Selective repression of RNA polymerase II by microinjected phosvitin.
We have used a microinjection technique to examine whether injected phosvitin, in its capacity as substrate for casein kinase NII, could compete out the endogenous phosphorylation of some nuclear phosphoproteins with regulatory potential and thereby interfere with the activity of RNA polymerase II. Phosphorylation, which utilizes ATP as phosphate donor, was separated from phosphorylation which uses GTP. Phosvitin introduced into nuclei of salivary gland cells becomes phosphorylated by the endogenous nuclear protein kinase(s) and incorporates phosphates from ATP as well as from GTP. The phosphorylation of nuclear proteins and phosvitin is heparin-sensitive, indicating that they are phosphorylated by casein kinase NII. Microinjected phosvitin does not seem to affect the incorporation of phosphate groups from ATP into nuclear proteins, but protein phosphorylation by GTP is influenced. Apart from a minor overall reduction of 32P-incorporation, the phosphorylation of a 42 kDa nuclear protein, a putative transcription stimulatory factor, and of a 115 kDa nuclear protein was competed out by 70%-80% compared with the control value obtained in the absence of phosvitin. Parallel analyses of DNA transcription in phosvitin-injected nuclei showed that the RNA polymerase II-mediated synthesis of hnRNA and Balbiani ring RNA was diminished by 80% and 90%, respectively. In contrast, the transcription of nucleolar pre-ribosomal 38 S RNA by RNA polymerase I remained unaffected. The inhibitory effect of injected phosvitin could be reversed by in vitro phosphorylation of phosvitin prior to injection, using isolated nuclei as source of protein kinase(s). Taken together, the results suggest a causal relationship between the modification of the GTP-dependent phosphorylation of specific non-histone proteins and the activity of RNA polymerase II.
Egyházi
E
E
Pigon
A
A
D000818:Animals; D047388:Casein Kinases; D002364:Caseins / Q000378:metabolism; D002683:Chironomidae / Q000235:genetics / Q000378:metabolism; D004527:Egg Proteins / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008845:Microinjections; D010750:Phosphoproteins / Q000096:biosynthesis; D010766:Phosphorylation; D010774:Phosvitin / Q000378:metabolism / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors*; D012332:RNA, Heterogeneous Nuclear / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects*
D002364:Caseins; D004527:Egg Proteins; D010750:Phosphoproteins; D012332:RNA, Heterogeneous Nuclear; D006160:Guanosine Triphosphate; D010774:Phosvitin; D011494:Protein Kinases; C026813:protein kinase NII; D047388:Casein Kinases; D012319:RNA Polymerase II