EKLF is a erythroid cell-specific transcription factor which is required for the production of Beta-globin. Regulation of EKLF by phosphorylation will be explored by: 1. Mapping its phosphorylation sites by the combination of HPLC, mass spectrometry and chemical sequencing. 2. Testing the effects of phosphorylation on its molecular properties by monitoring the effects of phosphorylation mutants on either DNA binding affinity or transcriptional activity. 3. Testing the effects of phosphorylation on its biological function by comparing the abilities of wild type or phosphorylation mutant EKLFs to rescue Beta-globin transcription in embryoid bodies that have been differentiated in vitro from EKLF -/- embryonic stem cells. These studies will provide a basis for us to identify protein kinnases which can phosphorylate EKLF and will contribute to the understanding of the mechanisms of both normal erythroid cell gene regulation during development and potential mechanisms for its disruption in leukemia.
