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

Questions???