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Involvement of PP6 in Dephosphorylation of Bcl11b (an Anti-Tumorigenic Transcription Factor)

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.

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Involvement of PP6 in Dephosphorylation of Bcl11b (an Anti-Tumorigenic Transcription Factor)

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  1. 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

  2. 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 T-cell acute lymphoblastic leukemia (T-ALL)

  3. 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? Bcl11b Transcription Factor

  4. Background Research Time of P/A Treatment (min.) 0 30 60 120 240 Fold Induction of mRNA B P/A-induced phosphorylation-dephosphorylation of Bcl11b in thymocytes 5 - 4 - 3 - 2 - 1 - Courtesy of Drs. Ling Zhang and Mark Leid, Dept of Pharmaceutical Sciences, OSU 0 - 1 2 3 4 5

  5. Id2 Regulation

  6. Bcl11b Phosphorylation Sites

  7. HEK-293T Cells • Human Embryonic Kidney cell line • Easily transfected • No endogenous Bcl11b HEK293T cells http://www.sigmaaldrich.com

  8. 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.

  9. 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

  10. Experimental Design A

  11. Results A Bcl11b immunoprecipitation followed by Western Blot using indicated antibodies

  12. Experimental Design B

  13. Results B Bcl11b immunoprecipitation followed by Western Blot using indicated antibodies

  14. Experimental Design C

  15. Results C

  16. 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?

  17. 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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