Presentation format4 posttranslational modification in cell adhesion and migration
- 1. Posttranslational modification in cell adhesion and migration with emphasis on acetylation Dipl.-Ing. Birgit Kastberger Department of Cellular Physiology and Metabolism, University Medical Center, University of Geneva Oral exam: November 7, 2014
- 2. Cell Extracellular matrix (ECM) Integrins are involved in cell adhesion and migration provide a link from the ECM to the actin cytoskeleton a b1A Harvard University, BioVisions-The Inner Life Of A Cell, 2011 Integrins Actin
- 3. adaptor and b1A integrin PTMs are involved in cell adhesion & migration Filopodial contacts first contact with ECM spreading Lamellipodia adhesion, maturation, signalling Focal adhesions ECM remodelling Fibrillar adhesions W. Hu et al, Plos One, 2014, Geiger, et al., Nature Reviews Molecular Cell Biology, 2001 Fibrillar adhesions: ↓phospho-Tyrosine, ↑Tensin Tensin Focal adhesions: ↑phospho-Tyrosine pTyr … integrins … weblike/remodeling actin … adaptor proteins … bundled/contractile actin stress fibres
- 4. b1A integrin tail is phosphorylated and acetylated Extracellular domain: 21-728 aa Transmembrane domain: 729-751 aa Cytoplasmic tail: 752-798 aa β1A LLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK What role do PTMs play? PP P PPP Ac
- 5. Over 200 distinct covalent PTMs diversify the proteome KL Vermillion et al, JCB, 2014; Prabakaran et al, Wiley P., 2012, Thermo Scientific: post-translational-modification Most frequent PTMs experimentally observed Khoury et al., Sci. Rep. 1, 2014 (2011) P h o s p h o ry la tio n A c e ty la tio n M e th y la tio n O th e rs P P P P Ac Ac Me AcAc
- 6. 3 examples of more widely studied reversible PTMs : phosphorylation t1/2=20min Donor: ATP Kinase Phosphatase x Okadaic acid, H2O2 x Imatinib, Dasatinib, Staurosporin EGF, PDGF experimental phosphoryl. frequencynorm to aa frequ. pY/pT/pS: 1/1/4 +PO4 -3 provoke charge modification Kinases > high Kd for ATP > makes them operate close to independently from ATP concentrations Arena et al, CMLS, 2005; S. Prabakaran et al., Wiley Periodicals, 2012 phosphorylation modulates • activity • conformation • docking sites
- 7. Phosphorylation in cell adhesion and migration: Cofilin K. Rottner et al 2011 Curr. Opin. Cell Biol; Y. Zhang, Genes Dev, 2012, Mizuno, Cellular Signalling, 2013; Moriyama, Genes Cells, 1996; Nagai et al, Genes & Cancer, 2012; Harvard University, BioVisions-The Inner Life Of A Cell, 2011 Cofilin ↑pSer3 inactive Cofilin does not bind actin (phospho mimic S3D mutation) β1 integrin triggers downstream phosphorylation of pSer3 in Cofilin ↓pSer3 active Cofilin binds to actin found at the leading edge of mobile cells is required for directional migration severs actin filaments Cofilin active Cofilin Ser-3 P inactive Ser-3 ↑dephospho mimic S3A mutant > ↑cell migration and invasiveness invading astrocytoma brain cancer cell number through the matrigel-precoated membrane activity regulated by phosphorylation > local K/P concentration
- 8. 3 examples of widely studied reversible PTMs: methylation S. Prabakaran et al., Wiley Periodicals, 2012; S. Lanouette et al, Mol. Syst. B., 2014, Vermillion et al, JCB, 2014 Methyl Donor: SAM Lysine Methyl-Transferase (HMT) t1/2= very stable GSK J4 HCl x only small mass change ↑ protein stability modulates interactions between protein/DNA, protein/protein: docking site no change of positive charge crosstalks with PTMs x Demethylase (HDM) Mono- Di- Tri-methylation ↓ hydrogen bond formation not erasable by hydrolysis but via oxidative conversion > into hydrolytically labile imine methylation Competitive inhibitors for SAM: Trifluoroacetate salt, UNC0631 each methyl group > ↑ hydrophobicity of hydrophilic Lys
- 9. Lysine-Methylation in cell adhesion and migration: elongation factor 1-a1 (EF1a1) methylation is required for neural crest migration K.L. Vermillion et al, JCB, 2014 IF Control chicken embryo no change in neural crest migration (at somite 9 stage) EF1a1 binds to the actin cytoskeleton at the leading edge of migratory neural crest cells Sox10: Protein specifically present in migrating neural crest cells GFP GFP GFP GFP 6x di-/tri-methylated K (found by MS) to methylation resistant A EF1a1 prevents or promotes actin polymerization (conc. dep.) methylation resistant EF1a1 expression > ↓ cell migration distance
- 10. 3 examples of more widely studied reversible PTMs: acetylation t1/2= 1-60min Acetyl transferase (HAT) or equilibrium reaction Deacetylase (HDAC) Glucose, EtOH x SBHA, TSA, SAHA Anacardic acid Resveratrol, SRT-501 (are well tolerated in humans) Lundby et al, Cell Reports, 2012; Friis et al, Nucleic Acids Research 2009; Walsh et al, Angew. Chem. Int. Ed., 2005; Okanishi et al, J. proteome research, 2013; S. Prabakaran et al., Wiley Periodicals, 2012 Lys-e amine acetylation neutralizes its positive charge • alters hydrogen bonds • alters stability • docking site • hampers electrostatic interaction • increases hydrophobicity of hydrophilic Lys • tissue-type acetylation pattern • cell compartment specific Lys acetylation x Donor: AcCoA
- 11. Lysine is the most heavily modified amino acid • Lys acetylation blocks other PTMs • lengthens protein’s lifetime (e.g. p53) XJ Yang, Mol. Cell., 2008
- 12. 18 human HDAC enzymes classified into 4 groups based on yeast homology Kelly et al, Ashley Publications, 2002; Kim et al, Am J Transl Res, 2011; Sadoul et al, J. Biomed. and Biotech., 2011; Grant, Academic Press, p89, 2012; Michishita et al, MBC, 2005 HDACs class III SIRT 1/2cytop/3/4/5/6/7 NAD+ ubiquitously expressed high expression in brain, testis HDACs class I 1/2/3cytop/8cytop Zn2+ ubiquitously expressed TSA HDACs class II IIa: 4cytop/5cytop/7cytop/9cytop IIb: 6cytop/10cytop Zn2+ predominant in heart, smooth muscle, brain, kidney TSA HDACs class IV 11cytop Zn2+ predominant in heart, smooth muscle, brain, kidney TSA
- 13. Acetylation can mingle with other PTMs to form complex regulatory programs XJ Yang, Mol. Cell., 2008; Rao et al, BMC Bioinformatics, 2013; Z. Lu et al, Plos One, 2011; Latham et al, Nat Struct Mol Biol, 2007 46.0% of studied Ac-Lys are predicted to have an effect on phosphorylation sites: • creation/destruction of a phosphorylation site • alterations in kinase binding Z. Lu et al, Plos One, 2011 #ofalteredphosphorylationsites Distance from K-Ac Distance distribution of altered phosphorylation site to K-Ac (Q) in silico perturbation of the microenvironment through substitution of Lys (K) (+) with Glu (Q) (neutral), mimicking a neutral Ac-Lys
- 14. autophagy amino acid degradation Acetyl-CoA: the crossroads of fat, sugar and protein catabolism J. Patel et al., Nutrition & Metabolism, 2011; Choudhary et al, Nature reviews, 2014; Wellen et al, Nature reviews, 2012; Mariño et al, cell press, 2014 acetate can be produced by • deacetylation reactions • ethanol metabolism in the liver • by bacteria in the colon Citrate-Shuttle nutrient starvation > depletes AcCoA provokes autophagy caloric intake elevates Acetyl-CoA concentrations acetyl-CoA concentration influences • HAT activity • overall acetylation level ↑↓
- 15. 2 examples of protein acetylation in cell adhesion and migration: a-tubulin acetylation reduces cell motility XJ Yang et al, cell press, 2008; Joo et al, nature communications, 2014; Glozak, science direct, 2005; Harvard University, BioVisions-The Inner Life Of A Cell, 2011 welkescience Microtubles • α- and β-tubulin dimers form long, hollow cylinders • component of cytoskeleton • platforms for intracellular transport (secretory vesicles, organelles) Microtubules Deacetylation: HDAC6, SIRT2 ↑microtubule acetylation • ↓reduces cell migration • ↑increased stability ↓ microtubule acetylation • found at leading edge • prone to ↑depolymerization • ↑HDAC6 >↓microtubule acetylation > ↑cell migration speed
- 16. 2 examples of protein acetylation in cell adhesion and migration: Cortactin acetylation reduces cell motility X. Zhang et al, Cell Press, 2007; Y. Zhang et al, Oncogene, 2009; XJ Yang et al, cell press, 2008; Kirkbridge et al, Cell Adhesion and Migration, 2011;p ; Harvard University, BioVisions-The Inner Life Of A Cell, 2011 Cortactin Cortactin • cytoplasmic, monomeric • regulates branched actin assembly & stabilization at leading edge • abnormally expressed in many human tumours ↑ overexpression > ↑invasiveness and ↑migration ↓ depletion > ↓ impairs cell migration Acetylation: p300 Deacetylation: HDAC6 and Sirt1 deacetylated cortactin • ↑ cells migrate faster • translocates to cell periphery • ↑ actin binding • ↑ high levels of SIRT1 • observed in tumours hyperacetylated cortactin (up to 10 residues) • ↓ translocation to the cell periphery • ↓ association with actin • ↓cell motility
- 17. Drug effects on cell acetylation state and disease: HDAC inhibitors Singh et al, ERAT, 2010; Choudhary et al, Science, 2009; Wikipedia: SAHA, 2014; stocan.weebly.com, 2014; penn-medicine-lung-transplant.blogspot.ch, 2014 SAHA (Vorinostat) • FDA approved • treatment for cutaneous T-cell lymphoma (CTCL) • anti-cancer drug with low toxicity • competes with Zn2+ > blocks active site of HDACs • affects growth/survival of tumor cells • ↑ overall protein acetylation • provokes tumour selective ↑ pro-apoptotic gene ↓ pro-survival gene HDAC-1 gastric cancer lung cancer HDAC-5/10 HDAC inhibitors are anticancer drugs • ↑ cell cycle arrest/apoptosis/autophagy • ↓ metastasis/angiogenesis • ↑ acetylation mediated p53 activation > ↑ p53’s half life SAHA HDACs are often deregulated in cancer
- 18. Drug effects on cell acetylation state and disease: Glucose Miller et al, MBoC, 2014; Choudhary et al; Nature reviews, 2014 ↑SIRT1 consequences of high glucose conditions for adhesion/matrix ↑ increase integrin cell adhesion ↑ stimulate FN polymerization diabetics > ↑ higher plasma glucose concentrations > may entail greater ↑overall protein acetylation E.g. diabetic nephropathy comes with ECM expansion ↓glucose restriction ↑ provokes glucose production from fatty acids > maintains blood glucose levels ↑ SIRT1 > ↓ acetylation of proteins? ↑high concentrations of glucose ↑ acetyl-CoA levels ↑ overall protein acetylation ↑ fatty acid production
- 19. Drug effects on cell acetylation state and disease: EtOH Blythe et al, alcoholism: Clinical and Experimental research, 2010; Kannarkat, Journal of Hepatology, 2006; Lieberman et al, Wolters Kluwer, 2012 3 male rats fed 5 weeks with 36% ETOH containing diet Arrows: 2 fold or greater increase of acetylation Acetyl-CoA liver’s EtOH metabolism EtOH transformed into acetate (liver) > acetate metabolized to AcCoA Anti-acetyl Lysine antibody of rat livers highly reactive with Lys not toxic treatment chronic liver disease > ↓ overall acetylation HDAC activator Resveratrol attenuates fatty liver in alcohol-exposed mice • chronic EtOH consumption provokes liver protein ↑hyperacetylation (3x ↑): tubulin, actin, cortactin, p53 • key factor in liver injury mitochondrial dysfunction, altered protein trafficking • after EtOH withdrawal > acetylation remains • 0.04 ‰ endogenous EtOH in human blood bacterial fermentation caloric intake > 0.06 ‰ • EtOH exposure to cells > ↑protein acetylations, phosphorylations, methylations
- 20. Conclusion: cell adhesion and migration are crucial for organisms • structural organization embryogenesis body growth • body function immune responses repair after injury • constant dynamics tissue remodelling tissue renewal (adult’s cell age: 7-10 years) • maintain body structure connective tissue epithelial tissue • allow cell movement matrix modelling adhesion making/breaking Spalding et al, Cell, 2005; Alberts, 2008 Cell-Matrix adhesions: via integrins Cell migration: via cytoskeleton PTMs drive integrin function PTMs drive the function of cofilin, a-tubulin, cortactin, EF1a1 Cell adhesion and migration are essential for the structure and maintenance of multicellular organisms & PTMs are involved in their regulation
- 21. Thank you for your attention! Now it’s time for questions..
- 22. PTMs crosstalking evidence: the p53 example DNA damage> p53 activation • via methylation-acetylation-phosphorylation cascade • Lys372 methylation required for p53 acetylation • Lys370 and Lys372 acetylation on p53 change DNA binding specificity forms docking sites for transcriptional co-activators enhances nuclear localization impairs methylation, ubiquitination > ↑ p53 half-life • impacts on phosphorylation of Ser371 contributes to p53 stabilization cell cycle arrest through targeted protein expression Acetyltransferases (HAT): p300 Deacetylases (HDACs) : HDAC1, HDAC3, SIRT1, SIRT7 p53-transcription factor, tumour suppressor • very unstable • may be ac, p, m, ub,.. • gene mutated in 50% of human cancers (eg Lys 120) • in nucleus/cytoplasm at low levels (unstressed cells) • activated p53 protein induces cell-cycle arrest/apoptosis mice with 7xKR mutations at the C-term including Lys370/372: viable and phenotypically normal Patel et al, Nutrition & Metabolism, 2011; XJ Yang et al, Cell, 2008; Walsh et al, Angew. Chem., 2005; Ashcroft, et al Mol. Cell. Biol., 1999
Editor's Notes
- #3: May I list the functions of integrins with the help of this scheme Integrins serve as mechanical link to ECM & provide signalling in response to mechanical tension b1A Integrin -b1A synthesises (fibrillogenesis) and migrates on the fibronectin scaffold knock out of b1A is lethal in mice embryos (Baudoin et al, 1998) Since integrin signaling is also bidirectional (outside the cell to inside the cell and vice versa)
- #4: Filopodia are spike like projections of the plasma membrane that allow a cell explore its environment
- #5: which HDACs are involved siRNA: small interfering RNA, =transient 46 aa in tail
- #6: Within the last few decades, scientists have discovered that the human proteome is vastly more complex than the human genome. While it is estimated that the human genome comprises between 20,000 and 25,000 genes (1), the total number of proteins in the human proteome is estimated at over 1 million (2). These estimations demonstrate that single genes encode multiple proteins. Genomic recombination, transcription initiation at alternative promoters, differential transcription termination, and alternative splicing of the transcript are mechanisms that generate different mRNA transcripts from a single gene
- #7: phosphoryl frequency: pY/pT/pS: 1/2/8 ; aa frequency: Y/T/S: 1/2/2 ILK: pseudo kinase; FAK functions without kinase activity, ckit has also kinase unrelated functions perphosphate group SH2 +PTB domain binds pTyrosine; 14-3-3 +WD40 domain binds pS/pT Protein Kinases constitute the largest single enzyme family in the human genome 1/3 of all proteins in mammalian cells can undergo phosphorylation Kinases have a big variety spectrum of substrates> reflecting the true complexity of signal transduction cascades. Of the hundreds of PTMs identified, the most intensively studied is protein phosphorylation, in which protein kinases (PK) attach phosphate moieties to Ser, Thr, or Tyr residues. Protein phosphorylation appears to play an essential role in many diverse and critical cellular processes, and inhibitors of their function have emerged as important modes of therapy for malignant diseases. Phosvitin is one of the egg (commonly hen’s egg) yolk[1][2] phosphoproteins known for being the most phosphorylated protein found in nature.[3][4][5] Phosvitin isolation was first described by Mecham and Olcott in the year 1949 Casein (/ˈkeɪs.ɪn/ or /ˈkeɪˌsiːn/, from Latin caseus, "cheese") is the name for a family of related phosphoproteins (αS1, αS2, β, κ). These proteins are commonly found in mammalian milk, making up 80% of the proteins in cow milk and between 20% and 45% of the proteins in human milk NaVO4: Sodium orthovanadate NaF: Sodium Fluoride EGF: epidermal growth factor PDGF: platelet-derived growth factor tyrosine-kinase inhibitors (TKIs) operate by four different mechanisms: they can compete with adenosine triphosphate (ATP), the phosphorylating entity, the substrate or both or can act in an allosteric fashion, namely bind to a site outside the active site, affecting its activity by a conformational change Left panel: Protein kinases mediate phosphorylation at serine, threonine and tyrosine side chains, and phosphatases reverse protein phosphorylation by hydrolyzing the phosphate group. Right panel: Phosphorylation causes conformational changes in proteins that either activate (top) or inactivate (bottom) protein function. How to increase phosphorylation: activating drugs from Masspec England Phosphorylation in cancers
- #8: PIP2 regulates cofilin activity by binding cofilin and inhibiting cofilin from binding to F-actin Serine residue at position 3 Aspartic acid (S3D) (B) Results of cell migration assays. EV, WT, S3A, and S3D cells were seeded through a cell sedimentation manifold to establish a circular confluent monolayer on substrate-coated wells (bovine serum albumin [BSA] or laminin [LN]). The cells were allowed to migrate for 24 hours, and photographs were taken before and after migration. Bar graphs show average migration rate calculated as the change in the diameter of the circle circumscribing the cell population over a 24-hour period. Asterisks indicate statistically significant differences (*P = 0.006, **P = 0.005). (C) Results of cell invasion assay. (Left) Bar graphs showing the average invading cell number through the Matrigel-precoated membrane with 8-mm pores within 24 hours in 6 random light microscopic fields magnified at 200 times. Asterisk indicates statistically significant difference (*P = 0.011). (Right) Photomicrographs of the membranes with the invading cells stained with 0.5% crystal violet. Original magnification 200×.
- #9: Chromo domain binds methyl Lys Methylation is a regulator of ubiquitination SAM: S-adenosylmethionine made out of ATP and aa methionine HMT do not only methylate Histones but alsol nonhistone proteins How to increase methylation: traumata, decrease: Psychopharmaca, histones (acetylation, methylation but in cytoplasmic proteins)?
- #10: All evidence suggests that EF1a1 methylation is not required for translation (Sherman et al, 1989; Cavalius et al, 1997; Polevoda et al, 2007) EF1a1 is primarily known as a component of the translational machinery: it shuttles tRNA into the ribosomal position; it binds both actin and actin mRNA at the leading edge, is thought to enable localized actin translation to facilitate actin polymerization for motility EF1a1 can either prevent or promote actin polymerization depending on its cellular concentration, EF1a1 levels are developmentally regulated At 4-6 somites the EF1a1 6mm mutant elicited a similar range of neural crest specification> so the mutant does not disrupt EF1a1 activity during specification much as a methylation-resistant EF1a1 does not affect translation in yeast. Methylation is a regulator of ubiquitination SAM: S-adenosylmethionine HMT do not only methylate Histones but alsol nonhistone proteins How to increase methylation: traumata, deacrease : Psychopharmaca, histones (acetylation, methylation but in cytoplasmic proteins)?
- #11: for Bromo domains SAHA: suberoylanilide hydroxamic acid transcription factor/ tumour suppressor protein: p53 E/Glu: Glutamic acid, D/Asp: Aspartic acid The acetylation of histone neutralizes the positive charge of lysine residues resulting in a decrease in the binding affinity for the negatively charged phosphate groups of DNA.
- #13: require a zinc molecule as cofactor in their active site 10 cytoplasmic found HDACs
- #14: Q: Glutamine Cartoon illustrating crosstalk between phosphorylation and acetylation. Different signals act on Ser (S), Thr (T) or Tyr (Y) phosphorylation, which in turn affects acetylation of a neighboring Lys. Acetylation might also regulate phosphorylation. The Lys can be adjacent to or far away from the phosphorylation site, which can be either N-terminal or C-terminal from the acetylation site.
- #15: from citrate ATP citrate lyase (ACL): Citrat shuttle: Ac-CoA+Oxalacetat from acetate by acetyl-CoA synthetase (ACS) from pyruvate by the pyruvate dehydrogenase complex (PDC) tricarboxylic acid (TCA) cycle (also known as the citric acid cycle). In mammalian cells, Ac-coA is synthesized in two pathways. In the first pathway, Ac-coA synthetase condenses acetate and coenzyme A into Ac-coA. In mammals, acetate can be produced by deacetylation reactions, by ethanol metabolism in the liver and by bacteria residing in the colon; acetate can then be used to produce acetyl-CoA In the second pathway, energy from hydrolyzed ATP is utilized by ATP-citrate lyase (ACL) to convert citrate, a TCA cycle intermediate, and coenzyme A into Ac-coA and oxaloacetate. Compartmentalized synthesis of acetyl-CoA. a | In mammalian cells, mitochondrial acetyl-coenzyme A (acetyl-CoA) is produced from pyruvate by the pyruvate dehydrogenase complex (PDC) or by the β-oxidation of fatty acids, and then feeds into the tricarboxylic acid (TCA) cycle (also known as the citric acid cycle). Citrate can be exported from mitochondria into the cytoplasm, where it freely diffuses into and out of the nucleus. The extra-mitochondrial pool of acetyl-CoA, which is used for protein acetylation or for lipogenesis, is synthesized from citrate by ATP citrate lyase (ACL) or from acetate by acetyl-CoA synthetase (ACS). ACS is localized in the cytoplasm and ACL is localized in the cytoplasm and the nucleus60, which implies that acetyl-CoA is produced in both compartments. Because these enzymes use different substrates for generating acetyl-CoA, the relative importance of ACL and ACS in non-mitochondrial acetylation may depend on differences in carbon source utilization.
- #16: Microtubulin, Yamada paper, which HDACs are involved
- #17: which HDACs are involved
- #18: which HDACs are involved Cutaneous T cell lymphoma (CTCL) is a type of cancer of the immune system. Unlike most non-Hodgkin's lymphomas (which are generally B-cell related), CTCL is caused by a mutation of T cells. The malignant T cells in the body initially migrate to the skin, causing various lesions to appear. These lesions change shape as the disease progresses, typically beginning as what appears to be a rash which can be very itchy and eventually forming plaques and tumors before metastasizing to other parts of the body.
- #19: Occlusion of the arterioles ↑ provokes glucose production from fatty acids (gluconeogenesis) which HDAC, in vitro fertilisation s are involved Mesangial cells are specialized cells around blood vessels in the kidneys, at the mesangium.
- #20: which HDACs are involved
- #21: Connective tissue (plentiful ECM-sparsely distributed cells, rare cell-cell contacts) Epithelial tissue (close-lined up cells bound to basement membrane by hemidesmosomes a6b4 integrin (Laminin las ECM ligand) Immune system: Macrophages, Neutrophils
- #22: For their great support
- #23: -proteins regulating kinase signalling are enriched for acetylation (MS data) > suggesting crosstalk between acetylation and phosphorylation -Acetylation of histone H3 at Lys9 and Lys27 crosstalks with phosphorylation of Ser10 and Ser28, respectively (Latham and Dent, 2007) Positive crosstalk has been reported between acetylation of Lys14 and phosphorylation of Ser10 in histone H3, and our findings correctly predict this interaction. multiple enzymes appear to phosphorylate histone protein H3 at residue Ser10 which appears to stimulate acetylation at Lys14 (positive crosstalk). Conversely, this phosphorylation blocks acetylation and methylation at Lys9 in the same protein (negative crosstalk) (Latham et al, Nat Struct Mol Biol, 2007)