Heat shock of HeLa cells inactivates a nuclear protein phosphatase specific for dephosphorylation of the C-terminal domain of RNA polymerase II

Reversible phosphorylation of the C-terminal domain (CTD) of the largest RN A polymerase II (RNAP II) subunit plays a key role in gene expression. Stre sses such as heat shock result in marked changes in CTD phosphorylation as well as in major alterations in gene expression. CTD kinases and CTD phosph atase(s) contribute in mediating differential CTD phosphorylation, We now r eport that heat shock of HeLa cells at temperatures as mild as 41 degrees C results in a decrease in CTD phosphatase activity in cell extracts. The ob servation that this CTD phosphatase interacts with the RAP74 subunit of the general transcription factor TFIIF suggests that it corresponds to the pre viously characterized major CTD phosphatase, This conclusion is also suppor ted by the finding that the distribution of the 150 kDa subunit of CTD phos phatase in cells is altered by heat shock Although CTD phosphatase is found predominantly in low salt extracts in unstressed cells, immunofluorescence microscopy indicates that its intracellular localization is nuclear, The d ecrease in CTD phosphatase activity correlates with a decrease in amount of 150 kDa phosphatase subunit in the extracts. During heat shock, CTD phosph atase switches to an insoluble form which remains aggregated to the nuclear matrix fraction. In contrast, heat shock did not result in a redistributio n of RAP74, indicating that not all nuclear proteins aggregate under these conditions. Accordingly, the heat-inactivation of both the CTD phosphatase and the TFIIH-associated CTD kinase might contribute to the selective synth esis of heat-shock mRNAs.