Characterization of the SATB1 phosphorylations upon ionization radiation
EMSL Project ID
16695
Abstract
A major goal of my laboratory is to understand the molecular signaling mechanisms involving nuclear architectural protein, SATB1. SATB1 is known to be a global gene regulator by organizing higher order chromatin structure in restricted cell types. SATB1 is essential for proper T cell development. SATB1 exhibits a cage-like nuclear distribution in thymocyte nuclei onto which genomic sequences are anchored and assembled with chromatin remodeling complexes and transcription factors. SATB1 appears to be a phosphorylated proteins and there are distinct populations of SATB1 within thymocytes exhibiting different affinity to its target DNA sequences. SATB1 knockout mice show DNA repair deficiency. We wish to characterize the post-translational modification of SATB1 in thymocytes before and after cells are exposed to ionizing radiation. We developed a biochemical method using DNA affinity column and we can purify SATB1 from thymocytes*(Ref.). This system will allow us to perform large-scale proteomics to characterize post-translational modification of SATB1. We will use 1E9cells for these studies to purify SATB1. The hallmark cellular response to ionization radiation is phosphorylation of proteins involved in signaling transduction pathway. Therefore, our objective is to characterize the phosphorylation sites responsible for ionization radiation in SATB1 protein. SATB1 from thymocytes will be immunoaffinity purified with DNA affinity column and enriched for phosphopeptides using an IMAC column and then analyzed using the NCRR high sensitivity, high resolution LC-MS/MS to identify phosphorylaton sites before and after ionization irradiation. Functional testing of key phosphorylation sites will then be performed by site-directed mutagenesis followed by transfection into wild-type and SATB1 knockout thymocytes in our laboratory. Results from our study will provide valuable molecular basis of the important role of SATB1 for cell development and gene regulation upon DNA damage, and help evaluate the biological effects of ionization radiation.
Project Details
Start Date
2006-02-02
End Date
2009-02-08
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members