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Cdk7 Is Essential for Mitosis and for in Vivo Cdk-Activating Kinase Activity

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Cdk7 Is Essential for Mitosis and for in Vivo Cdk-Activating Kinase Activity Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Cdk7 is essential for mitosis and for in vivo Cdk-activating kinase activity Ste´phane Larochelle,1 Judit Pandur,1 Robert P. Fisher,2 Helen K. Salz,3 and Beat Suter1,4 1Department of Biology, McGill University, Montreal, PQ, Canada H3A 1B1; 2Program in Cell Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 USA; 3Department of Genetics, Case Western Reserve University, Cleveland, Ohio 44106-4955 USA Cdk7 has been shown previously to be able to phosphorylate and activate many different Cdks in vitro. However, conclusive evidence that Cdk7 acts as a Cdk-activating kinase (CAK) in vivo has remained elusive. Adding to the controversy is the fact that in the budding yeast Saccharomyces cerevisiae, CAK activity is provided by the CAK1/Civ1 protein, which is unrelated to Cdk7. Furthermore Kin28, the budding yeast Cdk7 homolog, functions not as a CAK but as the catalytic subunit of TFIIH. Vertebrate Cdk7 is also known to be part of TFIIH. Therefore, in the absence of better genetic evidence, it was proposed that the CAK activity of Cdk7 may be an in vitro artifact. In an attempt to resolve this issue, we cloned the Drosophila cdk7 homolog and created null and temperature-sensitive mutations. Here we demonstrate that cdk7 is necessary for CAK activity in vivo in a multicellular organism. We show that cdk7 activity is required for the activation of both Cdc2/Cyclin A and Cdc2/Cyclin B complexes, and for cell division. These results suggest that there may be a fundamental difference in the way metazoans and budding yeast effect a key modification of Cdks. [Key Words: Drosophila; Cdk; CAK; mitosis; cell cycle] Received November 7, 1997; revised version accepted December 2, 1997. The orderly succession of DNA synthesis and cell divi- 1993; Solomon et al. 1993; Fisher and Morgan 1994; sion is known to be largely regulated by the successive Ma¨kela¨ et al. 1994). A third subunit, MAT1, has also activity of different cyclin-dependent kinases (Cdks) been found to associate with Cdk7 and cyclin H and to (Nigg 1995). The activity of Cdks is regulated by their serve as an assembly factor (Devault et al. 1995; Fisher et association with positive and negative regulatory sub- al. 1995; Tassan et al. 1995a). However, unlike most units, and by multiple phosphorylation events (Morgan other Cdks, Cdk7 was found to be active throughout the 1995). Complete activation of Cdks requires the phos- cell cycle with no detectable oscillation in its activity phorylation of a conserved threonine residue located (Brown et al. 1994; Matsuoka et al. 1994; Poon et al. within the T-loop, a substructure common to all Cdks 1994; Tassan et al. 1994). These results suggest that the and many other protein kinases. In monomeric inactive CAK activity of Cdk7 could be sufficient to provide the Cdk molecules, the T-loop blocks the catalytic site and activating Thr-161 (or equivalent) phosphorylation to all hinders substrate binding (De Bondt et al. 1993). X-ray Cdks throughout the cell cycle (Fesquet et al. 1993; Poon structural analysis of the Cdk2/Cyclin A complex sug- et al. 1993; Solomon et al. 1993; Fisher and Morgan 1994, gests that the T-loop is displaced by cyclin binding, 1996; Matsuoka et al. 1994). In addition to its putative thereby opening up the active site for substrate binding. role in cell cycle regulation, Cdk7 is also able to phos- Phosphorylation of the T-loop threonine then allows full phorylate the carboxy-terminal domain (CTD) of RNA activation of the complex (Jeffrey et al. 1995; Russo et al. polymerase II (Pol II) as part of the TFIIH basic transcrip- 1996). Because this threonine phosphorylation of the dif- tion factor complex (Roy et al. 1994; Serizawa et al. 1995; ferent Cdks is a crucial step in their activation (Morgan Shiekhattar et al. 1995). 1995), much effort has been directed toward identifying What appears to be a functional as well as a sequence and characterizing the kinases responsible for this event. homolog to Cdk7 has been found in the fission yeast An enzyme complex has been identified that is able to Schizosaccharomyces pombe (Buck et al. 1995; Damag- phosphorylate a number of different Cdks on their acti- nez et al. 1995). The S. pombe Mop1/Crk1 gene is es- vating threonine residue in vitro and is known as Cdk- sential and its product behaves biochemically as a CAK. Activating Kinase (CAK). CAK itself is a Cdk/Cyclin However, mutations in the S. pombe Mop1/Crk1 do not complex: Cdk7/Cyclin H (Fesquet et al. 1993; Poon et al. lead to a uniform cell cycle arrest, presumably because its activity is also required for TFIIH to regulate the tran- scriptional activity of RNA Pol II (Buck et al. 1995; Da- 4Corresponding author. magnez et al. 1995). In Saccharomyces cerevisiae, the E-MAIL BEAT [email protected]; FAX (514) 398-8051. gene product with the highest sequence similarity to 370 GENES & DEVELOPMENT 12:370–381 © 1998 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/98 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Cdk7 requirement for Cdc2 activation in vivo Cdk7 is Kin28. Although Kin28 was shown to be part of of Cdc2 Thr-161 phosphorylation was shown to oscillate the TFIIH transcription factor (Feaver et al. 1994) and to during the late preblastoderm embryonic cycles (Edgar et be required for the phosphorylation of the CTD of RNA al. 1994). This indicates that the target site for CAK is Pol II, it is not involved in the phosphorylation of Cdc28, regulated at least during some cell cycles. the budding yeast Cdc2 homolog (Cismowski et al. Here we report the identification of the Drosophila 1995). The protein responsible for CAK activity in S. cdk7 gene. By creating null and temperature-sensitive cerevisiae was identified as CAK1/Civ1 (Espinoza et al. mutations of Dmcdk7 we were able to analyze the in 1996; Kaldis et al. 1996; Thuret et al. 1996). Surprisingly, vivo molecular and cellular requirements for Cdk7. Al- CAK1/Civ1 shares only limited sequence similarity though our analysis does not reveal a Cdk7 requirement with Cdk7 and other Cdks. The identification of this in for Cdk2/Cyclin E activity, it demonstrates that Cdk7 is vivo CAK in budding yeast and the demonstration that it required for mitosis and for the activation of Cdc2 in is not closely related to the vertebrate Cdk7 led to the vivo. postulation that Cdk7/Cyclin H may in fact not repre- sent a physiologically relevant CAK activity (Cimowski Results et al. 1995; Espinoza et al. 1996; Kaldis et al. 1996; Isolation of the Dmcdk7 gene Thuret et al. 1996). Besides the two yeast, Drosophila has become a sys- We isolated a Drosophila melanogaster sequence ho- tem of choice for an in vivo analysis of the cell cycle mologous to the vertebrate cdk7 genes using a degener- (Edgar and Lehner 1996; Follette and O’Farrell 1997). ate PCR-based approach. This Drosophila cdk7 gene One of its major values is that it allows the genetic codes for a predicted polypeptide of 353 amino acids with analysis of cell cycle events in a multicellular organism. a calculated molecular mass of 39 kD. Drosophila and Like vertebrates, but contrary to the unicellular yeast, human Cdk7 proteins share 65% identity over the entire Drosophila cells use distinct Cdks at the different cell polypeptide (Fig. 1A), a sequence similarity higher than cycle transitions. Interestingly, although the activity of to any other Cdk. A single 1.6-kb Dmcdk7 poly(A+) RNA Cdk7 has been shown in different systems to be constant species is present throughout development and accumu- throughout the cell cycle (Brown et al. 1994; Matsuoka lates most strongly in ovaries and young embryos where et al. 1994; Poon et al. 1994; Tassan et al. 1994), the level it is probably maternally deposited (Fig. 1B). Figure 1. Identification and characterization of the Drosophila cdk7 gene. (A) Comparison of Drosophila and vertebrate Cdk7 proteins. (B) poly(A+) RNA blot showing the develop- mental profile of accumulation of the Dm- cdk7 message. Embryonic stages (E) are in hours. (L1) First instar larvae; (L2) second in- star larvae; (eL3) early third instar larvae; (lL3) late third instar larvae; (eP) early pupae; (lP) late pupae. A single 1.6-kb transcript accumu- lates predominantly in samples containing the female germ line and in the early embryos where it is contributed maternally. (Bottom) An autoradiograph of the same filter after hy- bridization with the small ribosomal subunit protein gene DL11. GENES & DEVELOPMENT 371 Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Larochelle et al. Dmcdk7 is an essential gene The Dmcdk7 gene is located in cytological interval 4F and is separated by ∼0.4 and 3 kb from its proximal neighbors sans fille (snf) and deadhead (dhd), respec- tively (Fig. 3). snf and Dmcdk7 are oriented head to head. To create a Dmcdk7 null mutation, we took advantage of a P-element insertion at the dhd locus (dhdP8). Impre- cise excision of this P-element produced a number of lethal mutations (Flickinger and Salz 1994). From this screen we identified a 4.4-kb deletion that removes the entire Dmcdk7 and snf coding regions, as well as part of Figure 2. (A) Cdk7 immunoblot on total 0- to 4-hr embryonic the dhd gene (Fig. 3). This new deficiency is designated lysate. (B) DmCdk7 protein phosphorylates and activates Cdk2/ Df(1)JB254.
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