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GSK3B and congenital heart defects. Eva Dimitrova Image retrieved from : http://www.smashingapps.com/. Congenital heart defects introduction. 35,000 babies with congenital heart defects Li treatment for bipolar disorder contravercies Li and GSK3B. - PowerPoint PPT Presentation
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GSK3B and congenital heart defects
Eva DimitrovaImage retrieved from : http://www.smashingapps.com/
Congenital heart defects introduction
• 35,000 babies with congenital heart defects
• Li treatment for bipolar disorder contravercies
• Li and GSK3B
Bruneau, B. (February 2008). Review Article The developmental genetics of congenital heart disease. Nature. 451, 943-948
GSK3B – serine/threonine kinase
Glycogen Synthase Kinase 3 – S/T kinase
Other substrates similar mechanism
Two known isoforms GSK3α and GSK3β
Key regulator of Wnt/beta-catenin pathway
GSK3B GS
P
GS P Inhibition
Image retrieved from: http://www.ebi.ac.uk
GSK3B key regulator in Wnt/ beta-catenin pathway
1. Target processes• Differentiation• Proliferation• Cell migration• Cell polarity 2. Wnt /beta-catenin
molecular mechanism
• Resting cells– beta-catenin degradation
• Activated cells – beta-catenin accumulation, gene transcription
Image retrieved from: David M Roberts, Kevin C Slep & Mark Peifer Nature Structural & Molecular Biology 14, 463 - 465 (2007)
Wnt pathway in resting cells Wnt pathway in activated cells
GSK3B – homology and phylogeny
Muscle
Clustal W method
Data created using http://www.phylogeny.fr/
GSK3B mutations and related phenotypes
Mice Gsk3b-/- KO gene (et al Kerkela, R. 2008)
Zebrafish GSK3-beta MO silencing
Xenopus Xgsk3K/R (et al Pierce, S 1995)
Drosophila – sgg-/- loss of function (et al Heslip,T.1997)
Arabidopsis thalianaAtSK3-2 R178A(et al Claisse, G 2007)
Homo sapiens GSK3α
Homo sapiens GSK3β
Pan troglodytes GSK3-like protein (predicted)
Canis familiaris GSK3-like protein (predicted)
Drosophila melanogaster Sgg
Danio rerio GSK3β
S/T Kinase domainATP binding site and Active site
Arabidopsis thaliana AtSK3-2
Caenorhabditis elegans Gsk-3
Images retrieved from PROSITE: http://www.expasy.ch/prosite
Conserved sites in the serine/threonine kinase domain of GSK3B homologues
ATP-binding site and active site homology
Homo sapiens GSK3α
Homo sapiens GSK3β
Pan troglodytes GSK3-like protein (predicted )
Canis familiaris GSK3-like protein (predicted)
Danio rerio GSK3β
Drosophila melanogaster Sgg
Caenorhabditis elegans Gsk-3
Images retrieved from PROSITE: http://www.expasy.ch/prosite
Arabidopsis thaliana AtSK3-2
ATP-binding site and Active site Conserved distance between the two sites
GSK3B interactions with other proteins
Homo sapiensMus musculus
Conclusion: players in Wnt pathway
Data retrieved from :http://string.embl.de/
GSK3B interactions with other proteins
Homo sapiensMus musculus
Conclusion: players in Wnt pathway
Data retrieved from :http://string.embl.de/
GSK3B relationship to cardiac hypertrophy
GSK3B - suggested to phosphorylate:
1. GATA4 2. NF-ATc3. cMyc4. Cyclin D1
Inhibition andnuclear exit
Repression of cardiac hypertrophy
Kerkela, R. (2008). Deletion of GSK-3 beta in mice leads to hypertrophic cardiomyopathy secondary to cardiomyoblast hyperproliferation. The journal of clinical investigation, 118, iss:11, 3609 -3618
DNA Motifs on cDNA sequence
Motif Name Description Consensus Position Score
GATA-2 GATA- binding GGGGATAGTG 1007..1016 97
factor 2 NNNGATRNNN
NF-ATc Nuclear factor CACAGGAAAAAC 1556..1567 96
of activated T-cells NNNWGGAAAANN
GATA-1 GATA binding GGGGATAGTG 1007..1016 94
factor 1 SNNGATNNNN
GATA -3 GATA- binding GGGATAGTG 1008..1016 91
factor 3 NNGATARNG
Data retrieved from Motif search :http://motif.genome.jp/
DNA Motifs on cDNA sequence
Motif Name Description Consensus Position Score
GATA-2 GATA- binding GGGGATAGTG 1007..1016 97
factor 2 NNNGATRNNN
NF-ATc Nuclear factor CACAGGAAAAAC 1556..1567 96
of activated T-cells NNNWGGAAAANN
GATA-1 GATA binding GGGGATAGTG 1007..1016 94
factor 1 SNNGATNNNN
GATA -3 GATA- binding GGGATAGTG 1008..1016 91
factor 3 NNGATARNG
Data retrieved from Motif search :http://motif.genome.jp/
GATAs definition, structure, function
GATA family – zinc finger transcription factors. They recognize the GATA motif in the promoter
GATA4 -regulate genes involved in embryogenesis and in myocardial differentiation and function. Mutations in this gene have been associated with cardiac septal defects.
GATA3 - Transcriptional activator which binds to the enhancer of the T-cell receptor alpha and delta genes.
GATA2 - expressed in hematopoietic progenitors, promotes proliferation at the expense of differentiation
GATA1 - erythroid development by regulating the switch of fetal hemoglobin to adult hemoglobin
Data retrieved from GeneCards: http://www.genecards.org
GSK3B interactions with GATA factors(Cyclin D1)
Data retrieved from :http://string.embl.de/
Text miningHomologyDatabasesExperiments
GSK3B interactions with GATA factors(Cyclin D1)
Data retrieved from :http://string.embl.de/
Text miningHomologyDatabasesExperiments
GATA protein motifs and homology
GATA1
GATA2
GATA4
GATA3
Data retrieved from SMART:http://smart.embl.de
Domains
Zinc Finger DNA binding
Pfam GATA-Ntranscription activation
Microarray- GATA 1 knockdown and GSK3B expression
Title: GDS1245 / 1437001_at / Gsk3b / Mus musculusSummary: Analysis of megakaryocytes lacking the transcription factor GATA-1. GATA-1 is required for the development of megakaryocytes and erythroid cells. Megakaryocytes obtained from 13.5 day C57BL/6 mutant embryos. Results provide insight into the role of GATA-1 in megakaryopoiesis.
Data retrieved from : http://www.ncbi.nlm.nih.gov/geo/
Future approachesExperiments for GSK3B and GATA4 interactionsTAP-tag – specific protein interactionsY2H – no need to force nuclear entry
- can even identify specific site interactionsDNA-expression plasmid arraysGATA4 knock out or knock down experiments compare to GSK3BModel organisms:
MO (morpholino) – early stages of development; easy to use
RNAi – for early stages, knock out genes – zygotic expression
References1.Frame, S. (2001).GSK3 takes centre stage more than 20 years after its discovery.The Biochemical journal, 359, 1 -16.
2. Kerkela, R. (2008). Deletion of GSK-3 beta in mice leads to hypertrophic cardiomyopathy secondary to cardiomyoblast hyperproliferation. The journal of clinical investigation, 118, iss:11, 3609 -3618
3. Science daily (2008, October 3). Loss of the protein target of lithium disrupts normal mouse embryonic heart development. Retrieved February 1, 2009 from http://www.sciencedaily.com
4. Congenital heart defects. Retrieved January 31, 2009 from http://www.nhlbi.nih.gov/health/dci/index.html
5. National center for biotechnology information http://www.ncbi.nlm.nih.gov/sites
6. Brady, H. and Horgan, J.(1998, January). Lithium and the heart. Chest, 93.1,166-169.
7. Wnt/ beta-catenin signaling. Retrieved February 3, 2009 from http://www.cellsignal.com/reference/pathway/Wnt_beta_Catenin.html
8.BLAST :http://blast.ncbi.nlm.nih.gov/Blast.cgi
9. Homologene:http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene
10 .Phylogeny fr: http://www.phylogeny.fr
11. SMART: http://smart.embl-heidelberg.de
12. Pfam: http://pfam.sanger.ac.uk
13.Doble, B. & Woodgett, J. (2003). GSK-3: tricks of the trade for multi-tasking kinase. Journal of cell science 116, 1175-1186
14.Lee, H. & Tsai, J. (August, 3 2007). Glycogen synthase kinase 3α and 3β have distinct functioning during cardiogenesis of Zebrafish embryo. BMC developmental biology, 7:93.
15. Claisse, G. & Charrier, B. (February, 15 2007). The Arabidopsis thaliana GSK3/Shaggy like kinase AtSK3-2 modulates floral cell expansion. Plant Mol Biol 64:113-124.
16. Pierce, S. & Kimelman, D. (1995). Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3. Development 121, 755-765.
17. PROSITE: http://www.expasy.ch/prosite
18. Bax,B.(December 2001).The structure of phosphorylated GSK-3 beta complexed with a peptide, FRATtide, that Inhibits beta-catenin phosphorylation. Structure. 9, 1143-1152
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