GSK3B and congenital heart defects Eva Dimitrova Image retrieved from :

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,

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 P Inhibition Image retrieved from:

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, (2007) Wnt pathway in resting cellsWnt pathway in activated cells

GSK3B – homology and phylogeny Muscle Clustal W method Data created using

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 thaliana AtSK3-2 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 domain ATP binding site and Active site Arabidopsis thaliana AtSK3-2 Caenorhabditis elegans Gsk-3 Images retrieved from 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: Arabidopsis thaliana AtSK3-2 ATP-binding site and Active site Conserved distance between the two sites

GSK3B interactions with other proteins Homo sapiens Mus musculus Conclusion: players in Wnt pathway Data retrieved from :

GSK3B interactions with other proteins Homo sapiens Mus musculus Conclusion: players in Wnt pathway Data retrieved from :

GSK3B relationship to cardiac hypertrophy GSK3B - suggested to phosphorylate: 1. GATA4 2. NF-ATc 3. cMyc 4. Cyclin D1 Inhibition and nuclear 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,

DNA Motifs on cDNA sequence Motif NameDescriptionConsensusPositionScore GATA-2GATA- binding GGGGATAGTG factor 2 NNNGATRNNN NF-ATcNuclear factor CACAGGAAAAAC of activated T-cells NNNWGGAAAANN GATA-1GATA binding GGGGATAGTG factor 1 SNNGATNNNN GATA -3GATA- binding GGGATAGTG factor 3 NNGATARNG Data retrieved from Motif search :

DNA Motifs on cDNA sequence Motif NameDescriptionConsensusPositionScore GATA-2GATA- binding GGGGATAGTG factor 2 NNNGATRNNN NF-ATcNuclear factor CACAGGAAAAAC of activated T-cells NNNWGGAAAANN GATA-1GATA binding GGGGATAGTG factor 1 SNNGATNNNN GATA -3GATA- binding GGGATAGTG factor 3 NNGATARNG Data retrieved from Motif search :

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:

GSK3B interactions with GATA factors (Cyclin D1) Data retrieved from : Text mining Homology Databases Experiments

GSK3B interactions with GATA factors (Cyclin D1) Data retrieved from : Text mining Homology Databases Experiments

GATA protein motifs and homology GATA1 GATA2 GATA4 GATA3 Data retrieved from SMART: Domains Zinc Finger DNA binding Pfam GATA-N transcription activation

Microarray- GATA 1 knockdown and GSK3B expression Title: GDS1245 / _at / Gsk3b / Mus musculus Summary: 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.GDS1245 Data retrieved from :

Future approaches Experiments for GSK3B and GATA4 interactions TAP-tag – specific protein interactions Y2H – no need to force nuclear entry - can even identify specific site interactions DNA-expression plasmid arrays GATA4 knock out or knock down experiments compare to GSK3B Model organisms: MO (morpholino) – early stages of development; easy to use RNAi – for early stages, knock out genes – zygotic expression

References 1.Frame, S. (2001).GSK3 takes centre stage more than 20 years after its discovery.The Biochemical journal, 359, 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, Science daily (2008, October 3). Loss of the protein target of lithium disrupts normal mouse embryonic heart development. Retrieved February 1, 2009 from 4. Congenital heart defects. Retrieved January 31, 2009 from 5. National center for biotechnology information 6. Brady, H. and Horgan, J.(1998, January). Lithium and the heart. Chest, 93.1, Wnt/ beta-catenin signaling. Retrieved February 3, 2009 from 8.BLAST : 9. Homologene: 10.Phylogeny fr: SMART: Pfam: 13.Doble, B. & Woodgett, J. (2003). GSK-3: tricks of the trade for multi-tasking kinase. Journal of cell science 116, Lee, H. & Tsai, J. (August, ). Glycogen synthase kinase 3α and 3β have distinct functioning during cardiogenesis of Zebrafish embryo. BMC developmental biology, 7: Claisse, G. & Charrier, B. (February, ). The Arabidopsis thaliana GSK3/Shaggy like kinase AtSK3-2 modulates floral cell expansion. Plant Mol Biol 64: Pierce, S. & Kimelman, D. (1995). Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3. Development 121, PROSITE: Bax,B.(December 2001).The structure of phosphorylated GSK-3 beta complexed with a peptide, FRATtide, that Inhibits beta-catenin phosphorylation. Structure. 9,

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