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