O-Glycosylation of Transcription Factor BCL11b: Truth or Myth?
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1 O-Glycosylation of Transcription Factor BCL11b: Truth or Myth? Elizabeth Pendergrass Dr. Theresa Filtz Dept Of Pharmaceutical Sciences College Of Pharmacy Oregon State University
2 T-cell Leukemia T-cell Leukemia: Cancer of the white blood cells, or T-cells. Affects approximately 50,000 people per year About 4,000 people die from it annually Recovery Rate: ~90% in children Relapse Rate: ~50% in adults Looking for potential drug targets Leukemia - Chronic T-Cell Lymphocytic - Statistics. Cancer.Net. N.p., n.d. Web. 20 Aug
3 BCL11b: a haploinsufficient tumor suppressor Loss or mutation of BCL11b results in T-cell leukemia BCL11b mutation is associated with ~15% of T-cell leukemia cases
4 TCR-dependent TCR-independent Periphery BM Thymus BCL11b: Essential for Thymocyte Development B m DC NK Progenitor DN1 Specification DN2 Commitment DN3 -selection DN4 Bcl11b is upregulated at DN2 stage Bcl11b needed for proliferation of immature myeloid T cell progenitors NKT Treg ISP DP Positive selection CD4 CD8 Bcl11b needed for progression into CD4 or CD8 SP cells Bcl11b known to be modified by phosphorylation and sumoylation CD4 CD8 itreg Th1 Th2 Th17 CTL Scheme c/o Ellen Rothenberg, Cal Tech
5 Genetic Regulation Transcription Factors Control gene expression by activating or inhibiting transcription of DNA sequences Regulated by localization, synthesis/degradation, and post-translational modifications.
6 Post Translational Modification PTM Major Targeted Amino Acids Phosphorylation Ser, Thr, Tyr Yes Sumoylation Lys Yes O-Glycosylation Ser, Thr? Modifies BCL11b
7 Methods for Detecting O-Glycosylation Antibodies targeted to O-glycosylation residues Beta-N-Acetylhexosaminidase to remove glycosylated residues Wheat germ agglutinin to precipitate glycosylated proteins Mass Spectrometry to characterize location on protein
8 PUGNAc Inhibits O-GlcNAc-β-N-acetylglucosaminidase (the enzyme for deglycosylation) in the cells Hypothesis: If BCL11b is O-glycosylated, PUGNAc treatment will cause a significant increase in O-glycosylation signal.
9 HEK293T Cells Human Embryonic Kidney Cells Transformed Can easily be transfected to express BCL11b Can also easily be treated with PUGNAc
10 Experimental Methods Cell Culture Immunoprecipitation Western Blot
11 Ratio of O-Gly:Total Protein Changes in O-Glycosylation caused by PugNAc In BCL11b-Transfected HEK cells HEK Cells Treated with PUGNAc 130 kda IB: BCL11b IB:O-Glycosylation Overlay min 15 min 30 min 45 min Treatment Duration
12 Beta-N-acetylhexosaminidase An enzyme that removes an O-linked oligosaccharide from a glycoprotein Designing a negative control for the O-glycosylation antibodies
13 Ratio of Glycosylation to Total BCL11b Beta-N-acetylhexosaminidase 1 HEK 203T cells Treated with Beta-N-acetylhexosaminidase over time IB: BCL11b IB: O-Glycosylation Overlay Beta0 Beta5- Beta5+ Beta10- Beta10+ Beta30- Beta30+ Treatment Type (min)
14 Mouse Thymocytes Why are they a better model? Natively express BCL11b Phosphorylation and sumoylation change dynamically in thymocytes upon stimulation Is BCL11b glycosylated? Does it also change?
15 P/A (Phorbal Ester and A23187) Mimics T-cell receptor activated signaling and phosphorylation cascades MAPK pathway stimulated by Phorbol ester Ca2+ pathway stimulated by A23187 Image from Walter Vogel; Data from JBC vol 287:26971
16 Ratio of Glycosylation to Total BCL11b Changes in Glycosylation (P/A) On BCL11b in Thymocytes 0.14 Thymocytes Stimulated with P/A IB:BCL11b IB:O-Glycosylation Overlay 0 no drug 7 min 30 min 60 min 3 hr Treatment Type and Duration -Non-specific binding of the antibody
17 Wheat Germ Agglutinin (WGA) Binds to N-acetyl-D-glucosamine Procedure is identical to the immunoprecipitation described previously Pulls down all glycosylated proteins Gel is then scanned for specific proteins AKT1 is used as a positive control, as it is known to be glycosylated in mouse thymocytes
18 Wheat Germ Agglutinin Blot 130 kda Red = IB:BCL11b Green = IB:O-Gly 72 kda Result: WGA did not bind BCL11b AKT1 was present in the WGA lane Conclusion: BCL11b is not directly glycosylated Green = IB:AKT1 Overlay*
19 Thymocytes Scanned for Phosphorylation 130 kda 30 min Treatments Result: Phosphorylation levels of BCL11b decreased when PUGNAc was added Conclusion: Glycosylation levels affect the activity of BCL11b, but not directly.
20 Ratio of Phosphorylation to Total BCL11b Changes in Phosphorylation caused by PUGNAc In BCL11b-Transfected HEK293T cells IB: BCL11b Signal 0.5 Phosphorylation of HEK cells Treated with PUGNAc 0.45 Phosphorylation Signal Overlay min 20 min 45 min Series
21 Determining the effects of glycosylation on other proteins in the NuRD complex and along the MAPK and Ca2+ dependent pathways. Next Up:
22 Funding Sources College of Pharmacy URISC In-Lab Assistance Dr. Theresa Filtz Wisam Selman Heather Wisner Xuemei Wan Dr. Chrissa Kioussi Arun Singh Vera Chang Elahe Esfandiari Special Thanks
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