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

Abstract

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 ∼10-fold by csk1 deletion, and Cdk9 complexes from csk1Δ but not csk1⁺ cells can be activated by Csk1 in vitro. A cdk9 ^T212A mutant is viable but phenocopies conditional growth defects of csk1Δ 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.

Supplemental material for this article may be found at http://mcb.asm/org/.

We are grateful to Matthew Gamble, Hilary Gerber, and Stéphane Larochelle for critical review of the manuscript; to Karen Lee and Julia Saiz for providing strains, reagents, and advice; and to Hediye Erdjument-Bromage and Paul Tempst for mass spectrometric identification of proteins. S.S. is an American Cancer Society Research Professor.

This work was supported by NIH grant GM52470 to S.S. and by American Cancer Society grant RSG-99-043-044-CCG and NIH grant GM56985 to R.P.F.