REGULATION OF C-MYB EXPRESSION BY PHOSPHORYLATION

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Truncation of c-Myb is associated with its oncogenic activation and differentially truncated forms of Myb result in the transformation of phenotypically distinct hematopoietic cell types. Thus, structural modifications alter the function of c-Myb. During normal differentiation structural modifications could be mediated by phosphorylation or differential RNA splicing. It is clear the c-Myb is a phosphoprotein and we have recently found that pp42mapk phosphorylates c-Myb in vitro at multiple sites. Interestingly, these sites appear to be located in the negative regulatory domain which has been shown to affect c-Myb sequence specific DNA binding, transcription transactivation, transformation and to potentially be involved in protein/protein interactions. The regulation of transcription factor function by phosphorylation is an attractive hypothesis as it would allow for the integration of a number of signalling pathways via protein kinases and phosphatases at the nuclear level. It is the purpose of this proposal to explore the regulation of c-Myb function by phosphorylation. Thus, this proposal has three specific aims. First, we will use two dimensional phosphopeptide mapping to characterize c-Myb phosphorylation in vivo with particular emphasis on determining conditions under which the pp42mapk sites are phosphorylated. Second, we will use four measures of c-myb activity to determine the role of phosphorylation in regulating c-myb function including: 1) sequence specific DNA binding, 2) transcription transactivation, 3) transformation and 4) the ability to block terminal differentiation in murine erythroleukemia cells. Third, we will identify and functionally characterize novel sites of phosphorylation c-Myb. In our first specific aim we expect to identify changes in sites of phosphorylation aside from the pp42mapk phosphorylation sites. This will allow us to move beyond the pp42mapk sites and begin to understand the impact of other kinases on c-Myb function. The broad goal of this proposal is to understand the role played by phosphorylation in regulating c-Myb function.