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Involvement of PP6 in Dephosphorylation of Bcl11b
(an Anti-Tumorigenic Transcription Factor)
Chelsea Parker
Dr. Theresa FiltzDept. Pharmaceutical Sciences
Oregon State UniversityHoward Hughes Medical Institute
Undergraduate Summer Research Program
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
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?
Background Research
1 -
2 -
3 -
4 -
5 -
0 -
1 2 3 4 5
0 30 60 120 240
Time of P/A Treatment (min.)
Fo
ld In
du
ctio
n o
f m
RN
A
P/A-induced phosphorylation-dephosphorylation of Bcl11b in thymocytes
B
Courtesy of Drs. Ling Zhang and Mark Leid, Dept of Pharmaceutical Sciences, OSU
Id2 Regulation
Bcl11b Phosphorylation Sites
HEK-293T Cells
Human Embryonic Kidney cell line
Easily transfected No endogenous Bcl11b
HEK293T cells http://www.sigmaaldrich.com
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.
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
Experimental Design A
HEK293 Treatment Group A
(- PMA)
HEK293 Treatment Group B
(+ PMA)
Plate # 1 2 1 2
Bcl11b * * * *
PP6 * *pCDNA vector * *
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
Experimental Design B
HEK293 cell Treatment Groups
DNAA (-PMA) B (+PMA)
1 2 3 1 2 3
Bcl11b * * * * * *
PP6 * *
H114A * *
pCDNA * *
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
Experimental Design C
DNAadded
Plate #(HEK293 cells)
1, 2 3, 4, 5 6, 7 8, 9, 10
βgal * * * *
Id2 CAT * * * *
Bcl11b * *
PP6 * *
pCDNA * * *
Results C
βgal Assay Results
Sample # βgal unit1 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
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?
Special Thanks
Howard Hughes Medical Institute
OSU Dept. of Pharmaceutical Sciences
Dr. Theresa Filtz
Dr. Kevin Ahern
Xiao Liu
Dr. Mark Leid for Bcl11b constructs and antibodies
Dr. David Brautigan for PP6 constructs and antibodies