Expression of the ETS proteins during the cell cycle has been examined in cells synchronized by centrifugal elutriation or mitotic block using the microtubule-destabilizing agent, nocodazole. Both methods reveal the presence of a hyperphosphorylated isoform of ETSI during the early mitotic phase of the cell cycle. This isoform exhibits a strong mobility shift in SDS-PAGE and has been observed during mitosis in each of four cell lines (three human T-cell lines and one human astrocytoma line) which express c-ETSI. This isoform is distinguishable from previously described phosphorylated isoforms of ETS1 and appears to arise due to multiple phosphorylations on serine in the domain of the protein encoded by exon 7 of the ETSI gene. Treatment of unsynchronized cells with the phosphatase inhibitor, okadaic acid, results in a stoichiometric shift of ETS1 to this hyperphosphorylated state, suggesting that cellular phosphatase activity is important for regulation of ETSI. The potential correlation of the different ETS1 phosphorylation states with functional activity of the protein in sequence-specific DNA binding is currently under investigation. Carbachol-induced stimulation of ETS1 phosphorylation in 132INI astrocytoma cells is also being studied; the relation to intracellular Ca++ levels and enzymes involved during this stimulation is being compared to the mitotic ETSI phosphorylation.
