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

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 transcriptional 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

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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 represent 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 developmental profile of accumulation of the Dm- cdk7 message. Embryonic stages (E) are in hours. (L1) First instar larvae; (L2) second instar 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 hybridization with the small ribosomal subunit protein gene DL11.

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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), respectively (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. Cyclin A in vitro. Mock immunoprecipitation (lanes 1,2)or To study the effects of lack of Dmcdk7 alone, the two immunoprecipitation using anti-DmCdk7 antibodies were in- other genes disrupted by Df(1)JB254, snf and dhd, were cubated with wild-type (wt) recombinant human HA-Cdk2/Cy- T160A reintroduced into this deficiency background by P-ele- clin A (lanes 1,3) or mutant HA–Cdk2 /Cyclin A (lanes 2,4). + + + After incubation with DmCdk7 immunoprecipitates, the wt ment-mediated transformation (Pw [snf ,dhd ]; Fig. 3). + + + (lane 3) but not the mutant (lane 4) HA–Cdk2 is phosphorylated To show that the Pw [snf ,dhd ] transgene is active, we as observed by a mobility shift (HA–Cdk2p) and by incorpora- tested its ability to complement the lethal phenotype tion of radiolabeled phosphate (32P-Cdk2p). The protein kinase associated with Df(1)J210, a 3.0-kb deletion with the activity of the CDK2/Cyclin A complex toward histone H1 is same proximal breakpoint as Df(1)JB254 but that affects stimulated by incubation with DmCdk7 (H1k). Incubation of only the snf and dhd genes (Fig. 3; Flickinger and Salz Cdk2/Cyclin A with larger amounts of DmCdk7 results in the 1994). Pw+[snf+,dhd+] fully rescues the lethal phenotype complete conversion of Cdk2 into the fast migrating isoform of Df(1)J210 (not shown), but fails to rescue the lethality and further increases histone H1 kinase activity (not shown). associated with Df(1)JB254, indicating that this chromosome lacks at least one additional essential function. This function is Dmcdk7 because the introduction of a DmCdk7 exhibits CAK activity in vitro wild-type Dmcdk7 gene (Pw+[Dmcdk7+]) into the Df(1)JB254-Pw+[snf+,dhd+] background results in com- To be able to analyze the biochemical activity of Dm- plete rescue of the lethality. These results demonstrate Cdk7, we raised antibodies against the DmCdk7 protein that Df(1)JB254 disrupts the Dmcdk7, snf, and dhd and used them to isolate the active enzyme from tissue genes, that the defects caused by lack of snf and dhd are homogenates. On immunoblots prepared from fly tis- rescued by Pw+[snf+,dhd+], and that the absence of Dm- sues, monoclonal antibodies raised against the full- cdk7 results in zygotic lethality. Therefore, we refer to length recombinant DmCdk7 recognize with high affin- animals carrying the Pw+[snf+,dhd+] transposon in a ho- ity a single polypeptide species, with a relative mobility mozygous Df(1)JB254 background as cdk7null mutants. of ∼40 kD (Fig. 2A). This is the first indication that these anti-DmCdk7 antibodies react specifically with Dm- Cdk7. The second one is that Drosophila Cdc2 and Cdk2 proteins, which both share a high degree of similarity with DmCdk7, cannot be detected in immunoprecipitates performed with anti-DmCdk7 antibodies (not shown). To demonstrate that the identified Drosophila protein possesses CAK activity, we used DmCdk7 immunoprecipitated from embryos 0–4 hr old to activate recombinant human HA–Cdk2/Cyclin A complexes (Fisher and Morgan 1994). HA–Cdk2/Cyclin A is strongly phosphorylated when incubated with a DmCdk7 immunoprecipitate, indicating that DmCdk7 can act as a Cdk kinase (Fig. 2B). The DmCdk7-mediated phosphoryla- Figure 3. Dmcdk7 locus and mutants. Both Df(1)J210 and tion of Cdk2 seems to occur specifically at threonine Df(1)JB254 were obtained by imprecise excision of dhdP8, aP- residue 160. This is demonstrated by the ability of Dm- element inserted into the dhd gene (Flickinger and Salz 1994). Cdk7 to phosphorylate the wild-type Cdk2 but not the The cdk7–P1 and dhd–P5 primers were used for PCR amplifica- tion and precise mapping of the Df(1)JB254 breakpoints. Below Cdk2T160A mutant protein (Fig. 2B). DmCdk7 also acts the genomic map, the fragments used for the different rescue as a CAK as it can stimulate the histone H1 kinase ac- constructs are indicated. The Pw+[snf+, dhd+] construct was tivity of Cdk2/Cyclin A (Fig. 2B). These results confirm used to rescue snf and dhd in Df(1)JB254. The Pw+ [cdk7x] con- that Dmcdk7 codes for a protein that exhibits CAK ac- struct was used to introduce the wild-type and the temperature- tivity in vitro and likely represents a functional homolog sensitive Dmcdk7 genes into the Df(1)JB254–Pw+[snf+,dhd+] of the vertebrate cdk7 genes. background.

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Cdk7 requirement for Cdc2 activation in vivo

Lack of zygotically expressed cdk7 results in death before or soon after the initiation of pupation. cdk7null animals often remain in a larval state, noticeably longer that their cdk7+ siblings and, under optimal conditions, up to 50% of the mutant larvae form a puparium, the remaining die as larvae. cdk7null larvae exhibit defects consistent with a disruption of mitotic activity. In cdk7null animals, the imaginal discs fail to proliferate and do not reach their normal size, whereas the polyploid tissue, which proceeds through replicative S- phases without mitosis, appear normal (not shown). This phenotype resembles the one seen in loss-of-function cdc2 mutants (Stern et al. 1993).

Figure 4. Effects of the P140S mutation on Cdk7. (A) cdk7ts A temperature-sensitive allele of Dmcdk7 flies were incubated at 18°C (a), or 29°C for 1 hr (b),2hr(c), or To be able to study the requirements for cdk7 activity in 3hr(d), before the immunoprecipitation and measurement of ts different tissues and at different times during develop- Cdk7 activity. (e) Mock immunoprecipitation. (B) The amount ment, we created a temperature-sensitive allele. Tem- of Cdk7 present in ovaries and embryos was measured by im- perature-sensitive mutations in the related cdc2 and munoblot (top) from similar amounts of total protein extracts (bottom). There is a progressive decrease in Cdk7P140S level Mek (Carr et al. 1989; Hsu and Perrimon 1994; Sigrist et with time spent at the restrictive temperature. Ovaries: (a) wild al. 1995) suggested that substituting the conserved pro- type ; (b) cdk7null/+; (c) cdk7null/+; Pw+[cdk7ts]; (d) cdk7ts 18°C; line residue 140 for a serine was likely to confer tem- (e) cdk7ts 29°C for 3 days; (f) cdk7ts 29°C for 5 days; (g) cdk7ts perature sensitivity to cdk7. Therefore, we introduced 29°C for 8 days. Embryos: (h) Wild type; (i) cdk7ts 18°C; (j) cdk7ts this mutation into cdk7 by site-directed mutagenesis, 29°C for 36 hr; (k) cdk7ts 29°C for 60 hr. In the case of embryos, and reintroduced the modified gene into flies. Animals the indicated times represent time the mothers were kept at the carrying a cdk7+ transgene in the cdk7null background restrictive temperature before the eggs were laid. were found to be 100% viable at all temperatures tested, whereas those carrying the cdk7P140S allele in the null ture retains significant activity until turned over. The background proved to be fully viable at 18°C but not described properties of the P140S mutation can explain viable at 27°C or above. These results demonstrate that the existence of a lag phase between the shift of tem- the P140S mutation results in a temperature-dependent perature-sensitive mothers to the restrictive tempera- inactivation of cdk7. Flies carrying only the cdk7P140S ture and the appearance of the mutant phenotype in em- transgene as a source of Cdk7 protein therefore will be bryos (see below). Similar lag phases have also been ob- referred to as cdk7ts mutants. served, for example, before cell cycle arrest occurs in the After transfer of mutant mothers to the restrictive cak1-22 mutant of S. cerevisiae (Kaldis et al. 1996). temperature, many of the embryos laid during the first Loss of either cdk7 or cdc2 causes similar cell 36 hr will eclose as larvae. Most of the embryos that fail proliferation defects to eclose during that period exhibit defects late in development. Mutant embryos exhibit gradually earlier devel- Like their cdk7+ siblings, mutant adults allowed to de- opmental arrest to a point where the embryonic nuclear velop at the permissive temperature can live for >40 days division program fails to be initiated (data not shown). after transfer to the restrictive temperature. The produc- When immunoprecipitated Cdk7P140S is used in CAK tion of gametes, however, stops in cdk7ts animals as fe- assays, a significant amount of activity is still present in males transferred to the restrictive temperature cease to embryos from females kept at the restrictive tempera- lay eggs after 3–4 days as a result of compromised cell ture for 1 day. Some activity can still be detected after 2 division in the germ line and supporting somatic tissue. days at the restrictive temperature (data not shown). In Drosophila, the ovary consists of a number of tube- Similarly, the Cdk7P140S protein fails to be inactivated like structures called ovarioles at the tip of which two to rapidly in vivo by incubation at high temperature (Fig. three mitotically active germ-line stem cells continu- 4A). We observe a similar failure to inactivate the Dm- ously divide. The asymmetric division of a stem cell Cdk7 protein in vitro after immunoprecipiation (data not gives rise to another stem cell and a cystoblast that then shown). What then is the basis of the temperature sen- goes through four incomplete mitotic divisions, result- sitivity of cdk7ts? Embryos and ovaries isolated from ing in a cyst of 16 germ cells connected to each other by Cdk7ts animals kept at the restrictive temperature show cytoplasmic bridges (for review, see Spradling 1993). As a progressive reduction of Cdk7 protein levels with time the 16-cell cyst moves posteriorly down the ovariole, it (Fig. 4B, see also Fig. 7, below). becomes enveloped by a continuous monolayer of so- Taken together, these data suggest that the P140S mu- matic follicle cells that are also supplied by dividing tation interferes with the stability of the mutant protein stem cells (Fig. 5A; Margolis and Spradling 1995). When synthesized at the restrictive temperature, whereas the pupae are transferred to the restrictive temperature, vi- mutant protein synthesized at the permissive tempera- able cdk7ts mutant adults continue to eclose for up to 3

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Larochelle et al. days after the temperature shift to 29°C. These animals the reduction in the number of follicle cells surrounding exhibit normal adult viability when kept at the restric- each egg chamber, the effect on the mitotic activity of tive temperature, but the mitotically active tissues ex- the germ line becomes apparent when cysts are found hibit progressively stronger defects with increasing time that possess fewer than the normal number of 16 germ spent at 29°C. When females eclose shortly after transfer cells (Fig. 5D,E). to 29°C, the first abnormality that is observed in cdk7ts Cyclin E was shown previously to function during en- ovaries is the depletion of the somatic follicle cells (Fig. doreplication of polyploid tissues (Sauer et al. 1995; Lilly 5C,D). A rapid depletion of follicle cells is predicted to and Spradling 1996), and its activity is thought to be occur if cell division is compromised as each daughter of mediated through the activation of Cdk2 (Knoblich et al. the follicle stem cells must divide approximately nine 1994; Sauer et al. 1995). Therefore, we were surprised times to produce the 1200 follicle cells surrounding each that although cell proliferation is arrested completely in germ-line cyst (Mahowald and Kambyselis 1980; Margo- cdk7ts ovaries, the capability of these cells to endorepli- lis and Spradling 1995). The depletion of follicle cells in cate their DNA is not affected (Fig. 5E–G). This result the cdk7ts mutant shows that cdk7 is required for mi- may suggest that the activity of Cdk2 is not affected by totic division of these somatically derived cells. After loss of cdk7 activity.

Figure 5. Phenotype of cdk7 during oogenesis. (A) Schematic view of the early stages of oogenesis in one ovariole. The germarial regions 1, 2, and 3 are indicated (1,2,3); region 3 contains the stage 1 egg chamber. (tfc) Terminal filament cells; (inc) inner sheath cells; (gsc) germ-line stem cells; (fsc) follicle stem cells; (dcc) dividing cystocytes; (nc) polyploid nurse cell nuclei; (fc) follicle cells; (Oo) oocyte; (Stg 2) stage 2 egg chamber. The germ- line part of the egg chamber in a 16-cell cyst contains 1 diploid oocyte and 15 nurse cells, which are in the process of becoming polyploid. The cyst is surrounded completely by still dividing somatic follicle cells. (A) Anterior; (P) posterior. Effects of lack of cdk7 in the ovary. Nuclear staining with Hoechst 33258 (B) wild-type egg chambers. (C) Dmcdk7ts animals were transferred to the restrictive temperature shortly (<1 day) before eclosion and ovaries dissected 3 days after eclosion. At this stage, although some follicle cells are present, their number is too low to surround completely each egg chamber. (D–G) Ovaries from cdk7ts females that eclosed 3 days after pupae were transferred to the restrictive temperature and dissected 3 days (D), 5 days (E,F), or 7 days (G) after eclosion. (D) Two cysts with fewer than 16 germ-line cells and no somatic follicle cells. (E) ‘‘Egg chamber’’ with a single polyploid germ-line cell. (F) View of an entire ovary completely populated by large polyploid cells. (G) Ovari- ole containing only seven large polyploid cells. (H–K) Effects of lack of cdc2 activity during oogenesis. Transfer of cdc2ts females to restrictive temperature causes a rapid depletion of somatic follicle cells. (H) cdc2ts ovaries transferred to the restrictive temperature 1 day after eclosion and dissected 2 days later. (I–K) Germarial region from (I) wild-type ovary, (J) Dmcdc2ts, (K) Dmcdk7ts ovaries from female transferred to the restrictive temperature 2 days before and dissected 3 days after eclosion. The region in which mitotic proliferation of the germ line occurs in the wild type is entirely occupied by large polyploid cells in both mutants, resulting in an enlargement of the germarium. cdc2 and cdk7 mutant ovaries eventually empty out of germ line (not shown).

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Cdk7 requirement for Cdc2 activation in vivo

If Cdk7 is required specifically for mitosis, we would that the lack of cdk7 does not cause a noticeable reduc- expect the ovarian phenotype resulting from lack of cdk7 tion in cyclin levels. Cyclins A and B are expressed to be similar to the one resulting from lack of cdc2. uniquely in mitotically active cells (Lehner and O’Farrell Therefore, we analyzed the cdc2 phenotype using the 1990). In total lysates from embryos at different stages of temperature-sensitive transgene Dmcdc2A171T (Sigrist et development, the level of mitotic cyclin proteins is al. 1995). Females carrying two copies of this tempera- greatly reduced in later development (Lehner and ture-sensitive allele in the Dmcdc2B47 background O’Farrell 1989; Edgar et al. 1994). The high level of Cy- showed a rapid depletion of follicle cells when trans- clins A and B still observed in cdk7ts embryos aged >6 hr ferred to the restrictive temperature after eclosion (Fig. (Fig. 6) is attributable to the fact that these embryos ar- 5H). This depletion of follicle cells is identical to the one rested development at a stage when mitotic cyclin levels observed in cdk7ts ovaries. Also, as noted for the cdk7ts are still high. Another observation suggesting that tran- mutant ovaries, mitotic proliferation of the germ line scription in general is not disrupted is the fact the germ- stops but the capacity of the germ-line cells to replicate line cells can still endoreplicate their DNA in mutant their DNA is not affected by the loss of cdc2 activity cdk7ts ovaries. Endoreplication has been proposed to re- (Fig. 5J). Polyploidization of the germ cells usually occurs quire the pulse transcription of cyclin E in embryonic only when the mitotic division program is terminated tissues (Sauer et al. 1995), and presumably also in the and the 16-cell cyst is formed. In both cdc2 and cdk7 ovary (Lilly and Spradling 1996). mutants the polyploidization of the germ line occurs pre- maturely (Fig. 5J,K). Because Cdc2 mediates this block of cdk7 mutant embryos are deficient in physiological endoreplication in mitotic tissues (Hayashi 1996), these CAK activity results also suggest that the premature endoreplication observed in cdk7 mutant ovaries may be attributable to To test whether cdk7 is essential for CAK activity, we lack of Cdc2 activity. incubated cdk7ts animals at the restrictive temperature for different amounts of time and measured the CAK Cyclin levels are not reduced in cdk7ts mutant activity in total cell lysates from their embryos. A embryos gradual reduction of CAK activity, down to background level, was observed (Fig. 7A,B). This indicates a genetic Because Cdk7 has also been implicated in the phos- requirement for cdk7 for most or all of the cellular CAK phorylation the CTD of RNA Pol II as part of the TFIIH activity that can be measured in vitro. Because immu- complex (Roy et al. 1994; Akoulithchev et al. 1995; Ser- nodepletion of Cdk7 protein from embryonic homog- izawa et al. 1995; Shiekhattar et al. 1995), the cell cycle enates can effectively eliminate CAK activity from wild- arrest observed in the cdk7 mutants could be an indirect type extracts (Fig. 7C,D), it can be concluded that the result of reduced RNA Pol II activity, which in turn Cdk7 protein itself provides all of the measurable CAK would result in low cyclin levels (Cismowski et al. activity in Drosophila embryos. 1995). To determine whether reduced cdk7 activity re- If Cdk7 also acts in vivo as a CAK, we would expect sults in a decrease in cyclin levels, the amount of the levels of Cdk T-loop phosphorylation and Cdk activity to different cyclins present in the mutant and wild-type be reduced in cdk7 mutant tissues. Therefore, we iso- embryos was determined by immunoblot (Fig. 6). The lated different Cdk/Cyclin complexes from mutant and level of all three cyclins is high in embryos from cdk7ts wild-type embryos using antibodies directed against Cy- parents during stages when they are also present at high clins A, B, and E. In Drosophila, Cyclin A (as well as levels in wild-type embryos. These observations indicate Cyclin B) associates uniquely with Cdc2 and not with Cdk2 (Knoblich et al. 1994; data not shown). Although Cyclin A can be precipitated equally from either wild- type or mutant embryos, the amount of Cdc2 protein recovered in the Cyclin A immunoprecipitates from mutant embryos is severely decreased (Fig. 8A). In both wild-type and mutant embryos, only the fast migrating isoform of Cdc2 can be found associated with Cyclin A in a stable complex (Fig. 8B). This indicates that Cdk7 activity is required for the formation of a stable Cdc2/ Cyclin A complex in vivo. On the contrary, Cdc2 can still form a stable complex with Cyclin B in cdk7 mutant embryos, but the amount of Thr-161 phosphorylated isoform of Cdc2 associated with Cyclin B is reduced (Fig. 8B). The addition of recombinant Cdk7/Cyclin H to the Figure 6. Cyclins in cdk7ts embryos. Before collecting embryos, cdk7ts parents were kept at 29°C for 60 hr. Total embry- mutant extracts before immunoprecipitation results in onic extracts from wild-type or cdk7ts embryos were analyzed an increase in the amount of fast migrating isoform of by Western blotting using monoclonal antibodies directed Cdc2, confirming its identity as T-loop phosphorylated against cyclins A, B, and E. The embryonic age is indicated in (Fig. 8B). hours. After immunoprecipitation from both wild-type and

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Figure 7. Decay of CAK activity in cdk7ts embryos. (A) H1k activity of Cdk2 after incubation with embryonic extracts; (left)0-to 4-hr-old embryos; (right) 2- to 6-hr-old embryos. The embryos were obtained from mothers kept at the restrictive temperature for 20 hr (lanes 1,4), 32 hr (lanes 2,5), 48 hr (lanes 3,6). (B) Quantitation of data shown in A. (C) H1k activity of Cdk2 after incubation with embryonic extracts subjected to immunodepletion using either protein G–agarose (Mock) or monoclonal anti-Cdk7 (19E7) coupled to protein G–agarose. Total CAK activity was measured from the same extracts after three consecutive rounds of depletion. (D) Quan- titation of data presented in C. The backround level of Cdk2 activity is measured after incubation with BSA instead of cellular extracts. cdk7ts embryos, the kinase activity toward histone H1 of tween the time at which there is apparently no active Cdc2/Cyclin A, Cdc2/Cyclin B, and Cdk2/Cyclin E Cdk7 protein remaining (Fig. 7) and the loss of Cdc2, complexes was measured. Although the total amount of Thr-161 phosphorylation (and the appearance of early ar- Cdc2 associated with Cyclin B is similar in both mutant rest phenotype) may be attributable to the fact that Cdc2 and wild-type embryos (Fig. 8D), the Cdc2/Cyclin B is phosphorylated maternally starting from mid-oogen- complex isolated from mutant embryos is less active esis. Therefore, this pool of active Cdc2 must be used up than the one isolated from wild-type embryos (Fig. 8D). before the effect of lack of Cdk7 can be clearly observed. This loss of activity correlates with the observed de- In the wild-type situation, Cdc2 Thr-161 does not appear crease in Thr-161 phosphorylation of Cdc2 (Fig. 8B). If to be significantly dephosphorylated until nuclear cycle the reduction in activity of Cyclin B-bound Cdc2 iso- 11 (Edgard et al. 1994). lated from cdk7ts embryos is attributable uniquely to In contrast to the above findings, the histone H1 ki- reduced Thr-161 phosphorylation, normal activity nase activity of the Cdk2/Cyclin E complex, as well as should be restored by treatment of this complex with the level of Thr-163 (equivalent to Thr-160 in mamma- CAK. To test this, the Cyclin B immunoprecipitates lian Cdk2) phosphorylation of Cdk2 are not affected sig- were incubated with active human recombinant Cdk7/ nificantly in mutant extracts as compared to wild type Cyclin H after the initial measurement of the histone H1 (Fig. 8E). kinase activity. This treatment results in the restoration of the activity (Fig. 8D) and Thr-161 phosphorylation Discussion (Fig. 8B) of the Cdc2 isolated from mutant embryos to a level equivalent to the one isolated from wild-type em- Cdk7 has been isolated previously as a Cdk-activating bryos. Therefore, it appears that the major reason why kinase (Fesquet et al. 1993; Poon et al. 1993; Solomon et the activity of the Cyclin B-bound Cdc2 is lower in cdk7 al. 1993; Fisher and Morgan 1994; Ma¨kela¨ et al. 1994), mutant embryos (as compared to the control) is that this and as a CTD kinase as part of the TFIIH basic transcrip- Cdc2 is hypophosphorylated on Thr-161. These results tion factor complex (Roy et al. 1994; Akoulichev et al. indicate that cdk7ts embryos are deficient in physiologi- 1995; Ossipow et al. 1995; Serizawa et al. 1995; Shiekhat- cal CAK activity. The slight delay that is observed be- tar et al. 1995; Adamczewski et al. 1996). Although the

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cdk7 is required for mitotic proliferation In addition to sharing a high degree of sequence similarity with vertebrate cdk7 genes, the Drosophila gene we have isolated in this study codes for a protein that can phosphorylate and activate Cdk2 in vitro. The terminal phenotype of the cdk7null mutant is similar to the strongest loss-of-function cdc2 mutant: death before or during pupation with little imaginal tissue (Stern et al. 1993). A phenotype now considered a hallmark of genes required for mitotic proliferation (Gatti and Baker 1989). The creation of a temperature-sensitive allele of cdk7 allowed us to further strengthen the possible in vivo link between cdc2 and cdk7. The temperature-dependent inactivation of the cdk7ts allele in the ovary results in the ts Figure 8. cdk7 embryos are defective in CAK activity. (A,B) rapid depletion of the somatic follicle cells, which need Phosphorylation states of Cdc2 in cyclin A and B immunopre- to be replenished continuously from a mitotically active cipitates. cdk7ts mutant embryos (0–4 hr) were obtained from population of stem cells (Margolis and Spradling 1995). mothers kept at 29°C for 56 hr (A)or48hr(B). (A) Cdc2 is ts precipitated efficiently from wild-type but not cdk7ts mutant Proliferation of the germ line is also disrupted in cdk7 embryos using anti-Cyclin A antibodies. (B) Similar amounts of mutants, resulting in the formation of cysts with fewer Cdc2 can be precipitated from either wild-type or mutant em- than the normal complement of 16 cells. We found these bryos using anti-Cyclin B antibodies but the fast migrating (Thr- phenotypes to be very similar to the temperature-sensi- 161 phosphorylated) isoform of Cdc2 (Edgar et al. 1994) is re- tive ovarian cdc2 phenotype we have described in this duced in the cdk7ts embryos as compared to wild type. The study. Similar mitotic defects in the ovary were reported amount of fast migrating isoform isolated from mutant embryos recently using a different allele of cdc2 (Reed and Orr- can be increased by the addition of recombinant Cdk7/Cyclin H Weaver 1997). These results are consistent with a model to the extracts before immunoprecipitation. This confirms the whereby Cdk7 acts in the same pathway as Cdc2 in pro- identity of the fast migrating isoform as T-loop phosphorylated. moting cell division. The last panel shows that the Cyclin A-bound Cdc2p corre- sponds to the fast migrating isoform. This loss of binding of Cyclin A to Cdc2 in embryos is observed relatively soon after transfer of the mothers to the restrictive temperature (not shown) at a time when there is still a sizable amount of CAK The CAK in cdk7 activity in embryonic extracts. (C–E) Mutant embryos were obtained from temperature-sensitive mothers kept at 29°C for 48– In cdk7 mutant embryos, the level of Thr-161 phos- 72 hr. The histone H1 kinase (H1k) activity of the different phorylation and activity of the Cyclin B-bound Cdc2 is Cdk/cyclin complexes were measured after immunoprecipita- reduced, and both can be restored by incubation with tion using anti-cyclin antibodies. (C) Lower amounts of H1k purified Cdk7/Cyclin H (Fig. 8). This indicates that the activity are recovered in Cyclin A immunoprecipitates from major difference between Cdc2 isolated from wild-type cdk7 mutant embryos than from wild type (top). The activity of and cdk7 mutant embryos is the extent of Thr-161 phos- the Cyclin A/Cdc2 complex cannot be restored by incubation phorylation. Therefore, Cdk7 is essential for in vivo with CAK (middle) as only a small amount of Cdc2 is present in CAK activity. Although Cdc2/Cyclin B complexes form anti-Cyclin A immunoprecipitates in the cdk7 mutant embryos normally in cdk7ts mutant embryos, Cdc2 and Cyclin A (bottom, see also B). (D) H1k activity of the Cyclin B-associated fail to form a stable complex in the cdk7 mutant. This is Cdk (Cdc2) is reduced in mutant embryos (top). After initial H1k activity measurement, the immunoprecipitates were incu- likely attributable to the fact that this event requires the bated with recombinant CAK and the H1k activity measured phosphorylation of Cdc2 on Thr-161, as even in the wild again (middle). After Cdk7 treatment, complexes isolated from type only the phosphorylated form is associated with both wild-type and mutant embryos have equivalent activity. Cyclin A (Fig. 8). These in vivo results correlate well The H1k activity is increased in complexes isolated from wild- with the finding that human Cdc2 needs to be phos- type as well as mutant embryos after CAK treatment (not phorylated by CAK to form a stable complex with Cyclin shown). The standard SDS-PAGE conditions used in those par- A in vitro, whereas stable Cdc2/Cyclin B and Cdk2/Cy- ticular experiments did not allow for the resolution of the dif- clin E complexes can form in the absence of Thr-161 (or ferent Cdc2 isoforms described in B. (E) The H1k activity of the 160) phosphorylation (Desai et al. 1995). The Cdc2/Cy- Cdk2/Cyclin E complex was measured as in C and D. clin A complex seems to be more sensitive to a reduction in CAK activity than the Cdc2/Cyclin B complex, as the concept that Cdk7 is involved in transcriptional regula- loss of Cyclin A binding occurs more rapidly than the tion has received general acceptance, thus far there has reduction of Thr-161 phosphorylation of Cyclin B-asso- been no satisfying demonstration that Cdk7 can also act ciated Cdc2. as a CAK in vivo (for review, see Fisher and Morgan Because Cdk7/Cyclin H is able to phosphorylate many 1996; Nigg 1996). With the analysis of the Drosophila Cdk/cyclin complexes in vitro, it was postulated that a cdk7 gene, we set out to clarify the cell cycle require- single CAK could be responsible for the activation of ments for cdk7 in vivo in a multicellular organism. all the different Cdks acting throughout the cell cycle

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(Fisher and Morgan 1994). This idea was further rein- sults in the abolition of in vivo CAK activity and reduced forced by the observation that Cdk7 activity is appar- Cdc2 T-loop phosphorylation and activity (this study) ently present throughout the cell cycle (Brown et al. and has a phenotype identical to that of loss of cdc2 (this 1994; Matsuoka et al. 1994; Poon et al. 1994; Tassan et study). These observations leave little room for an expla- al. 1994). Therefore, we were intrigued by the continuing nation other than that Cdk7 is active as an in vivo CAK. DNA synthesis observed in the germ cells of cdk7ts mu- The basic components of the cell cycle regulatory ma- tant ovaries. Two explanations could account for the chinery are, for the most part, shared by both yeast and lack of effect on endoreplication: (1) Cdk2 is not involved higher eukaryotes. It has been shown in numerous cases in DNA synthesis during endoreplication. This possibil- that the mechanisms, as well as molecules, that regulate ity cannot be excluded formally as no mutant phenotype the cell cycle in yeast are usually also conserved in has been described so far for cdk2. (2) Cdk2 may not higher eukaryotes. Therefore, it may come as a surprise require threonine phosphorylation for its DNA synthe- that yeast and metazoans would use entirely different sis-promoting activity during endoreplication. molecules, such as Cdk7 and CAK1, to carry out identi- Because no effect on the levels of Cdk2 Thr-163 phos- cal enzymatic reactions in such a basic mechanism as phorylation or activity could be detected in cdk7 mutant the activating phosphorylation of Cdks. Perhaps even embryos, it is possible that the threonine-phosphory- more surprising is that a ‘‘complex’’ multicellular organ- lated form of Cdk2 is highly stable or requires a lower ism would use a single enzyme to carry out two very threshold of CAK activity than Cdc2. Alternatively distinct functions, whereas the apparently much simpler Cdk2 may be activated by a CAK different from Cdk7. unicellular yeast would use two different ones. However, But as it appears that all of the measurable embryonic the analysis of all the data obtained in this study and CAK activity in Drosophila can be eliminated either ge- previously with Cdk7, Kin28, and CAK1 clearly point netically by the inactivation of cdk7 or biochemically by into this direction. immunodepletion of Cdk7, if such a second CAK exists, Now that the sequence of the whole genome of S. cere- its biochemical characteristics such as substrate speci- visiae is known, it is clear that of all yeast proteins ficity or solubility would have to be very different from Kin28 is the one with the highest sequence similarity to that of either Cdk7 or CAK1. Or it would have to be Cdk7. At the functional level, both proteins can be found absent from the stages analyzed. Figure 9 summarizes as subunits of TFIIH and are known to interact physi- the sum of accumulated data on CAKs and TFIIH ki- cally with related cyclin-like molecules, Cdk7 with Cy- nases in yeasts and metazoans. clin H (Fisher and Morgan 1994; Ma¨kela¨et al. 1994) and Kin28 with Ccl1 (Valay et al. 1993). Both Cdk7 and Kin28 can use the CTD of RNA Pol II as substrate in CAK in yeast and metazoan: a question of evolution? vitro. From these data it seems clear that Cdk7 and It is impossible to demonstrate with absolute certitude Kin28 are not only related by sequence, but also carry that Cdk7 phosphorylates Cdc2 in vivo. But the evidence out similar cellular functions in both organisms. How- to that effect is overwhelming; Cdk7 acts as an excellent ever, there is a major difference between the two mol- CAK in vitro and CAK activity can be entirely depleted ecules; Cdk7 is a very efficient CAK in vitro, whereas from a cellular extract with Cdk7 antibodies (this study; Kin28 has no detectable CAK activity either in vitro or Fesquet et al. 1997). The loss of cdk7 gene function rein vivo (Cismowski et al. 1995; Valay et al. 1995). The present work underlines another major difference between Cdk7 and Kin28, this time at the level of a genetic requirement. We clearly demonstrate that cdk7 activity is required for the production of CAK activity in vivo, whereas Kin28 is not (Cismowski et al. 1995; Valay et al. 1995). Our failure to detect a defect in transcription in cdk7 mutants could mean that the Cdk7ts protein per- sists longer as part of TFIIH, which may stabilize its activity. Alternatively, these observations may indicate that Cdk7 activity is not essential for transcription un- Figure 9. CAK in yeast and multicellular organisms. Simpli- der our experimental conditions. For example, it may be fied representation of TFIIH kinase, Cdk, and CAK activities in that a different protein acts redundantly with Cdk7 as a the unicellular yeasts S. pombe and S. cerevisiae, and Dro- CTD kinase. At least two other Cdks are known to be sophila incorporating the results of the present study, which able to act as CTD kinase in human cells, Cdk8 (Tassan provide strong genetic and biochemical evidence that metazo- et al. 1995b) and Cdk9(PITLARE) (Jones 1997). ans Cdk7 acts as an in vivo CAK for Cdc2. The well-character- Evidently unicellular organisms have continued to ized association of Cdk7 with TFIIH in vertebrates also indi- evolve just like metazoans did. Maybe it was advanta- cates a role for Cdk7 in transcription. In S. cerevisiae, CAK activity is provided by CAK1/Civ1, whereas the Cdk7-related geous for S. cerevisiae to use two distinct proteins to Kin28 is only active in transcriptional regulation. In combina- carry out functions for which only one has remained tion with our results, those available thus far in S. pombe sug- necessary in other organisms. The emergence of CAK1 gest that the Cdk7 homolog (Mop1/Crk1) may be active in vivo may then have lead to the evolution of Kin28, a Cdk7 as both a CAK and CTD kinase. that has lost its ability to act as a CAK. In this context it

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Cdk7 requirement for Cdc2 activation in vivo would be interesting to know whether metazoans have a was produced by cloning the entire coding region of the Dm- CAK1 homolog. Thus far none have been reported, but cdk7 cDNA into the NdeI–BamHI sites of the pET-3 (pAR) pro- whether there is or is not a CAK1 homolog in multicel- tein expression vector. BALB/c mice were immunized three lular organisms will be answered only with its discovery times at 5-week interval using 50 µg of protein emulsified in or the completion of the sequencing of a metazoan TiterMax synthetic adjuvant (CytRx corp., Atlanta, GA). Screening of the hybridoma lines was carried out by ELISA us- genome. ing a MBP–DmCdk7 fusion protein (New England Biolabs), and immunobloting using ovarian protein extracts from Drosophila. The antibodies used in this study (19E7 and 20H5) recognize a Materials and methods single polypeptide species with high affinity. For immunoblot- DNA cloning and deficiency analysis ting, standard SDS-PAGE was used except for the separation of the Thr-161 phosphorylated and nonphosphorylated form of The described Dmcdk7 cDNA was isolated during a screen for Cdc2 (Fig. 8A,B). For this purpose the acrylamide stock solution protein kinases (Larochelle and Suter 1995). Library screening, used was 30% T:1.67% C using Piperazine di-acrylamide (Bio- nucleic acid hybridizations, and sequencing were done accord- Rad) as crosslinker (Kumagai and Dunphy 1995). ing to standard protocols. Deletions produced by the imprecise excision of the dhdP8 P-element (Flickinger and Salz 1994) were tested by Southern hybridization using a Dmcdk7 cDNA probe. Ovary staining To map precisely the genomic region deleted by the Df(1)JB254 Ovaries were dissected in Ringer’s solution and fixed for 20 min. deficiency, PCR amplifications were carried out using primers in 200 µl of 4% paraformaldhehyde in PBS + 0.2% Tween 20, 20 cdk7–P1 (5 -ACAACTCATGTTGGGTGGCG-3 )intheDm- Ј Ј µl of DMSO, and 600 µl of n-heptane. Washed several times in cdk7 3 UTR region, and dhd–P5 (5 -GGATTGCTGCTTAC- Ј Ј PBST, and stained for 1 hr with 10 µg/ml of Hoechst 33258 in GCCTTC-3 ) followed by sequencing. Ј PBST.

Site-directed mutagenesis and transformation constructs Kinase assays The mutagenic primer used to create the temperature-sensitive For CAK assays on immunoprecipitates, ∼50–200 µl embryos (or mutation was P140S 5Ј-CGCGATTTGAAGTCCAACAATT- 20–100 pairs of ovaries) were homogenized in 1.0 ml of HoB TGC-3Ј. The rescue construct for dhd and snf genes was con- buffer [25 mM HEPES (pH 7.4); 150 mM NaCl, 5 mM NaF; 1 mM structed by cloning a 3.7-kb StuI–EcoRI fragment containing EDTA, 1 mM DTT, 0.1 mM Na VO , 0.1% Triton X-100, 2 µg/ ∼ 3 4 25% of the Dmcdk7 and the entire snf genomic region into ml of aprotinin; 2 mM PMSF, 1 µg/ml of leupeptin]. The ho- pBluescript KS− SmaI–EcoRI sites. A 4.8-kb XhoI fragment con- mogenate was centrifuged at 14,000 rpm for 20 min and the taining the dhd gene was then added at a single genomic XhoI cleared supernatant incubated with 20 µl of protein G plus aga- site within the 3.7-kb StuI–EcoRI fragment. A 5.5-kb XbaI–B- rose (Calbiochem) previously reacted with monoclonal anti- amHI fragment containing both entire snf and dhd genomic DmCdk7 19E7. The immunoprecipitate was washed three regions was then cloned into the pCaSpeR transformation times with the same buffer and three times with HD [25 mM vector. HEPES (pH 7.4), 1 mM DTT]. CAK activity was measured by adding 0.6 µg of recombinant human HA-Cdk2/Cyclin A (a gift Genetics and fly stocks from H. Espinoza and D.O. Morgan, University of California, San Francisco) in 20 µl [25 mM HEPES (pH 7.4), 10 mM MgCl2; The P-element excision screen that gave rise to Df(1)JB254 is 50 µM ATP; 10 µCi [␥-32P]ATP (3000 mCi/mmole), 1 mM DTT] described in Flickinger and Salz (1994). The Dmcdk7null chro- and incubated at room temperature for 20 min. The supernatant mosome was constructed by recombining an X chromosomal was removed from the immunoprecipitate and boiled twice in insert of Pw+[snf+,dhd+] with w Df(1)JB254. Dmcdk7ts were ob- one volume of sample buffer. For direct assessment of HA–Cdk2 tained by crossing males carrying a Sb Pw+[Dmcdk7-P140S] third phosphorylation the protein was run over a 10% polyacryl- chromosome to Dmcdk7null females. The females used for these amide gel and the dried gel exposed for 2 hr with intensifying crosses were either w Df(1)JB254 Pw+[snf+,dhd+]/ w Df(1)JB254 screens. For direct detection of Cdk2, samples were transferred Pw+[snf+,dhd+]; +/+; Pw+[Dmcdk7 P140S] Sb/+ for analysis of the to nitrocellulose and the HA–Cdk2 protein detected with anti- ovarian phenotype or w Df(1)JB254 Pw+[snf+,dhd+]/ w Df(1)JB254 HA (12CA5 or HA.11) antibody (BabCO) and SuperSignal Pw+[snf+,dhd+]; +/+; Pw+[Dmcdk7 P140S/Pw+[Dmcdk7 P140S]to (Pierce). obtain cdk7ts embryos. The Dmcdc2 alleles B47 and the tem- For histone H1 kinase assays on Cdk/cyclin complexes, 50 µl perature-sensitive Dmcdc2ts4x: w/w; Pw+[cdc2A171T], cdc2B47/ of control or mutant embryos (2–6 hr old) were homogenized in Pw+[cdc2A171T], cdc2B47; Pw+[cdc2A171T]/ Pw+cdc2A171T] lines 300 µl of HoB and incubated 3 hr with 15 µl of protein G– have been decribed before (Stern et al. 1993; Sigrist et al. 1995). agarose previously reacted with monoclonal anti-cyclins A, B, cdc2ts flies used in this study were obtained by crossing or E. The immunoprecipitates were washed three times with ts4x B47 + A171T Dmcdc2 to +/+; cdc2 /SM6A; +/+. w/+; Pw [cdc2 ], HoB, three times with HDS buffer [25 mM HEPES (pH 7.4), 1 mM B47 B47 + A171T cdc2 /cdc2 ; Pw [cdc2 ]/+ were used for phenotypic DTT, 150 mM NaCl] and once with HD buffer. The immuno- analysis of the ovary. precipitates were incubated with 30 µl of histone H1 kinase mix (3.0 µg of histone H1 (Boehringer), 25 mM HEPES (pH 7.4), 10 mM MgCl ,1mMDTT, 50 mM NaCl, 50 µM ATP, 10 µCi [␥- Antibodies and protein blotting 2 32P]ATP) and incubated at room temperature for 20 min. The Anti-DmCdk2 (Cdc2c) and anti-Cyclin A and B antibodies were reaction mixture was removed from the immunoprecipitate and provided by C. Lehner (Bayreuth), anti-DmCdc2 and anti- boiled in one volume of SDS sample buffer. For reactivation by PSTAIR antibodies by P. O’Farrell [University of California, San recombinant CAK, the immunoprecipitates from the previous Francisco (UCSF)] and M. Yamashita (Hokkaido University, step were washed several times with HoB and HD buffers as Sapporo, Japan), respectively. Recombinant DmCdk7 protein above and then incubated with 50 ng of recombinant human

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Cdk7/Cyclin H in 20 µl [25 mM HEPES (pH 7.4), 10 mM MgCl2, kinase, during Xenopus oogenesis and embryogenesis. Mol. 1mM DTT, 0.1 mM ATP] for 20 min. The beads were subse- Biol. Cell 5: 921–932. quently washed with HoB and HDS to remove the CAK. Finally, Buck, V., P. Russell, and J.B.A. Millar. 1995. Identification of a the histone H1 kinase activity was remeasured as described cdk-activating kinase in fission yeast. EMBO J. 14: 6173– above. 6183. For CAK assay on total lysates, samples (embryos or ovaries) Carr, A.M., S.A. MacNeill, J. Hayles, and P. Nurse. 1989. Mo- were homogenized in EB buffer [10 mM Tris (pH 7.5), 80 mM lecular cloning and sequence analysis of mutant alleles of

Na␤-glycerophosphate, 20 mM EGTA, 15 mM MgCl2,2mM the fission yeast cdc2 protein kinase gene: Implications for + Na3VO4,1mMbenzamidine, 1 mM meta-bisulfite, 2 µg/ml of cdc2 protein structure and function. Mol. & Gen. Genet. aprotinin, 2 mM PMSF, 1 µg/ml of leupeptin] (Edgar et al. 1994). 218: 41–49. The protein concentration in each sample was measured using Cismowski, M.J., G.M. Laff, M.J. Solomon, and S.I. Reed. 1995. Bradford protein assay (Bio-Rad) with BSA as standard, and the KIN28 encodes a C-terminal domain kinase that controls protein concentration in the lysates adjusted to ∼30 µg/µl. mRNA transcription in Saccharomyces cerevisiae but lacks Samples of 10 µl were mixed with 10 µl of assay mix [25 mM Cyclin-dependent kinase-activating kinase (CAK) activity.

HEPES (pH 7.4), 10 mM MgCl2, 0.1 mM ATP, 10 µCi of [␥- Mol. Cell. Biol. 15: 2983–2992. 32P]ATP (3000 mCi/mmole), 1 mM DTT, 0.1 µg of HA–Cdk2/ Damagnez, V., T.P. Ma¨kela¨, and G. Cottarel. 1995. Schizosac- Cyclin A]. The reaction was stopped after 25 min by the addi- charomyces pombe Mop1–Mcs2 is related to mammalian tion of 500 µl of HoB + 20 mM EDTA. HA–Cdk2 was immuno- CAK. EMBO J. 14: 6164–6172. precipitated using anti-HA antibodies/protein G–agarose and De Bondt, H.L., J. Rosenblatt, J. Jancarik, H.D. Jones, D.O. Mor- the activity of the precipitated HA–Cdk2/Cyclin A complexes gan, and S.-H. Kim. 1993. Crystal structure of cyclin-depen- was assayed on histone H1 as described above. Quantitative dent kinase 2. Nature 363: 595–602. data were obtained using a Fujix BASS 2000 TR PhosphorIm- Desai, D., H.C. Wessling, R.P. Fisher, and D.O. Morgan. 1995. ager. Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2. Mol. Cell. Biol. 15: 345–350. Devault, A., A.-M. Martinez, D. Fesquet, J.-C. Labbe´, N. Morin, Acknowledgments J.-P. Tassan, E.A. Nigg, J.-C. Cavadore, and M. Doreé. 1995. We thank Drs. C. Lehner for anti-Cdk2, cyclins A and B anti- MAT1 (‘‘meńage a`trois’’) a new RING finger protein subunit bodies, and for cdc2 fly stocks, P. O’Farrell for the anti-Cdc2 Ab, stabilizing cyclin H-cdk7 complexes in starfish and Xenopus H. Richardson for the anti-Cyclin E Ab, and M. Yamashita for CAK. EMBO J. 14: 5027–5036. anti-PSTAIR Ab, D.O. Morgan and H. Espinoza for HA–Cdk2/ Edgar, B.A. and C.F. Lehner. 1996. Developmental controls of Cyclin A, A.R. Knights for the preparation of recombinant cell cycle regulators: A fly’s perspective. Science 274: 1646– Cdk7/Cyclin H. Thanks to C. Cariles and J. Geiger for technical 1652. assistance, M. Miron for help with the PhosphorImager, and Edgar, B.A., F. Sprenger, R.J. Duronio, P. Leopold, and P.H. Anne Marcil for assistance during the preparation of anti-Dm- O’Farrell. 1994. Distinct molecular mechanisms regulate Cdk7 monoclonal antibodies. We also thank Dr. D.Y. Thomas cell cycle timing at successive stages of Drosophila embryo- for providing laboratory space during the hybridoma screening. genesis. Genes & Dev. 8: 440–452. Thanks to Akira Nakamura and Andrew Swan for critical read- Espinoza, F.H., A. Farell, H. Erdjument-Bromage, P. Tempts, ing of the manuscript. Work in the H.K.S. laboratory is sup- and D.O. Morgan. 1996. A Cyclin-dependent kinase-activat- ported by funds form the U.S. National Science Foundation. ing kinase (CAK) in budding yeast unrelated to vertebrate This work is supported by funds to B.S. from the National Can- CAK. Science 273: 1714–1717. cer Institute of Canada with funds from the Canadian Cancer Feaver, W.J., J.Q. Svejsrup, N.L. Henry, and R.D. Kornberg. Society and the Terry Fox Run. B.S. is a research scientist of the 1994. Relationship of CDK-activating kinase and RNA poly- National Cancer Institute of Canada supported by funds from merase II CTD kinase TFIIH/TFIIK. Cell 79: 1103–1109. the Canadian Cancer Society. Fesquet, D., J.-C. Labbe´, J. Derancourt, J.-C. Capony, S. Galas, F. The publication costs of this article were defrayed in part by Girard, T. Lorca, J. Shuttleworth, M. Doreé, and J.-C. Cava- payment of page charges. This article must therefore be hereby dore. 1993. The MO15 gene encodes the catalytic subunit for marked ‘‘advertisement’’ in accordance with 18 USC section a protein kinase that activates cdc2 and other cyclin-depen- 1734 solely to indicate this fact. dent kinases (CDKs) through phosphorylation of thr161 and its homologues. EMBO J. 12: 3111–3121. Fesquet, D., N. Morin, M. Doreé, and A. Devault. 1997. Is Note Cdk7/cyclin H/MAT1 the genuine cdk activating kinase in cyclin xenopus egg extracts? Oncogene 15: 1303–1307. The sequence reported in this manuscript has been deposited Fisher, R.P. and D.O. Morgan. 1994. A novel form of cyclin into GenBank (accession no. U56661). associates with MO15/CDK7 to from the CDK-activating kinase. Cell 78: 713–724. References ———. 1996. CAK in TFIIH: Crucial connection or confounding coincidence? Biochim. Biophys. Acta 1288: O7–O10. Adamczewski, J.P., M. Rossignol, J.P. Tassan, E.A. Nigg, V. Fisher, R.P., P. Jin, H.M. Chamberlin, and D.O. Morgan. 1995. Monocollin, and J.M. Egly. 1996. MAT1, cdk7 and cyclin H Alternative mechanisms of CAK assembly require an assem- form a kinase complex which is UV light sensitive upon bly factor or an activating kinase. Cell 83: 47–57. association with TFIIH. EMBO J. 15: 1877–1884. Flickinger, T.W. and H.K. Salz. 1994. The Drosophila sex deter- Akoulitchev, S., T.P. Ma¨kela¨, R.A. Weinberg, and D. Reinberg. mination gene snf encodes a nuclear protein with sequence 1995. Requirement for TFIIH kinase activity in transcription and functional similarity to the mammalian UIA snRNP by RNA polymerase II. Nature 377: 557–560. protein. Genes & Dev. 8: 914–925. Brown, A.J., T. Jones, and J. Shuttleworth. 1994. Expression and Follette, P.J. and P.H. O’Farrell. 1997. Cdks and the Drosophila activity of p40MO15, the catalytic subunit of cdk-activating cell cycle. Curr. Opin. Genet. Dev. 7: 17–22.

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Cdk7 requirement for Cdc2 activation in vivo

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GENES & DEVELOPMENT 381 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

Stéphane Larochelle, Judit Pandur, Robert P. Fisher, et al.

Genes Dev. 1998, 12:

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