Bcl11b Transcription Factor Dephosphorylation by PP6 in

Report
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
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
B
Time of P/A Treatment (min.)
Fold Induction of mRNA
0
P/A-induced phosphorylation-dephosphorylation
of Bcl11b in thymocytes
Courtesy of Drs. Ling Zhang and Mark Leid, Dept of Pharmaceutical Sciences, OSU
30
60
120
240
4
5
5-
4-
3-
2-
1-
01
2
3
Id2 Regulation
Bcl11b Phosphorylation Sites
HEK-293T Cells



HEK293T cells
http://www.sigmaaldrich.com
Human Embryonic
Kidney cell line
Easily transfected
No endogenous Bcl11b
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
Bcl11b immunoprecipitation followed by Western Blot using
indicated antibodies
1A
-PMA
AntiBcl11b
Antiphosphothreonine
Anti- PP6
2A
-PMA
+PP6
1B
+PMA
2B
+PMA
+PP6
Experimental Design B
HEK293 cell Treatment Groups
A (-PMA)
B (+PMA)
DNA
Bcl11b
1
2
3
1
2
3
*
*
*
*
*
*
*
PP6
*
H114A
pCDNA
*
*
*
*
Results B
Bcl11b immunoprecipitation followed by Western Blot using
indicated antibodies
1A
-PMA
Anti –
Bcl11b
Anti pThr
Anti –
PP6
1B
+PMA
2A
-PMA
+PP6
2B
+PMA
+PP6
3A
-PMA
+H114A
3B
+PMA
+H114A
Experimental Design C
DNA
added
Plate #
(HEK293 cells)
1, 2
3, 4, 5
6, 7
8, 9, 10
βgal
*
*
*
*
Id2 CAT
*
*
*
*
*
Bcl11b
*
PP6
pCDNA
*
*
*
*
*
Results C
Id2 gene Fold Repression
βgal Assay Results
Sample #
1
2
3
4
6
7
8
9
βgal unit
77.50
48.95
122.17
94.76
63.57
50.52
16.00
17.93
6
5
4
3
2
1
0
pCDNA
(1, 2)
Bcl11b
(3, 4)
PP6
(6, 7)
Bcl11b/PP6
(8, 9)
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

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