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†

Autor: Yi Pei, Selena R. Granitto, Hongyan Du, Juliet Singer, Stewart Shuman, Robert P. Fisher, Courtney V. St. Amour
Jazyk: angličtina
Rok vydání: 2006
Předmět:
Popis: Cyclin-dependent kinases (CDKs) first emerged as controllers of cell division but have also been implicated in processes not strictly coupled to the cell cycle, most notably transcription by RNA polymerase II (Pol II) (4). In metazoans, Cdk9 and cyclin T constitute positive transcription elongation factor b (P-TEFb), which phosphorylates both the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of Pol II, and Spt5, a subunit of the elongation factor DRB sensitivity-inducing factor, to overcome kinetic blocks to elongation (21). The requirement for Cdk9 in facilitating elongation is probably a general one (66) and is posited to be a quality control mechanism to ensure that nascent transcripts are not elongated unless and until mRNA-capping enzymes and other processing machinery can be recruited (9, 10, 49, 52). In the budding yeast Saccharomyces cerevisiae, the Bur1/Bur2 CDK-cyclin pair and a heterotrimeric CDK, CTDK-I, show roughly equal homology between their catalytic subunits (Bur1 and Ctk1, respectively) and metazoan Cdk9 (46). The BUR1 gene is essential (56), whereas neither BUR2 nor any of the genes encoding CTDK-I subunits is required for viability (68, 77). Cdk9 of the fission yeast Schizosaccharomyces pombe was identified in a two-hybrid interaction screen with Pct1, the RNA triphosphatase component of the mRNA-capping apparatus (52). Cdk9 can form an active kinase complex with the essential fission yeast cyclin Pch1, and the two proteins expressed together in S. cerevisiae complemented a bur1 deletion (19, 52), but the physiologic cyclin partner of Cdk9 in S. pombe remained to be identified. CDKs depend to various degrees on phosphorylation within the activation segment, or T loop, of the catalytic subunit by a CDK-activating kinase (CAK). The dedicated cell cycle CDKs, exemplified by Cdk1, absolutely require T-loop phosphorylation (22, 37), whereas CDKs involved in transcription need this modification for full catalytic activity and/or for stability but not for their essential functions (29, 31, 78). The CAKs fall into two classes. In metazoans, the major CAK is a heterotrimeric complex of Cdk7, cyclin H, and the RING finger protein Mat1, which is also part of general transcription factor IIH (TFIIH), which phosphorylates the Pol II CTD (23). In contrast, the sole CAK in budding yeast is Cak1, a monomeric enzyme related only distantly to the CDK family (27). Cak1 activates both Cdk1 and the Cdk7 ortholog Kin28 (17, 29), which unlike its metazoan counterpart is a dedicated TFIIH-associated CTD kinase that does not activate CDKs (12, 73). S. pombe has one CAK from each class: the Mcs6/Mcs2/Pmh1 complex, which is homologous to Cdk7/cyclin H/Mat1, and Csk1, a single-subunit enzyme most closely related to Cak1 (2, 5, 14, 24, 33, 34, 67). Both enzymes can activate Cdk1 (34, 63), a redundancy that probably explains why csk1+ is dispensable for viability (43) and why mutations in genes encoding Mcs6 complex subunits do not impair CDK activation or impede entry into mitosis unless combined with other mutations, such as csk1Δ (5, 14, 24, 33, 34, 43, 63). The Mcs6 complex is required for viability, however, suggesting it has another essential target, which is likely to be Pol II. The growth defects caused by csk1 deletion (3, 25, 63) might reflect specialized requirements for Csk1-mediated activation of Cdk1 and/or activity of Csk1 towards proteins that the Mcs6 complex does not phosphorylate. Activation of metazoan P-TEFb by a CAK has not been demonstrated, but mutating Thr-186 in the human Cdk9 T loop abolished activity and, paradoxically, binding to a ribonucleoprotein inhibitor (7). In budding yeast, a temperature-sensitive bur1 mutation was suppressed by overexpression of CAK1, and phosphorylation by Cak1 stimulated the kinase activity of Bur1 in vitro, dependent on the Thr-240 residue of the Bur1 T loop. That stimulation is apparently important in vivo; a bur1T240A allele only partially complemented bur1Δ (78). More recently, Cak1 was shown to activate Ctk1, and a mutation in ctk1 preventing T-loop phosphorylation caused a defect in the entry into stationary phase (50). Changing Thr-212 within the T loop of fission yeast Cdk9 to alanine abolished heterologous complementation of bur1Δ, whereas a mutation of the same residue to glutamic acid rendered it cold and temperature sensitive (52, 53). The regulation of Cdk9 by upstream kinases (CAKs) in fission yeast has not been investigated. Moreover, its role(s) in regulating gene expression and possibly coordinating mRNA-processing events with transcription remains to be elucidated. Here we show specificity within the CAK-CDK network of S. pombe; Csk1, but not the Mcs6 complex, activates Cdk9/Pch1 complexes in vitro and in vivo. The nonphosphorylatable T-loop mutant cdk9T212A grows poorly on minimal media and is cold sensitive, essentially phenocopying csk1Δ. We observe a synthetic interaction between cdk9T212A and the analogous mcs6S165A T-loop mutation, suggesting that the essential Cdk9 and Mcs6 complexes, which have partially overlapping substrate specificities in vitro, have redundant as well as unique functions in controlling gene expression in vivo. Finally, we provide support for the idea that Cdk9 couples transcription to mRNA capping (9, 10, 49, 52) by demonstrating (i) that the carboxyl-terminal, Pct1-interacting region of Cdk9 (52) is required for viability and (ii) that Cdk9 stably associates in vivo with the guanine-N7 methyltransferase component of the fission yeast mRNA-capping apparatus in ∼500-kDa complexes that are released from larger complexes by RNase digestion.
Databáze: OpenAIRE
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