Cyclin-dependent kinase 9 (Cdk9) of fission yeast is activated by the CDK-activating kinase Csk1, overlaps functionally with the TFIIH-associated kinase Mcs6, and associates with the mRNA cap methyltransferase Pcm1 in vivo.
Cyclin-dependent kinase 9 (Cdk9) of fission yeast is an essential ortholog of metazoan positive transcription elongation factor b (P-TEFb), which is proposed to coordinate capping and elongation of RNA polymerase II (Pol II) transcripts. Here we show that Cdk9 is activated to phosphorylate Pol II and the elongation factor Spt5 by Csk1, one of two fission yeast CDK-activating kinases (CAKs). Activation depends on Cdk9 T-loop residue Thr-212. The other CAK-Mcs6, the kinase component of transcription factor IIH (TFIIH)-cannot activate Cdk9. Consistent with the specificities of the two CAKs in vitro, the kinase activity of Cdk9 is reduced approximately 10-fold by csk1 deletion, and Cdk9 complexes from csk1Delta but not csk1+ cells can be activated by Csk1 in vitro. A cdk9(T212A) mutant is viable but phenocopies conditional growth defects of csk1Delta strains, indicating a role for Csk1-dependent activation of Cdk9 in vivo. A cdk9(T212A) mcs6(S165A) strain, in which neither Cdk9 nor Mcs6 can be activated by CAK, has a synthetic growth defect, implying functional overlap between the two CDKs, which have distinct but overlapping substrate specificities. Cdk9 forms complexes in vivo with the essential cyclin Pch1 and with Pcm1, the mRNA cap methyltransferase. The carboxyl-terminal region of Cdk9, through which it interacts with another capping enzyme, the RNA triphosphatase Pct1, is essential. Together, the data support a proposed model whereby Cdk9/Pch1-the third essential CDK-cyclin complex described in fission yeast-helps to target the capping apparatus to the transcriptional elongation complex.
Pei
Yi
Y
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY. r-fisher@ski.mskcc.org.
Du
Hongyan
H
Singer
Juliet
J
Stamour
Courtney
C
Granitto
Selena
S
Shuman
Stewart
S
Fisher
Robert P
RP
D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000235:genetics / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D004789:Enzyme Activation; D017353:Gene Deletion; D008780:Methyltransferases / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D035602:Transcriptional Elongation Factors / Q000378:metabolism; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D002868:Chromosomal Proteins, Non-Histone; D016213:Cyclins; C105853:Pch1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D035602:Transcriptional Elongation Factors; C069940:SPT5 transcriptional elongation factor; D013912:Threonine; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; D011494:Protein Kinases; C117097:Csk1 protein, S pombe; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase