Investigation of the cell cycle regulation of ets-1 and ets-related proteins remains a major focus of the project. A hyperphosphorylated isoform of ets-1 was discovered in mitotic cells after cell synchronization by centrifugal elutriation or nocodazole block. Structural and functional characterization of this novel isoform is ongoing. Two potential sites of phosphorylation, both within the exon 7- encoded domain of the protein, are being studied using site-directed mutagenesis and in vitro phosphorylation by Ca++/calmodulin kinase. The contribution of these sites to the mitotic and interphase phosphorylations of ets-1, which can be distinguished by differential mobility in SDS-PAGE, is being assessed. The effect of phosphatase activity on differential ets-1 phosphorylation is also being studied, since it was found that exposure of unsynchronized cells to the phosphatase inhibitor, okadaic acid, results in a shift of ets-1 to the hyperphosphory-lated state. The hyperphosphorylation does not appear to affect the affinity of sequence- specific binding by ets-1 to its target sequence, but the mobility of the protein/DNA complex was altered. The potential effect of this change on transcription regulation and other interactions of ets-1 will be explored. Studies on the role of ets proteins in astrocytic cells are also being continued. Differentiation of P19 mouse embryonal carcinoma cells in response to retinoic acid and other treatments, is utilized as a model for astrocyte differentiation. Expression of ets-1, ets-related proteins and ets-specific DNA-binding activity are being characterized in these cells in order to assess their correlation with the astrocyte lineage.
