Transcript Slide 1

GSK3B and congenital heart defects
Eva Dimitrova
mage 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
P
GSK3B
GS
GS
P
Inhibition
Other substrates similar mechanism
Two known isoforms GSK3α and GSK3β
Key regulator of Wnt/beta-catenin pathway
Image retrieved from:
http://www.ebi.ac.uk
GSK3B key regulator in Wnt/ betacatenin pathway
Wnt pathway in resting cells
Wnt pathway in activated cells 1. Target processes
• Differentiation
• Proliferation
• Cell migration
• Cell polarity
2. Wnt /beta-catenin
molecular
mechanism
• Resting cells– betacatenin degradation
• Activated cells – betacatenin accumulation,
gene transcription
Image retrieved from: David M Roberts, Kevin C Slep & Mark Peifer Nature Structural & Molecular Biology 14, 463 - 465 (2007)
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 thaliana
AtSK3-2 R178A
(et al Claisse, G 2007)
Conserved sites in the serine/threonine kinase domain of GSK3B
homologues
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
Arabidopsis thaliana AtSK3-2
S/T Kinase domain
ATP binding site and Active site
Images retrieved from PROSITE: http://www.expasy.ch/prosite
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
Arabidopsis thaliana AtSK3-2
ATP-binding site and Active site Conserved distance between the two sites
Images retrieved from PROSITE: http://www.expasy.ch/prosite
GSK3B interactions with other proteins
Homo sapiens
Data retrieved from :http://string.embl.de/
Mus musculus
GSK3B interactions with other proteins
Homo sapiens
Data retrieved from :http://string.embl.de/
Mus musculus
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, 3609 -3618
DNA Motifs on cDNA sequence
Motif Name
Description
Consensus
GATA-2
GATA- binding
GGGGATAGTG
factor 2
NNNGATRNNN
Nuclear factor
CACAGGAAAAAC
of activated T-cells
NNNWGGAAAANN
GATA binding
GGGGATAGTG
factor 1
SNNGATNNNN
GATA- binding
GGGATAGTG
factor 3
NNGATARNG
NF-ATc
GATA-1
GATA -3
Position
Score
1007..1016
97
1556..1567
96
Data retrieved from Motif search :http://motif.genome.jp/
1007..1016
94
1008..1016
91
DNA Motifs on cDNA sequence
Motif Name
Description
Consensus
GATA-2
GATA- binding
GGGGATAGTG
factor 2
NNNGATRNNN
Nuclear factor
CACAGGAAAAAC
of activated T-cells
NNNWGGAAAANN
GATA binding
GGGGATAGTG
factor 1
SNNGATNNNN
GATA- binding
GGGATAGTG
factor 3
NNGATARNG
NF-ATc
GATA-1
GATA -3
Position
Score
1007..1016
97
1556..1567
96
Data retrieved from Motif search :http://motif.genome.jp/
1007..1016
94
1008..1016
91
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)
Text mining
Homology
Databases
Experiments
Data retrieved from :http://string.embl.de/
GSK3B interactions with GATA factors
(Cyclin D1)
Text mining
Homology
Databases
Experiments
Data retrieved from :http://string.embl.de/
GATA protein motifs and homology
Domains
GATA1
Zinc Finger
GATA2
DNA binding
GATA3
Pfam GATA-N
transcription activation
GATA4
Data retrieved from SMART:http://smart.embl.de
Microarray- GATA 1 knockdown and
GSK3B expression
Title: GDS1245 / 1437001_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.
Data retrieved from : http://www.ncbi.nlm.nih.gov/geo/
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, 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 64:113124.
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???