Three RNA polymerase II carboxyl-terminal domain kinases display distinct substrate preferences.
CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, we tested the hypothesis that the kinases differ in their ability to phosphorylate CTD heptad arrays. Our data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO(4). This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs.
Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
Y
Y
Ramanathan
S M
SM
Rajpara
S M
SM
Reza
E
E
Lees
S
S
Shuman
M B
MB
Mathews
T
T
Pe'ery
D000595:Amino Acid Sequence; D056850:Cyclin-Dependent Kinase 8; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D017346:Protein Serine-Threonine Kinases; C095686:CDK8 protein, human; C467969:CDK9 protein, human; D056850:Cyclin-Dependent Kinase 8; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human