Partial purification and characterization of two distinct protein kinases that differentially phosphorylate the carboxyl-terminal domain of RNA polymerase subunit IIa.
RNA polymerase II is a multisubunit enzyme composed of two large subunits of molecular weight in excess of 100,000 and a collection of 8-10 smaller subunits. The largest subunit, designated IIa, contains at its carboxyl terminus a highly repetitive domain consisting of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Extensive phosphorylation within this COOH-terminal domain (CTD) gives rise to subunit IIo which has a markedly reduced mobility in SDS-polyacrylamide gel electrophoresis (PAGE) relative to subunit IIa. Recent evidence suggests that RNA polymerase IIA, containing an unphosphorylated CTD, is involved in preinitiation complex assembly, whereas RNA polymerase IIO is involved in elongation. Consequently, CTD phosphorylation is thought to occur after RNA polymerase II has bound to the promoter by a protein kinase that stably associates with the preinitiation complex. We present here the partial purification and characterization of two distinct CTD kinases from a HeLa cell transcription extract. These CTD kinases, designated CTDK1 and CTDK2, are fractionated by chromatography on Mono Q. CTDK1 catalyzes the incorporation of approximately 33 pmol of phosphate/pmol of calf thymus RNA polymerase subunit IIa, almost exclusively on serine. CTDK2 catalyzes the incorporation of approximately 50 pmol of phosphate/pmol of calf thymus subunit IIa, predominantly on serine; appreciable phosphate transfer onto threonine is also observed. Phosphorylation by CTDK2, but not CTDK1, results in a complete mobility shift in SDS-PAGE of subunit IIa to the position of IIo. CTDK1 can utilize ATP, dATP, or GTP as phosphate donor, whereas CTDK2 can utilize only ATP or dATP. The apparent Km for ATP is 30 microM for CTDK1 and 60 microM for CTDK2. CTDK1 and CTDK2 also differ in their protein substrate specificity. CTDK1 phosphorylates casein whereas CTDK2 does not. Neither kinase phosphorylates phosvitin or histone H1 to an appreciable extent. CTDK1 and CTDK2 do not appear to be related to cdc2 kinases as determined by their inability to phosphorylate H1 and their failure to react with antibodies directed against the cdc2 kinase. These results establish that a partially fractionated HeLa transcription extract contains two distinct CTD kinases that differ in their nucleotide requirements and in their patterns of CTD phosphorylation.
Payne
J M
JM
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Dahmus
M E
ME
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology
D000596:Amino Acids; D007527:Isoenzymes; D046911:Macromolecular Substances; D009711:Nucleotides; D011494:Protein Kinases; C060137:carboxy-terminal domain kinase; D012319:RNA Polymerase II