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 RNA polymerase II (RNAP II) subunit plays a key role in gene expression. Stresses such as heat shock result in marked changes in CTD phosphorylation as well as in major alterations in gene expression. CTD kinases and CTD phosphatase(s) contribute in mediating differential CTD phosphory-lation. We now report that heat shock of HeLa cells at temperatures as mild as 41 degreesC results in a decrease in CTD phosphatase activity in cell extracts. The obser-vation that this CTD phosphatase interacts with the RAP74 subunit of the general transcription factor TFIIF suggests that it corresponds to the previously charac-terized major CTD phosphatase. This conclusion is also supported by the finding that the distribution of the 150 kDa subunit of CTD phosphatase 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 decrease in CTD phosphatase activity correlates with a decrease in amount of 150 kDa phosphatase subunit in the extracts. During heat shock, CTD phosphatase switches to an insoluble form which remains aggregated to the nuclear matrix fraction. In contrast, heat shock did not result in a redistribution 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 synthesis of heat-shock mRNAs.
Dubois
M F
MF
Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France.
Marshall
N F
NF
Nguyen
V T
VT
Dahmus
G K
GK
Bonnet
F
F
Dahmus
M E
ME
Bensaude
O
O
D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D018869:Heat-Shock Response; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D019473:Transcription Factors, TFII