SDS/PAGE, transferred to nitrocellulose, and blotted with either EDTA/1 mM dithiothreitol/l mM phenylmethylsulfonyl flu- anti-p42IaPk antibody 1798 (Upper) or anti-phosphotyrosine anti- oride and lysed by three passages through a chilled French body (Lower). pressure cell. The cell extracts were centrifuged at 12,000 x g for 20 min at 40C. p42maPk in the supernatant was then an autophosphorylation reaction. To test this hypothesis, we purified sequentially by batch absorption to phenyl- generated and expressed a kinase-defective mutant of Sepharose/Mono Q chromatography and phenyl-Superose p421Pk, with a substitution of arginine for lysine at position chromatography as described (13). 52 (in the ATP binding site) (6). Although IPTG also induced Protein Kinase Assay. Autophosphorylation of recombi- the appearance of this mutant protein (Fig. 1 Upper), the nant p42 Pk was performed by incubation in "autokinase protein did not become detectably phosphorylated on tyro- buffer" [25 mM Hepes/10 mM MgCl2/2 mM MnCl2/1 mM sine (Fig. 1 Lower). Thus, the appearance ofphosphotyrosine dithiothreitol, pH 7.5/50 ,uM [y-32P]ATP (5000 cpm/pmol)] at on bacterially expressed p42maPk requires a kinase-active 30°C. The amounts of protein and the time of autophosphor- enzyme, indicating that this tyrosine phosphorylation is a ylation are specified in the figure legends. The reaction was consequence of autophosphorylation. stopped by adding SDS electrophoresis
Load more

Recommended publications
  • 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]
  • HER2 Stabilizes EGFR and Itself by Altering Autophosphorylation Patterns in a Manner That Overcomes Regulatory Mechanisms and Pr
    Oncogene (2013) 32, 4169–4180 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE HER2 stabilizes EGFR and itself by altering autophosphorylation patterns in a manner that overcomes regulatory mechanisms and promotes proliferative and transformation signaling Z Hartman1, H Zhao1 and YM Agazie1,2 One of the causes of breast cancer is overexpression of the human epidermal growth factor receptor 2 (HER2). Enhanced receptor autophosphorylation and resistance to activation-induced downregulation have been suggested as mechanisms for HER2-induced sustained signaling and cell transformation. However, the molecular mechanisms underlying these possibilities remain incompletely understood. In the current report, we present evidence that show that HER2 overexpression does not lead to receptor hyper-autophosphorylation, but alters patterns in a manner that favors receptor stability and sustained signaling. Specifically, HER2 overexpression blocks epidermal growth factor receptor (EGFR) tyrosine phosphorylation on Y1045 and Y1068, the known docking sites of c-Cbl and Grb2, respectively, whereas promoting phosphorylation on Y1173, the known docking site of the Gab adaptor proteins and phospholipase C gamma. Under these conditions, HER2 itself is phosphorylated on Y1221/1222, with no known role, and on Y1248 that corresponds to Y1173 of EGFR. Interestingly, suppressed EGFR autophosphorylation on the Grb2 and c-Cbl-binding sites correlated with receptor stability and sustained signaling, suggesting that HER2 accomplishes these tasks by altering autophosphorylation patterns. In conformity with these findings, mutation of the Grb2-binding site on EGFR (Y1068F–EGFR) conferred resistance to ligand-induced degradation, which in turn induced sustained signaling, and increased cell proliferation and transformation.
    [Show full text]
  • Understanding and Exploiting Post-Translational Modifications for Plant Disease Resistance
    biomolecules Review Understanding and Exploiting Post-Translational Modifications for Plant Disease Resistance Catherine Gough and Ari Sadanandom * Department of Biosciences, Durham University, Stockton Road, Durham DH1 3LE, UK; [email protected] * Correspondence: [email protected]; Tel.: +44-1913341263 Abstract: Plants are constantly threatened by pathogens, so have evolved complex defence signalling networks to overcome pathogen attacks. Post-translational modifications (PTMs) are fundamental to plant immunity, allowing rapid and dynamic responses at the appropriate time. PTM regulation is essential; pathogen effectors often disrupt PTMs in an attempt to evade immune responses. Here, we cover the mechanisms of disease resistance to pathogens, and how growth is balanced with defence, with a focus on the essential roles of PTMs. Alteration of defence-related PTMs has the potential to fine-tune molecular interactions to produce disease-resistant crops, without trade-offs in growth and fitness. Keywords: post-translational modifications; plant immunity; phosphorylation; ubiquitination; SUMOylation; defence Citation: Gough, C.; Sadanandom, A. 1. Introduction Understanding and Exploiting Plant growth and survival are constantly threatened by biotic stress, including plant Post-Translational Modifications for pathogens consisting of viruses, bacteria, fungi, and chromista. In the context of agriculture, Plant Disease Resistance. Biomolecules crop yield losses due to pathogens are estimated to be around 20% worldwide in staple 2021, 11, 1122. https://doi.org/ crops [1]. The spread of pests and diseases into new environments is increasing: more 10.3390/biom11081122 extreme weather events associated with climate change create favourable environments for food- and water-borne pathogens [2,3]. Academic Editors: Giovanna Serino The significant estimates of crop losses from pathogens highlight the need to de- and Daisuke Todaka velop crops with disease-resistance traits against current and emerging pathogens.
    [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]
  • Molecular Basis of Tank-Binding Kinase 1 Activation by Transautophosphorylation
    Molecular basis of Tank-binding kinase 1 activation by transautophosphorylation Xiaolei Maa,1, Elizabeth Helgasonb,1, Qui T. Phungc, Clifford L. Quanb, Rekha S. Iyera, Michelle W. Leec, Krista K. Bowmana, Melissa A. Starovasnika, and Erin C. Dueberb,2 Departments of aStructural Biology, bEarly Discovery Biochemistry, and cProtein Chemistry, Genentech, South San Francisco, CA 94080 Edited by Tony Hunter, Salk Institute for Biological Studies, La Jolla, CA, and approved April 25, 2012 (received for review December 30, 2011) Tank-binding kinase (TBK)1 plays a central role in innate immunity: it the C-terminal scaffolding/dimerization domain (SDD), a do- serves as an integrator of multiple signals induced by receptor- main arrangement that appears to be shared among the IKK mediated pathogen detection and as a modulator of IFN levels. family of kinases (3). Deletion or mutation of the ULD in TBK1 Efforts to better understand the biology of this key immunological or IKKε severely impairs kinase activation and substrate phos- factor have intensified recently as growing evidence implicates phorylation in cells (22, 23). Furthermore, the integrity of the aberrant TBK1 activity in a variety of autoimmune diseases and ULD in IKKβ is not only required for kinase activity (24) but was fi cancers. Nevertheless, key molecular details of TBK1 regulation and shown also to confer substrate speci city in conjunction with the β substrate selection remain unanswered. Here, structures of phos- adjacent SDD (25). Recent crystal structures of the IKK phorylated and unphosphorylated human TBK1 kinase and ubiq- homodimer demonstrate that the ULD and SDD form a joint, uitin-like domains, combined with biochemical studies, indicate three-way interface with the KD within each protomer of the a molecular mechanism of activation via transautophosphorylation.
    [Show full text]
  • Cysteine-179 of Iκb Kinase Β Plays a Critical Role in Enzyme Activation by Promoting Phosphorylation of Activation Loop Serines
    EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 38, No. 5, 546-552, October 2006 Cysteine-179 of IκB kinase β plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines Mi-Sun Byun, Jin Choi and interaction with ATP. Dae-Myung Jue1 Keywords: cysteine; IκB kinase; NF-κB; phosphor- Department of Biochemistry, College of Medicine ylation; protein serine-threonine kinases The Catholic University of Korea Seoul 137-701, Korea 1Corresponding author: Tel, 82-2-590-1177; Introduction Fax, 82-2-596-4435; E-mail, [email protected] Nuclear factor-κB (NF-κB) is a transcription factor that regulates expression of a wide range of cellular Accepted 20 September 2006 and viral genes that play pivotal roles in immune and 12-14 inflammatory responses (Barnes and Karin, 1997). Abbreviations: 15dPGJ2, 15-deoxy-∆ -PGJ2; GST, glutathione In unstimulated cells, NF-κB is associated with S-transferase; HA, hemagglutinin; IKK, IκB kinase; MAPKKK, mito- inhibitory IκB proteins that inhibit nuclear localization gen-activated protein kinase kinase kinase; MEKK, MAPK/ and DNA binding of NF-κB. In response to stimuli extracellular signal-regulated kinase kinase; NIK, NF-κB-inducing including TNF, IL-1, LPS, or viruses, the IκBs are kinase phosphorylated and subsequently degraded, releasing NF-κB to bind DNA and induce expression of specific Abstract target genes. Phosphorylation of IκB is one of the primary IκB kinase β (IKKβ) subunit of IKK complex is points of regulation in NF-κB activation pathway, and essential for the activation of NF-κB in response to occurs by IκB kinase (IKK). IKK is present as a various proinflammatory signals.
    [Show full text]
  • Tyrosine Kinases Play a Permissive Role in Glucose-Induced Insulin Secretion from Adult Rat Islets
    19 Tyrosine kinases play a permissive role in glucose-induced insulin secretion from adult rat islets S J Persaud, T E Harris, C J Burns and P M Jones Cellular and Molecular Endocrinology Group, Biomedical Sciences Division, King’s College London, Campden Hill Road, London W8 7AH, UK (Requests for offprints should be addressed to S J Persaud) ABSTRACT The role(s) played by protein tyrosine kinases (TA47) inhibited islet tyrosine kinase activities (PTKs) in the regulation of insulin secretion from and glucose-, 4á ketoisocaproic acid (KIC)- and pancreatic â cells is not clear. We have examined the sulphonylurea-stimulated insulin release, without effects of glucose, the major physiological insulin affecting glucose metabolism. GS and TA47 also secretagogue, on the tyrosine phosphorylation state inhibited protein serine/threonine kinase activities of islet proteins, and assessed â cell insulin secretory to a limited extent, but had no effect on Ca2+, cyclic responses in the presence of PTK inhibitors. Under AMP- or phorbol myristate acetate (PMA)-induced basal conditions islets contained many proteins insulin secretion from electrically permeabilised phosphorylated on tyrosine residues, and glucose islets. These results suggest that PTK inhibitors (20 mM; 5–15 min) was without demonstrable effect exert their inhibitory effects on insulin secretion on the pattern of tyrosine phosphorylation, in either proximal to Ca2+ entry and it is proposed that the absence or presence of the protein tyrosine they act at the site of the voltage-dependent Ca2+ phosphatase (PTP) inhibitor, sodium pervanadate channel which regulates Ca2+ influx into â cells (PV). PV alone (100 µM) increased tyrosine following nutrient- and sulphonylurea-induced phosphorylation of several islet proteins.
    [Show full text]
  • SNT, a Differentiation-Specific Target of Neurotrophic Factor-Induced Tyrosine Kinase Activity in Neurons and PC12 Cells STUART J
    MOLECULAR AND CELLULAR BIOLOGY, Apr. 1993, p. 2203-2213 Vol. 13, No. 4 0270-7306/93/042203-11$02.00/0 Copyright ) 1993, American Society for Microbiology SNT, a Differentiation-Specific Target of Neurotrophic Factor-Induced Tyrosine Kinase Activity in Neurons and PC12 Cells STUART J. RABIN, VAUGHN CLEGHON, ANtD DAVID R. KAPLAN* Eukaryotic Signal Transduction Group, Molecular Mechanisms of Carcinogenesis Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702-1201 Received 23 October 1992/Returned for modification 26 November 1992/Accepted 26 January 1993 To elucidate the signal transduction mechanisms used by ligands that induce differentiation and the cessation of cell division, we utilized p13 uc1agarose, a reagent that binds p34C2/cdk2 By using thi re t identified a 78- to 90-kDa species in PC12 pheochromocytoma cells that is rapidly phosphorylated on tyrosine following treatment with the differentiation factors nerve growth factor (NGF) and fibroblast growth factor but not by the mitogens epidermal growth factor or insulin. This species, called SNT (suc-associated neurotrophic factor-induced tyrosine-phosphorylated target), was also phosphorylated on tyrosine in primary rat cortical neurons treated with the neurotrophic factors neurotrophin-3, brain-derived neurotrophic factor, and fibroblast growth factor but not in those treated with epidermal growth factor. In neuronal and fibroblast cells, where NGF can also act as a mitogen, SNT was tyrosine phosphorylated to a much greater extent during NGF-induced differentiation than during NGF-induced proliferation. SNT was phosphorylated in vitro on serine, threonine, and tyrosine in pl3sucl-agarose precipitates from NGF-treated PC12 cells, indicating that this protein may be a substrate of kinase activities associated with p13suc1-p34cdc21cdk2 complexes.
    [Show full text]
  • Tyrosine Phosphorylation and Activationof STAT5, STAT3
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8705-8709, September 1995 Immunology Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15 JAMES A. JOHNSTON*tt, CHRIS M. BACON*t§, DAVID S. FINBLOOMI, ROBERT C. REES§, DAVID KAPLANII, KYO SHIBUYAII, JOHN R. ORTALDO**, SANJAY GUPTAtt, YI QING CHEN*, JUDY D. GIRItt, AND JOHN J. O'SHEA* *Lymphocyte Cell Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892; IDivision of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD; **Laboratory of Experimental Immunology, Biological Response Modifiers Program, Division of Cancer Treatment and I1Advanced BioScience Laboratories, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201; ttlmmunex Research and Development Corporation, Seattle, WA 98101; ttDepartment of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032; and §Institute for Cancer Studies, University of Sheffield Medical School, Sheffield, South Yorkshire, S10 2RX, United Kingdom Communicated by Henry Metzger, National Institutes of Health, Bethesda, MD, June 15, 1995 (received for review March 23, 1995) ABSTRACT The cytokines interleukin 2 (IL-2) and IL-15 important to examine whether IL-15 might also activate JAK3 have similar biological effects on T cells and bind common and JAK1. hematopoietin receptor subunits. Pathways that involve Janus Studies of the mechanism by which hematopoietin receptors kinases (JAKs) and signal transducers and activators of activate gene transcription indicate that STAT proteins be- transcription (STATs) have been shown to be important for come rapidly tyrosine-phosphorylated in the receptor complex hematopoietin receptor signaling.
    [Show full text]
  • HER2/Neu: an Increasingly Important Therapeutic Target
    Clinical Trial Outcomes NELSON HER2/neu: an increasingly important therapeutic target. Part 1 4 Clinical Trial Outcomes HER2/neu: an increasingly important therapeutic target. Part 1: basic biology & therapeutic armamentarium Clin. Invest. This is the first of a comprehensive three-part review of the foundation for and Edward L Nelson therapeutic targeting of HER2/neu. No biological molecule in oncology has been more University of California, Irvine, extensively or more successfully targeted than HER2/neu. This review will summarize School of Medicine; Division of Hematology/Oncology; Department the pertinent biology of HER2/neu and the EGF receptor family to which it belongs, of Medicine; Department of Molecular with attention to the biological foundation for the design and clinical development Biology & Biochemistry; Sprague Hall, of the entire range of HER2/neu-targeted therapies, including efforts to mitigate Irvine, CA 92697, USA resistance mechanisms. In conjunction with the subsequent two parts (HER2/neu tissue [email protected] expression and current HER2/neu-targeted therapeutics), this comprehensive survey will identify opportunities and promising areas for future evaluation of HER2/neu- targeted therapies, highlighting the importance of HER2/neu as an increasingly important therapeutic target. Keywords: c-erbB2 • EGF receptor • EGFR • EGFR ligand • expression modulation • HER2/neu • monoclonal antibody • signaling network • targeted therapeutic • tyrosine kinase inhibitor • vaccine The history of the molecule known as c-erbB2 = neu, c-erbB3 = EGFR-3, and HER2/neu dates back to the earliest stud- eventually, c-erbB4 = EGFR-4) were estab- 10.4155/CLI.14.57 ies of virus-associated oncogenes. In 1979, lished [13] . The fact that the neu molecule studies of avian erythroblastosis virus iden- and coding sequence was originally identi- tified two putative viral oncogenes, v-erbA fied in the rat species and only recently has and v-erbB [1–3] .
    [Show full text]
  • Phospho-EGF Receptor (Tyr1068) (D7A5) XP Rabbit
    Revision 1 C 0 2 - t Phospho-EGF Receptor (Tyr1068) a e r ® o t (D7A5) XP Rabbit mAb S Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) 7 7 Web: [email protected] 7 www.cellsignal.com 3 # 3 Trask Lane Danvers Massachusetts 01923 USA For Research Use Only. Not For Use In Diagnostic Procedures. Applications: Reactivity: Sensitivity: MW (kDa): Source/Isotype: UniProt ID: Entrez-Gene Id: WB, IHC-P, IF-IC, F H M R Mk Endogenous 175 Rabbit IgG P00533 1956 Product Usage Information phosphorylated by CaM kinase II; mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation (10). Application Dilution 1. Hackel, P.O. et al. (1999) Curr Opin Cell Biol 11, 184-9. 2. Zwick, E. et al. (1999) Trends Pharmacol Sci 20, 408-12. Western Blotting 1:1000 3. Cooper, J.A. and Howell, B. (1993) Cell 73, 1051-4. Immunohistochemistry (Paraffin) 1:400 4. Hubbard, S.R. et al. (1994) Nature 372, 746-54. 5. Biscardi, J.S. et al. (1999) J Biol Chem 274, 8335-43. Immunofluorescence (Immunocytochemistry) 1:800 6. Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86. Flow Cytometry 1:1600 7. Levkowitz, G. et al. (1999) Mol Cell 4, 1029-40. 8. Ettenberg, S.A. et al. (1999) Oncogene 18, 1855-66. Storage 9. Rojas, M. et al. (1996) J Biol Chem 271, 27456-61. 10. Feinmesser, R.L. et al. (1999) J Biol Chem 274, 16168-73. Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.
    [Show full text]