Heat shock-induced alterations in phosphorylation of the largest subunit of RNA polymerase II as revealed by monoclonal antibodies CC-3 and MPM-2.
The phosphorylation of the carboxy-terminal domain of the largest subunit of RNA polymerase II plays an important role in the regulation of transcriptional activity and is also implicated in pre-mRNA processing. Different stresses, such as a heat shock, induce a marked alteration in the phosphorylation of this domain. The expression of stress genes by RNA polymerase II, to the detriment of other genes, could be attributable to such modifications of the phosphorylation sites. Using two phosphodependent antibodies recognizing distinct hyperphosphorylated forms of RNA polymerase II largest subunit, we studied the phosphorylation state of the subunit in different species after heat shocks of varying intensities. One of these antibodies, CC-3, preferentially recognizes the carboxy-terminal domain of the largest subunit under normal conditions, but its reactivity is diminished during stress. In contrast, the other antibody used, MPM-2, demonstrated a strong reactivity after a heat shock in most species studied. Therefore, CC-3 and MPM-2 antibodies discriminate between phosphoisomers that may be functionally different. Our results further indicate that the pattern of phosphorylation of RNA polymerase II in most species varies in response to environmental stress.
Lavoie
S B
SB
Centre de recherche sur la fonction, la structure et l'ingénierie des proteins et Département de médecine, Université Laval, Sainte-Foy, QC, Canada.
Albert
A L
AL
Thibodeau
A
A
Vincent
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012319:RNA Polymerase II / Q000187:drug effects / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D019384:Nucleic Acid Synthesis Inhibitors; D011500:Protein Synthesis Inhibitors; D004004:Dichlororibofuranosylbenzimidazole; D003513:Cycloheximide; D011494:Protein Kinases; C060137:carboxy-terminal domain kinase; D012319:RNA Polymerase II; D010749:Phosphoprotein Phosphatases; C116022:carboxy-terminal domain phosphatase