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 ϳ10-fold by csk1 deletion, and Cdk9 complexes from csk1⌬ but not csk1 ؉ cells can be activated by Csk1 in vitro. A cdk9 T212A mutant is viable but phenocopies conditional growth defects of csk1⌬ 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 yeasthelps to target the capping apparatus to the transcriptional elongation complex.

RNA polymerase II CTD phospho-sites Ser5 and Ser7 govern phosphate homeostasis in fission yeast

“…The cyclin-dependent kinase Cdk9 is a physiological target for activation by Csk1 (Pei et al 2006). Cdk9, in a complex with its cyclin partner Pch1, catalyzes serine phosphorylation of the Pol2 CTD .…”

“…Csk1 stimulates the CTD kinase activity of Cdk9•Pch1 by phosphorylating Cdk9 on residue Thr212 of the activating "Tloop" segment. The activated Cdk9•Pch1 enzyme phosphorylates the Pol2 CTD at positions Ser2 and Ser5 of the CTD heptad (Pei et al 2006). The Cdk9-T212A mutant is refractory to activation by Csk1.…”

“…The protein kinase Csk1 is a negative regulator of the phosphate response, as judged by the fact that pho1 expression is constitutively turned on in csk1Δ cells under phosphate-replete conditions (Henry et al 2011;Carter-O'Connell et al 2012;Schwer et al 2014). Csk1 is a CDK-activating kinase with several physiological targets, including the kinases Cdc2, Mcs6, and Cdk9 (Saiz and Fisher 2002;Pei et al 2006). Mcs6 and Cdk9 figure prominently in fission yeast transcription via their phosphorylation of the carboxy-terminal domain (CTD) of the Rpb1 subunit of RNA polymerase II (Pol2) and/or the carboxy-terminal domain (CTD) of the transcription elongation factor Spt5 (Pei et al 2006;Viladevall et al 2009).…”

“…Csk1 is a CDK-activating kinase with several physiological targets, including the kinases Cdc2, Mcs6, and Cdk9 (Saiz and Fisher 2002;Pei et al 2006). Mcs6 and Cdk9 figure prominently in fission yeast transcription via their phosphorylation of the carboxy-terminal domain (CTD) of the Rpb1 subunit of RNA polymerase II (Pol2) and/or the carboxy-terminal domain (CTD) of the transcription elongation factor Spt5 (Pei et al 2006;Viladevall et al 2009). …”

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

“…The Spt5/Spt4-induced elongation arrest in metazoans is alleviated by the Cdk9 protein kinase, a subunit of positive transcription elongation factor b (P-TEFb) (Wada et al 1998b). In the fission yeast Schizosaccharomyces pombe, the essential Cdk9 kinase exists as a stoichiometric complex in vivo with its cyclin partner (Pch1) and the methyltransferase component of the S. pombe capping apparatus Pei et al 2006;Guiguen et al 2007). S. pombe Cdk9 also interacts with the RNA triphosphatase component of the capping apparatus .…”