Phosphorylation of the RNA polymerase II largest subunit during heat shock and inhibition of transcription in hela cells

Abstract

The phosphorylation of the C-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II has been investigated in HeLa cells exposed to heat shock. In control cells, the phosphorylated subunit, IIo, and the dephosphorylated subunit, IIa, were found in similar amounts. During heat shock, however, the phosphorylated subunit, IIo, accumulated, whereas the amount of IIa subunit decreased. Since phosphorylation of the CTD had been suggested to play a role in the initiation of transcription and since heat shock was known to perturb gene expression at the level of transcription, the phosphorylation state of RNA polymerase II was examined in cells that had been treated with various inhibitors of transcription. Under normal growth temperature, actinomycin D (over 0.1 microgram/ml) and okadaic acid, a phosphatase inhibitor, were found to inhibit polymerase dephosphorylation. Whereas 5,6-dichlorobenzimidazole riboside (DRB), N-(2-[Methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), and actinomycin D (over 5 micrograms/ml) were found to inhibit polymerase phosphorylation. Actinomycin D concentrations, which inhibited the dephosphorylation process, were lower than those required to inhibit the phosphorylation process. In contrast, during heat shock or exposure to sodium arsenite, a chemical inducer of the heat-shock response, the phosphorylated subunit, IIo, accumulated even in the presence of inhibitors of transcription such as DRB, H-8, and actinomycin D. These experiments demonstrated the existence of a heat-shock-induced CTD-phosphorylation process that might contribute to the regulation of transcription during stress.

Hyperphosphorylation of RNA Polymerase II in Response to Topoisomerase I Cleavage Complexes and Its Association with Transcription- and BRCA1-dependent Degradation of Topoisomerase I

“…Besides Cdk7, Cdk9 (and Cdk8) can phosphorylate Pol II at residue Ser 5 40 and DRB and FLV are somewhat more selective for Cdk9 than for Cdk7. 28,29 However, downregulation of Cdk9 by siRNA failed to reduce CPTinduced Ser 5 -P (Fig. 3e).…”

“…To determine whether Pol II hyperphosphorylation is related to its normal phosphorylation by Cdks, 1,27 we examined whether CPT-induced Pol II hyperphosphorylation would be prevented by the commonly used Cdk inhibitors, 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) 28 and flavopiridol (FLV). 29 Figure 2a and b shows that both DRB and FLV prevented CPTinduced Pol II hyperphosphorylation.…”

Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains.

“…Exposure of cells to 100 /zM DRB for 3 h results in the appearance of multiple partially hypophosphorylated forms of Pol 1I LS (Dubois et al, 1994). The DRB-induced, hypophosphorylated forms of Pol II LS are nearly completely extracted in TD buffer as shown in the blots probed with mAbs H14 and 8WG16.…”

“…DRB is a CTD kinase inhibitor in vitro and in vivo (Zandomeni et al, 1986;Stevens and Maupin, 1989;Chodosh et al, 1989;Dubois et al, 1994). Exposure of cells to 100 /zM DRB for 3 h results in the appearance of multiple partially hypophosphorylated forms of Pol 1I LS (Dubois et al, 1994).…”

TFIIF-associating Carboxyl-terminal Domain Phosphatase Dephosphorylates Phosphoserines 2 and 5 of RNA Polymerase II

“…Treatment of cells with the transcription inhibitor actinomycin D or ␣-amanitin increases the ratio of RNAP IIO/IIA (46). Serum stimulation and heat shock increase the level of RNAP IIO via the cellular activation of MAPK2/ERK2 (47)(48)(49) and change the pattern of CTD phosphorylation (12)(13)(14). The increase in IIo signals, as indicated by increases in POL3/3 immunoreactivity relative to control extracts, confirms the above-mentioned studies that RNAP IIO levels are elevated in response to environmental stimuli (Fig.…”