DOCSLIB.ORG

Phosphorylation/Dephosphorylation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Phosphorylation/Dephosphorylation Proc. Nati. Acad. Sci. USA Vol. 80, pp. 2477-2480, May 1983 Biochemistry Regulation of three key enzymes in cholesterol metabolism by phosphorylation/dephosphorylation (3-hydroxy-3-methylglutaryl-coenzyme A reductase/acyl-coenzyme A:cholesterol acyltransferase/cholesterol 7a-hydroxylase/homeostasis) TERENCE J. SCALLEN* AND AJIT SANGHVIt *Department of Biochemistry, School of Medicine, University of New Mexico, Albuquerque, New Mexico 87131; and tDepartment of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Communicated by N. E. Bradbury, January 17, 1983 ABSTRACT Our laboratories have investigated the role of phosphorylation and activated by dephosphorylation (10-19) (Fig phosphorylation/dephosphorylation in the regulation of three key 2). Recently, the in vivo significance of the phosphorylation of enzymes in cholesterol metabolism. 3-Hydroxy-3-methylglutaryl- HMG-CoA reductase was demonstrated, both for rats receiving coenzyme A (HMG-CoA ) reductase (EC 1.1.1.34), the major reg- a single dose of mevalonic acid by intragastric tube (20) and for ulatory enzyme in cholesterol biosynthesis, is inhibited by phos- rats receiving a single meal containing 2% cholesterol (21). Re- phorylation. Acyl-CoA:cholesterol O-acyltransferase (ACATase; cent studies from several other laboratories also support the EC 2.3.1.26) and cholesterol 7a-hydroxylase (EC 1.14.13.7), key biological significance of phosphorylation/dephosphorylation regulatory enzymes in the utilization of cholesterol, are activated of by phosphorylation. In view of these results, we propose that short- HMG-CoA reductase as a short-term regulatory mechanism term regulation of the concentration of intracellular unesterified (22-27). cholesterol is achieved by a coordinate phosphorylation/dephos- Acyl-CoACholesterol Acyltransferase ACATase (EC2.3.1.26). phorylation of these three enzymes. For example, if cholesterol ACATase is a membrane-bound microsomal enzyme that cata- enters the liver cell, HMG-CoA reductase would be inhibited by lyzes the formation of long-chain fatty-acyl cholesterol esters in phosphorylation and biosynthesis of cholesterol would be re- rat liver and other tissues (Fig. 1). This enzyme is important in duced; however, reactions utilizing cholesterol would be acti- regulating the concentration of unesterified cholesterol within vated, due to the phosphorylation of ACATase and cholesterol 7a- the cell, and thus it provides a mechanism for the removal of hydroxylase. Thus, the phosphorylation/dephosphorylation of these a potentially harmful excess of unesterified cholesterol by con- three enzymes provides an elegant short-term mechanism for the version to a form that can be stored intracellularly without del- homeostasis of intracellular unesterified cholesterol. eterious effects to the cell (1). In a preceding article (28), evidence is presented that ACAT- As has been noted by Spector et aL (1) and also by Kandutsch ase is capable of being regulated by phosphorylation/dephos- et al. (2), it is crucial that the concentration of unesterified cho- phorylation. It is significant that, whereas HMG-CoA reduc- lesterol within the cell be regulated within narrow limits. Unes- tase is inactivated by phosphorylation (Fig. 2), ACATase is terified cholesterol, present in cell membranes, functions to activated by phosphorylation (Fig. 3A). ACATase is therefore modulate the fluidity of the phospholipid bilayer (3). It has been regulated by phosphorylation in a manner exactly opposite to demonstrated that changes in the membrane cholesterol con- that of HMG-CoA reductase. In addition to being regulated by tent, by altering fluidity, can affect permeability, as well as the phosphorylation/dephosphorylation, the rate of cholesterol es- properties of membrane-bound enzymes and transport systems ter formation in rat liver microsomes is also affected by the (4-7). The presence of a storage pool (cholesterol ester) and, in availability of endogenous cholesterol substrate (29, 30), pre- the case of liver cells, the utilization of cholesterol via bile acid sumably delivered to ACATase by sterol carrier protein2 (SCP2) formation provide a means not only for the storage and utili- (31, 32). zation of cholesterol, but also for the control of the amount of Cholesterol 7a-Hydroxylase (Cholesterol 7a-Monooxygen- unesterified cholesterol present within the cell. ase, EC 1.14.13.7). The conversion of cholesterol to 7a-hy- In this article, we briefly review evidence consistent with droxycholesterol (Fig. 1), a reaction catalyzed by cholesterol 7a- the concept that regulation of three key enzymes in cholesterol hydroxylase, is the major regulatory step in bile acid biosyn- metabolism by phosphorylation/dephosphorylation provides a thesis (33, 34). Recent studies (35) have indicated that short-term short-term mechanism for intracellular unesterified cholesterol rapid changes in the activity of this enzyme may involve phos- homeostasis. The evidence presented supports our proposal that phorylation/dephosphorylation, with the active enzyme being enzymes involved in the regulation of cholesterol synthesis- phosphorylated (Fig. 3B). It was observed that incubation of rat e.g., 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) re- liver microsomes with Escherichia coli alkaline phosphatase ductase-are inactivated by phosphorylation, whereas en- produced a loss of cholesterol 7a-hydroxylase activity in pro- zymes involved in cholesterol utilization or storage are acti- portion to the amount of phosphatase added (35). Much of this vated by phosphorylation. loss was recovered by removal of the phosphatase, followed by HMG-CoA Reductase (EC 1.1.1.34). HMG-CoA reductase the addition of ATP, Mg2+, and a cytosolic protein fraction. It catalyzes the conversion of HMG-CoA to mevalonic acid (Fig. was shown recently (36) that cholesterol 7a-hydroxylase activity 1). This enzyme is the major regulatory enzyme in cholesterol is lost when rat liver microsomes are incubated in the presence biosynthesis (8, 9). Initially, evidence from several laboratories of various divalent cations e.g., Mg2 . This inactivation was demonstrated that HMG-CoA reductase activity is inhibited by Abbreviations: HMG-CoA reductase, 3-hydroxy-3-methylglutaryl- The publication costs ofthis article were defrayed in part by page charge coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acyl- payment. This article must therefore be hereby marked "advertise- ating), EC 1.1.1.34]; ACATase, acyl-coenzyme A:cholesterol O-acyl- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. transferase (EC 2.3. 1.26). 2477 Downloaded by guest on September 28, 2021 2478 Biochemistry: Scallen and Sanghvi Proc. Nad. Acad. Sci. USA 80 (1983) Acetyl-CoA CHOLLESTEROL1 SYNMTHESIS 4 HMG-CoA I HMG-CoA v Reductase Mevalonate *f INHIBITS O -j- --- -- -| CHOLESTEROL 1-~----|PHOSPHORYLATION|\ . I I I -Arl%-ATCQ-tIAX I IVAI Lb- 7a-Hydroxylase ACAT CHOLESTEROL UTILIZATION V 7a-hydroxycholesterol Cholesterol esters Bile acids FIG. 1. Diagram of cholesterol synthesis and utilization in rat liver. blocked when the incubation was conducted in the presence of may regulate the amount of unesterified cholesterol present in sodium fluoride. These results are consistent with the presence the cell. All three enzymes-i.e., HMG-CoA reductase, ACAT- of an endogenous microsomal protein phosphatase, activated ase, and cholesterol 7a-hydroxylase-have a common intra- by divalent cations, that in turn inactivates cholesterol 7a-hy- cellular location, namely, the endoplasmic reticulum. HMG-CoA droxylase by dephosphorylation (Fig. 3B). The recent studies reductase is the major enzyme regulating cholesterol biosyn- by Goodwin et al. (37) confirm the observation (36) that cho- thesis (Fig. 1). In liver cells, cholesterol utilization is regulated lesterol 7a-hydroxylase is capable of being regulated by phos- phorylation/dephosphorylation. A It is significant to note that cholesterol 7a-hydroxylase is ac- tivated by phosphorylation (Fig. 3B), whereas HMG-CoA re- ductase is inactivated by phosphorylation (Fig. 2). Cholesterol 7a-hydroxylase is therefore regulated by phosphorylation in a ACTIVE manner exactly opposite to that of HMG-CoA reductase. In ad- Protein Protein Kinase dition to being regulated by phosphorylation/dephosphoryla- Phosphatase ATP, M92+ tion, the rate of 7a-hydroxycholesterol formation in rat liver Mg2+ microsomes is also affected by the availability of endogenous cholesterol substrate (29). Short-Term Intracellular Cholesterol Homeostasis by Phos- phorylation/Dephosphorylation of Three Key Enzymes. The INACTIVE above findings provide a basis for proposing a mechanism that HMG - CoA Reductase B | 76-Hydroxylase I OPO3- OPO3 INACTIVE ACTIVE Protein j Protein Kinase Protein Reductase Kinase Phosphatase ATP, Mg2+ Phosphatase ATP, Mg2+ Mg2+ |HMG-CoA Reductase |7cx-Hydroxylase| OH OH ACTIVE INACTIVE FIG. 2. Diagram showing the phosphorylation/dephosphorylation FIG. 3. Diagram showing the phosphorylation/dephosphorylation of rat liver HMG-CoA reductase, the major regulatory enzyme in cho- of enzymes in rat liver involved in the regulation of cholesterol utili- lesterol biosynthesis. zation. (A) ACATase (ACAT); (B) cholesterol 7a-hydroxylase. Downloaded by guest on September 28, 2021 Biochemistry: Scallen and Sanghvi Proc. Natl. Acad. Sci. USA 80 (1983) 2479 A. CHOLESTEROL EXCESS was demonstrated that sterol ester hydrolase was activated by phosphorylation, which would cause increased amounts of Phosphorylation
Load more

Recommended publications
  • The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression
    International Journal of Molecular Sciences Review The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression Tingting Zou and Zhenghong Lin * School of Life Sciences, Chongqing University, Chongqing 401331, China; [email protected] * Correspondence: [email protected] Abstract: The cell cycle is a collection of events by which cellular components such as genetic materials and cytoplasmic components are accurately divided into two daughter cells. The cell cycle transition is primarily driven by the activation of cyclin-dependent kinases (CDKs), which activities are regulated by the ubiquitin-mediated proteolysis of key regulators such as cyclins, CDK inhibitors (CKIs), other kinases and phosphatases. Thus, the ubiquitin-proteasome system (UPS) plays a pivotal role in the regulation of the cell cycle progression via recognition, interaction, and ubiquitination or deubiquitination of key proteins. The illegitimate degradation of tumor suppressor or abnormally high accumulation of oncoproteins often results in deregulation of cell proliferation, genomic instability, and cancer occurrence. In this review, we demonstrate the diversity and complexity of the regulation of UPS machinery of the cell cycle. A profound understanding of the ubiquitination machinery will provide new insights into the regulation of the cell cycle transition, cancer treatment, and the development of anti-cancer drugs. Keywords: cell cycle regulation; CDKs; cyclins; CKIs; UPS; E3 ubiquitin ligases; Deubiquitinases (DUBs) Citation: Zou, T.; Lin, Z. The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression. 1. Introduction Int. J. Mol. Sci. 2021, 22, 5754. https://doi.org/10.3390/ijms22115754 The cell cycle is a ubiquitous, complex, and highly regulated process that is involved in the sequential events during which a cell duplicates its genetic materials, grows, and di- Academic Editors: Kwang-Hyun Bae vides into two daughter cells.
    [Show full text]
  • PTEN-L Is a Novel Protein Phosphatase for Ubiquitin Dephosphorylation to Inhibit PINK1–Parkin-Mediated Mitophagy
    www.nature.com/cr www.cell-research.com ARTICLE OPEN PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1–Parkin-mediated mitophagy Liming Wang1, Yik-Lam Cho1, Yancheng Tang2, Jigang Wang1, Jung-Eun Park3, Yajun Wu4, Chunxin Wang 5, Yan Tong6, Ritu Chawla1, Jianbin Zhang1,7, Yin Shi1, Shuo Deng1, Guang Lu1, Yihua Wu8, Hayden Weng-Siong Tan1,9, Pornteera Pawijit1, Grace Gui-Yin Lim10, Hui-Ying Chan1,9, Jingzi Zhang11, Lei Fang11, Hanry Yu1,12,13, Yih-Cherng Liou6, Mallilankaraman Karthik1, Boon-Huat Bay4, Kah-Leong Lim1,10, Siu-Kwan Sze 3, Celestial T. Yap1 and Han-Ming Shen1,9 Mitophagy is an important type of selective autophagy for specific elimination of damaged mitochondria. PTEN-induced putative kinase protein 1 (PINK1)-catalyzed phosphorylation of ubiquitin (Ub) plays a critical role in the onset of PINK1–Parkin-mediated mitophagy. Phosphatase and tensin homolog (PTEN)-long (PTEN-L) is a newly identified isoform of PTEN, with addition of 173 amino acids to its N-terminus. Here we report that PTEN-L is a novel negative regulator of mitophagy via its protein phosphatase activity against phosphorylated ubiquitin. We found that PTEN-L localizes at the outer mitochondrial membrane (OMM) and overexpression of PTEN-L inhibits, whereas deletion of PTEN-L promotes, mitophagy induced by various mitochondria-damaging agents. Mechanistically, PTEN-L is capable of effectively preventing Parkin mitochondrial translocation, reducing Parkin phosphorylation, maintaining its closed inactive conformation, and inhibiting its E3 ligase activity. More importantly, PTEN-L reduces the level of phosphorylated ubiquitin (pSer65-Ub) in vivo, and in vitro phosphatase assay confirms that PTEN-L dephosphorylates pSer65-Ub via its protein phosphatase activity, independently of its lipid phosphatase function.
    [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]
  • Protein Phosphatase 1-Α Regulates AS160 Ser588 and Thr642
    2606 Diabetes Volume 65, September 2016 Pragya Sharma,1 Edward B. Arias,1 and Gregory D. Cartee1,2,3 Protein Phosphatase 1-a Regulates AS160 Ser588 and Thr642 Dephosphorylation in Skeletal Muscle Diabetes 2016;65:2606–2617 | DOI: 10.2337/db15-0867 Akt substrate of 160 kDa (AS160) phosphorylation on insulin resistance is crucial for whole-body insulin re- Thr642 and Ser588 by Akt is essential for insulin’s full ef- sistance and type 2 diabetes (1). Muscle insulin resistance fect on glucose transport. However, protein phosphory- is secondary, in large part, to defective GLUT4 transloca- lation is determined by the balance of actions by kinases tion and glucose transport (2). Insulin’s stimulation of glu- fi and phosphatases, and the speci c phosphatase(s) cose transport is triggered by a complex insulin-signaling controlling AS160 dephosphorylation is (are) unknown. pathway that begins with insulin’s binding to its receptor, Accordingly, we assessed roles of highly expressed leading to receptor autophosphorylation and activation of skeletal muscle serine/threonine phosphatases (PP1, receptor tyrosine kinase (2). The insulin receptor kinase PP2A, PP2B, and PP2C) on AS160 dephosphorylation. Preliminary screening of candidate phosphatases used phosphorylates insulin receptor substrate (IRS) proteins an AS160 dephosphorylation assay. Lysates from insu- on multiple tyrosine residues, resulting in IRS protein en- lin-stimulated skeletal muscle were treated with phar- gagement with phosphatidylinositol (PI) 3-kinase (PI3K), macological phosphatase inhibitors and assessed for that in turn, phosphorylates PI 4,5-bisphosphate to create AS160 Ser588 and Thr642 dephosphorylation. AS160 de- 3,4,5-trisphosphate (PIP3). The serine/threonine kinase phosphorylation on both phosphorylation sites was un- Akt is recruited to bind PIP3 and become activated second- altered by PP2B or PP2C inhibitors.
    [Show full text]
  • A Protein Phosphatase 2Cα–Ca
    1914 • The Journal of Neuroscience, February 23, 2005 • 25(8):1914–1923 Cellular/Molecular A Protein Phosphatase 2c␣–Ca2ϩ Channel Complex for Dephosphorylation of Neuronal Ca2ϩ Channels Phosphorylated by Protein Kinase C Dongjun Li, Fushun Wang, Meizan Lai, Yuan Chen, and Ji-fang Zhang Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107 Phosphorylation and dephosphorylation are primary means for rapid regulation of a variety of neuronal functions, such as membrane excitability, neurotransmitter release, and gene expression. Voltage-gated Ca 2ϩ channels are targets for phosphorylation by a variety of second messengers through activation of different types of protein kinases (PKs). Protein phosphatases (PPs), like PKs, are equally important in regulating Ca 2ϩ channels in neurons. However, much less is understood about whether and how a particular type of PP contributes to regulating neuronal Ca 2ϩ channel activities. This is primarily because of the lack of specific inhibitors/activators for different types of PPs, particularly the PP2c family. The functional roles of PP2c and its substrates in the brain remain virtually unknown. During our yeast two-hybrid screening, PP2c␣ was pulled out by both N- and P/Q-type Ca 2ϩ channel C termini. This raised the possibility that PP2c␣ might be associated with voltage-gated Ca 2ϩ channels for regulation of the Ca 2ϩ channel activity. Biochemical studies show ϩ that PP2c␣ binds directly to neuronal Ca 2 channels forming a functional protein complex in vivo. PP2c␣, unlike PP1, PP2a and PP2b, is more effective in dephosphorylation of neuronal Ca 2ϩ channels after their phosphorylation by PKC. In hippocampal neurons, disruption of the PP2c␣–Ca 2ϩ channel interaction significantly enhances the response of Ca 2ϩ channels to modulation by PKC.
    [Show full text]
  • Faqs for DNA End Modification: Dephosphorylation
    FAQs for DNA End Modification: Dephosphorylation 1. Does the DNA need to be purified after a restriction digest and prior to the dephosphorylation step? If the enzymes used in the restriction digest are heat inactivatable, then a purification step is not needed. If the enzymes used are not heat-inactivatable, then a clean-up step between the restriction digest and the dephosphorylation step is recommended. 2. Does the DNA need to be purified after the dephosphorylation step and prior to the ligation step? If you use rSAP or Antarctic Phosphatase, which are heat-inactivatable, then a clean- up step is not necessary. If the alkaline phosphatase used is CIP, which is not heat- inactivatable, then a clean-up step is necessary. 3. Which alkaline phosphatase, CIP, Antarctic or rSAP, works best? rSAP (NEB #M0371) is a superior choice because it combines the advantages of both CIP (NEB #M0290) and Antarctic Phosphatase (NEB #M0289). rSAP has a very high specific activity comparable to CIP, but CIP cannot be completely heat-inactivated. rSAP is heat-inactivated in 5 minutes, as is Antarctic Phosphatase, but rSAP is preferred over Antarctic Phosphatase because it does not require added zinc or any other co-factors. As a result, rSAP can be added directly to restriction enzyme digests. Additionally, after heat-inactivation of rSAP, it is not necessary to purify vector DNA prior to the ligation reaction. 4. Are the alkaline phosphatases active in NEBuffers? rSAP: rSAP is active in all restriction enzyme NEBuffers 1.1, 2.1, 3.1 and CutSmart™ Buffer, and can be added directly to digested DNA.
    [Show full text]
  • Dephosphorylation of Threonine-14 and Tyrosine-15
    Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3521-3524, April 1993 Biochemistry Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15 BYRON SEBASTIAN*t, AKIRA KAKIZUKAt, AND TONY HUNTER* *Molecular Biology and Virology Laboratory and tGene Expression Laboratory, The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92186; and tDepartment of Biology, University of California at San Diego, La Jolla, CA 92093 Communicated by Renato Dulbecco, January 4, 1993 (receivedfor review December 15, 1992) ABSTRACT Recent evidence has suggested that human in regulating entry into S phase is suggested by reports that cyclin-dependent kinase 2 (CDK2) is an essential regulator of cycin E-associated kinase activity peaks in G1 (23, 24) and cell cycle progression through S phase. CDK2 is known to that overexpression ofcyclin E decreases the length ofG1 and complex with at least two distinct human cyclins, E and A. The diminishes the dependency of proliferating human cells on kinase activity of these complexes peaks in G1 and S phase, growth factors (23). Although both CDK2 and CDC2 asso- respectively. The vertebrate CDC2/cyclin Bi complex is an ciate with cyclin E, the predominant complex appears to be essential regulator of the onset of mitosis and is inhibited by CDK2/cyclin E. A role for CDK2/cyclin A in regulating phosphorylation of CDC2 on Thr-14 and Tyr-15. In vitro, progression through S phase is suggested by observations CDC2/cyclin Bi is activated by treatment with the members of that microinjection of either anti-cyclin A antibodies or the Cdc25 family of phosphatases.
    [Show full text]
  • AMP-Activated Protein Kinase: the Current Landscape for Drug Development
    REVIEWS AMP-activated protein kinase: the current landscape for drug development Gregory R. Steinberg 1* and David Carling2 Abstract | Since the discovery of AMP-activated protein kinase (AMPK) as a central regulator of energy homeostasis, many exciting insights into its structure, regulation and physiological roles have been revealed. While exercise, caloric restriction, metformin and many natural products increase AMPK activity and exert a multitude of health benefits, developing direct activators of AMPK to elicit beneficial effects has been challenging. However, in recent years, direct AMPK activators have been identified and tested in preclinical models, and a small number have entered clinical trials. Despite these advances, which disease(s) represent the best indications for therapeutic AMPK activation and the long-term safety of such approaches remain to be established. Cardiovascular disease Dramatic improvements in health care coupled with identifying a unifying mechanism that could link these (CVD). A term encompassing an increased standard of living, including better nutri- changes to multiple branches of metabolism followed diseases affecting the heart tion and education, have led to a remarkable increase in the discovery that the AMP-activated protein kinase or circulatory system. human lifespan1. Importantly, the number of years spent (AMPK) provided a common regulatory mechanism in good health is also increasing2. Despite these positive for inhibiting both cholesterol (through phosphoryla- Non-alcoholic fatty liver disease developments, there are substantial risks that challenge tion of HMG-CoA reductase (HMGR)) and fatty acid (NAFLD). A very common continued improvements in human health. Perhaps the (through phosphorylation of acetyl-CoA carboxylase disease in humans in which greatest threat to future health is a chronic energy imbal- (ACC)) synthesis8 (BOx 1).
    [Show full text]
  • Cell Type-Specific Control of Protein Synthesis and Proliferation by FGF-Dependent Signaling to the Translation Repressor 4E-BP
    Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP Rachel Ruoffa, Olga Katsaraa, and Victoria Kolupaevaa,1 aDepartment of Microbiology, New York University Langone Medical Center, New York, NY 10016 Edited by Nahum Sonenberg, McGill University, Montreal, Canada, and approved May 16, 2016 (received for review April 7, 2016) Regulation of protein synthesis plays a vital role in posttranscrip- the proliferation and differentiation of chondrocytes (4). Chon- tional modulation of gene expression. Translational control most drocyte homeostasis is regulated by a number of signaling molecules commonly targets the initiation of protein synthesis: loading 40S and transcription factors, and genetic studies identified FGF ribosome complexes onto mRNA and AUG start codon recognition. signaling as a crucial player in these processes (5). FGF molecules This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the signal by activating FGF Receptors (FGFRs), and several FGFR m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding mutations have been linked to autosomal, dominant bone subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assem- morphogenetic disorders (6). Importantly, all of these mutations bly of active eIF4F complex. The ability of eIF4E to recognize the cap are “gain of function” mutations, resulting in excessive FGF is prevented by its binding to eIF4E binding protein (4E-BP), which signaling. It has been well established that the major effect of thereby inhibits cap-dependent translation by sequestering eIF4E. FGF signaling in chondrocytes is growth inhibition (7, 8), an The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mech- effect which is in striking contrast to a universal proliferative anistic target of rapamycin) complex 1] mediated phosphorylation.
    [Show full text]
  • Phosphorylation of Ubiquitin-Activating Enzyme in Cultured Cells
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3454-3457, April 1995 Biochemistry Phosphorylation of ubiquitin-activating enzyme in cultured cells JAMES C. COOK* AND P. BOON CHOCKt Laboratory of Biochemistry, Section on Metabolic Regulation, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 3, Room 202, Bethesda, MD 20892 Communicated by Earl R. Stadtman, National Institutes of Health, Bethesda, MD, January 3, 1995 ABSTRACT Ubiquitin-activating enzyme, El, is the first protein ligase. Isoforms of all three enzymes have been enzyme in the pathway leading to formation of ubiquitin- described (11-14, 19). protein conjugates. El exists as two isoforms in human cells Obviously, ubiquitin conjugation must be a regulated pro- which are separable by electrophoresis. These isoforms mi- cess. Only certain proteins are recognized by the conjugating grate with apparent molecular sizes of 110 kDa and 117 kDa enzymes, and some are targets for conjugation only under in SDS/polyacrylamide gels. Immunoprecipitation ofEl from certain environmental conditions (15, 16) or during specific lysates of HeLa cells metabolically labeled with [32P]phos- phases of the cell cycle (17). Yet little is known about how this phate indicated the presence of a phosphorylated form of El regulation is accomplished. which migrates at 117 kDa. Phospho amino acid analysis Previous reports demonstrated that El can undergo in vitro identified serine as the phosphorylated residue in El. Phos- phosphorylation (18) and that it exists as two isoforms in phorylated El was also detected in normal and transformed human cells (19). These were designated "ElllOkDa" and cells from another human cell line.
    [Show full text]
  • Ordered Dephosphorylation Initiated by the Selective Proteolysis of Cyclin
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.12.148742; this version posted June 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit James Holder, Shabaz Mohammed and Francis A. Barr Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU Please address correspondence to: [email protected] [email protected] Keywords: Cell cycle, proteolysis, phosphatase, kinase Running title: Protein dephosphorylation drives mitotic exit 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.12.148742; this version posted June 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT APC/C-mediated proteolysis of cyclin B and securin promotes entry into anaphase, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-opposing phosphatases PP1 and PP2A-B55 leading to dephosphorylation of substrates crucial for mitotic exit. Meanwhile, continued APC/C activity is required to target various proteins, including Aurora and Polo kinases, for degradation. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution mass spectrometry, we compare the relative extent of proteolysis and protein dephosphorylation.
    [Show full text]
  • Fcp1 Phosphatase Controls Greatwall Kinase to Promote PP2A-B55
    SHORT REPORT Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression Rosa Della Monica1,2†, Roberta Visconti3†, Nando Cervone1,2, Angela Flavia Serpico1,2, Domenico Grieco1,2* 1CEINGE Biotecnologie Avanzate, Naples, Italy; 2Dipartimento di Medicina molecolare e Biotecnologie mediche, University of Naples Federico II, Naples, Italy; 3Istituto per l’endocrinologia e l’oncologia "Gaetano Salvatore", Consiglio Nazionale delle Ricerche, Naples, Italy Abstract During cell division, progression through mitosis is driven by a protein phosphorylation wave. This wave namely depends on an activation-inactivation cycle of cyclin B-dependent kinase (Cdk) 1 while activities of major protein phosphatases, like PP1 and PP2A, appear directly or indirectly repressed by Cdk1. However, how Cdk1 inactivation is coordinated with reactivation of major phosphatases at mitosis exit still lacks substantial knowledge. We show here that activation of PP2A-B55, a major mitosis exit phosphatase, required the phosphatase Fcp1 downstream Cdk1 inactivation in human cells. During mitosis exit, Fcp1 bound Greatwall (Gwl), a Cdk1-stimulated kinase that phosphorylates Ensa/ARPP19 and converts these proteins into potent PP2A-B55 inhibitors during mitosis onset, and dephosphorylated it at Cdk1 phosphorylation sites. Fcp1- catalyzed dephosphorylation drastically reduced Gwl kinase activity towards Ensa/ARPP19 promoting PP2A-B55 activation. Thus, Fcp1 coordinates Cdk1 and Gwl inactivation to derepress PP2A-B55, generating a dephosphorylation switch that drives mitosis progression. DOI: 10.7554/eLife.10399.001 *For correspondence: domenico. [email protected] †These authors contributed equally to this work Results and discussion Competing interests: The We recently reported a critical, transcription-independent, role for the essential RNA polymerase II- authors declare that no carboxy-terminal domain (RNAP II-CTD) phosphatase Fcp1 in Cdk1 inactivation at the end of mitosis competing interests exist.
    [Show full text]