involvement of pp6 in dephosphorylation of bcl11b (an anti-tumorigenic transcription factor)
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Involvement of PP6 in Dephosphorylation of Bcl11b (an Anti-Tumorigenic Transcription Factor) . Chelsea Parker Dr. Theresa Filtz Dept. Pharmaceutical Sciences Oregon State University Howard Hughes Medical Institute Undergraduate Summer Research Program. - PowerPoint PPT PresentationTRANSCRIPT
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Involvement of PP6 in Dephosphorylation of Bcl11b
(an Anti-Tumorigenic Transcription Factor)
Chelsea ParkerDr. Theresa Filtz
Dept. Pharmaceutical SciencesOregon State University
Howard Hughes Medical InstituteUndergraduate Summer Research Program
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T-cell acute lymphoblastic leukemia (T-ALL)
Leukemia is a cancer characterized by the uncontrolled accumulation of blood cells
In 20% of T-ALL cases Bcl11b has been made incorrectly
Defects cause leukemia because Bcl11b is a crucial component of several stages of T-cell development
Findings could be used to develop better treatments for aggressive childhood and blood cell cancers
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Bcl11b Transcription Factor Also known as CTIP2 In thymocytes:
Acts as a tumor suppressor against Id2 gene Undergoes a cycle of phosphorylation followed by
dephosphorylation Dephosphorylation of Bcl11b happens concurrently
with derepression of the Id2 gene
Which phosphatase dephosphorylates Bcl11b?
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Background Research
1 -
2 -
3 -
4 -
5 -
0 -1 2 3 4 5
0 30 60 120 240
Time of P/A Treatment (min.)
Fold
Indu
ctio
n of
mR
NA
P/A-induced phosphorylation-dephosphorylation of Bcl11b in thymocytes
B
Courtesy of Drs. Ling Zhang and Mark Leid, Dept of Pharmaceutical Sciences, OSU
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Id2 Regulation
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Bcl11b Phosphorylation Sites
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HEK-293T Cells
Human Embryonic Kidney cell line
Easily transfected No endogenous Bcl11b
HEK293T cells http://www.sigmaaldrich.com
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Hypothesis The Bcl11b transcription factor is
dephosphorylated by PP6 in transfected HEK293 cells.
Co-expression of PP6 and Bcl11b will result in de-repression/increased expression of the reporter gene controlled by the Id2 promoter.
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Predictions HEK-293T cells will show less
phosphorylation of Bcl11b in samples where PP6 is also present, with or without PMA stimulation
HEK-293T cells will exhibit changes in levels of expression of the Id2 gene in CAT reporter assays
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Experimental Design AHEK293 Treatment Group
A (- PMA)
HEK293 Treatment Group B
(+ PMA)Plate # 1 2 1 2
Bcl11b * * * *PP6 * *pCDNA vector * *
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Results A
1A -PMA
2A -PMA +PP6
1B+PMA
2B+PMA+PP6
Anti-Bcl11b
Anti-phospho-threonine
Anti- PP6
Bcl11b immunoprecipitation followed by Western Blot using indicated antibodies
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Experimental Design B
HEK293 cell Treatment Groups
DNAA (-PMA) B (+PMA)
1 2 3 1 2 3
Bcl11b * * * * * *
PP6 * *
H114A * *
pCDNA * *
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Results B
1A-PMA
1B+PMA
2A-PMA+PP6
2B+PMA+PP6
3A-PMA
+H114A
3B+PMA
+H114A
Anti – Bcl11b
Anti -pThr
Anti – PP6
Bcl11b immunoprecipitation followed by Western Blot using indicated antibodies
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Experimental Design C
DNAadded
Plate #(HEK293 cells)
1, 2 3, 4, 5 6, 7 8, 9, 10
βgal * * * *
Id2 CAT * * * *
Bcl11b * *
PP6 * *
pCDNA * * *
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Results C
βgal Assay ResultsSample # βgal unit
1 77.502 48.953 122.174 94.766 63.577 50.528 16.009 17.93 pCDNA
(1, 2)Bcl11b
(3, 4)PP6
(6, 7)Bcl11b/
PP6 (8, 9)
00.5
11.5
22.5
33.5
4
Id2 gene Fold Repression
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Conclusion PP6 co-expression is associated with
dephosphorylation of Bcl11b in HEK293T. H114A is not a good negative control for PP6
PP6 may be a nucleating factor? May require regulatory subunits R1 and R3?
PP6 has Bcl11b-independent action in reporter gene assays
Next steps: Bcl11b phosphosite mutants, PP6 knockdown experiments PP2A?
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Special Thanks
Howard Hughes Medical InstituteOSU Dept. of Pharmaceutical SciencesDr. Theresa FiltzDr. Kevin AhernXiao LiuDr. Mark Leid for Bcl11b constructs and
antibodiesDr. David Brautigan for PP6 constructs and
antibodies