RESEARCH PLAN: The first Specific Aim is to purify, microsequence and clone pp100, a common cytosolic target for EPO, IL-3 and GM-CSF receptor activated PTK(s). This will be accomplished by first expressing a transforming form of EPO receptor in FDC-P1 cells to achieve high constitutive expression of pp100. The purification stepsinclude PEG precipitation, affinitycolumn with antiphosphotyrosine antibodies and reverse phase-HPLC. Two main questions which were not addressed in the preliminary studies are: (1) the identity of pp100 and pp97 that was identified by other groups to be induced following the same stimulation; and (2) the identity of pp100 tyrosine phosphorylated protein stimulated by EPO vs. pp100 constitutively expressed following transfection with transforming form of EPO receptor. The Applicant could at least consider the second possibility before starting the large scale purification. The second Specific Aim is to investigate the mechanisms of transcriptional activation by GATA-1, particularly the roles of R1 vs. R2 domains, and of (modulated) phosphorylation at conserved, consensus sites. For comparing the R1 vs. R2 domains of GATA-1, the Applicant proposed to test the ability of various forms of R1 and R2 generated in the baculovirus system to transcribe different GATA- containing promoters in HeLa cell extracts. The role of phosphorylation of GATA-1 will be analyzed by generating another set of site-specific mutants and analyze their ability to bind target DNA, to transcribe GATA-containing promoter and to affect nuclear translocation.It appears that the phosphorylation status of GATA-1 in EPO-mediated signaling was only by correlation and the fact that there are more than ten phosphorylated sites on the molecule will probably make it difficult to interpret the mutagenesis results.It may be more fruitful to concentrate on the deletion mutants that the he will utilize in the in vitro transcription system to narrow down the regions required for DNA binding, transactivation and nuclear localization.
Specific Aim 3 is to understand the possible roles for SCL in erythroid development. This will be accomplished by forced or inhibited expression of SCL in J2E or B6SUt.EPcells and by direct screening SCL binding target sequences using PCR-based binding and enrichment scheme. It is not clear what useful information he intended to obtain by over- or under-expression of SCL in these cell lines. The second approach will probably generate more useful information in terms of the ability of SCL to activate transcription.
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