Phosphorylation State of the RNA Polymerase II C‐Terminal Domain (CTD) in Heat‐Shocked Cells

Abstract

RNA polymerase (RNAP) II is a multisubunit enzyme composed of several different subunits. Phosphorylation of the C-terminal domain (CTD) of the largest subunit is tightly regulated. In quiescent or in exponentially growing cells, both the unphosphorylated (IIa) and the multiphosphorylated (IIo) subunits of RNAP II are found in equivalent amounts as the result of the equilibrated antagonist action of protein kinases and phosphatases. In Drosophila and mammalian cells, heat shock markedly modifies the phosphorylation of the RNAP II CTD. Mild heat shocks result in dephosphorylation of the RNAP II CTD. This dephosphorylation is blocked in the presence of actinomycin D, as the CTD dephosphorylation observed in the presence of protein kinase inhibitors. Thus, heat shock might inactivate CTD kinases which are operative at normal growth temperatures, as some protein kinase inhibitors do. In contrast, severe heat shocks are found to increase the amount of phosphorylated subunit independently of the transcriptional activity of the cells. Mild and severe heat shocks activate protein kinases, which then phosphorylate, in vitro and in vivo, the CTD fused to beta-galactosidase. Most of the heat-shock-activated CTD kinases present in cytosolic lysates co-purify with the activated mitogen-activated protein (MAP) kinases, p42mapk and p44mapk. The weak CTD kinase activation occurring upon mild heat shock might be insufficient to compensate for the heat inactivation of the already existing CTD kinases. However, under severe stress, the MAP kinases are strongly heat activated and might prevail over the phosphatases. A survey of different cells and different heat-shock conditions shows that the RNAP II CTD hyperphosphorylation rates follow the extent of MAP kinase activation. These observations lead to the proposal that the RNAP II CTD might be an in vivo target for the activated p42mapk and p44mapk MAP kinases.