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Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase).

The activity of cyclin-dependent kinases (cdks) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34cdc2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating kinase), and it was shown to contain p40MO15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40MO15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40MO15, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coli. These tools were then used to demonstrate that p40MO15 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40MO15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40MO15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34cdc2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).

Abstract. The activity of cyclin-dependent kinases (cdl~) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34 ~c2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating _kinase), and it was shown to contain p40 M°15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40 M°15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40 M°tS, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coll. These tools were then used to demonstrate that p40 M°~5 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40 u°15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40 u°15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34 ~c2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).
T HE cell cycle in all eukaryotes is governed by cyclindependent protein kinases (cdks) l (Pines and Hunter 1991a;Meyerson et al., 1992;Norbury and Nurse, 1992;van den Heuvel and Harlow, 1993). These kinases are regulated by multiple mechanisms, including phosphorylation-dephosphorylation (For reviews see Fleig and Gould, 1991;Clarke and Karsenti, 1991;Solomon, 1993;Draetta, 1993), and the binding to other proteins, notably cyclin regulatory subunits (reviewed in Hunt, 1991;Sherr, 1993) and small inhibitory polypeptides (Xiong et al., 1993;Serrano et al., 1993;Gu et al., 1993;Harper et al., 1993;EI-Deiry et al., 1993;Polyak et al., 1994). The complex regulation imposed on cdks is likely to reflect the fact that orderly progression through the cell cycle requires the integration of a multitude of positive and negative signals.
These are thought to include signals relayed from extracellular agents such as mitogenic hormones and growth factors, as well as signals generated by intracellular feedback mechanisms that monitor the quality of individual cell cycle events and their orderly succession (for review see Murray, 1992).
The prototypic and best characterized member of the cdk family, p34cd% functions in association with cyclins of the A and B subfamilies to trigger entry into mitosis. Consistent with its function as an inducer of M phase, its activity increases sharply at the G2/M transition and drops abruptly as cells exit from M phase (reviewed in Murray and Kirschner, 1989;Mailer, 1991;Norbury and Nurse, 1992). Several phosphorylation sites have been mapped within the p34 ~2 molecule (Gould and Nurse, 1989;Gould et al., 1991;Nigg, 1991a, 1992;Russo et al., 1992). In all species examined, p34 ~c2 is phosphorylated on tyrosine 15 (Tyrl5), a residue located within the ATP-binding site of the kinase, and multiple kinases (weel, mikl) and phosphatases (cdc25, pyp3) acting on this residue have been characterized in several species (reviewed in Fleig and Gould, 1991;Labib and Nurse, 1993;Solomon, 1993). In both fission yeast and vertebrates, dephosphorylation of Tyrl5 represents the rate-limiting step for activation of p34 c.2 at the G2/M transition (Gould and Nurse, 1989;Nigg, 1991b, Norbury et al., 1991). It occurs in response to intracellular signalling pathways that monitor the completion of DNA replication, thereby preventing the premature initiation of mitosis (Enoch and Nurse, 1991;Smythe and Newport, 1992). The situation is somewhat different in budding yeast, where the temporal separation between DNA replication and certain aspects of mitosis is less distinct (Sorger and Murray, 1992;Amon et al., 1992). In vertebrates, additional phosphorylation of threonine 14 (Thr14) has been demonstrated (Krek and Nigg, 1991a;Norbury et al., 1991), and it is attractive to speculate that Thr14 may represent the sensor for a second checkpoint control pathway, monitoring the completion of another, as yet unidentified physiological event. Thrl4 can be dephosphorylated by the same phosphatase (cdc25) that also acts on Tyr15 (Honda et al., 1993;Sebastian et al., 1993), but the kinase responsible for its phosphorylation has not yet been identified.
Not all phosphorylations occurring on p34 ~2 lead to inactivation of the kinase. In particular, phosphorylation of Thr161 (Thr 167 in Schizosaccharomycespombe) was shown to be positively required for the function of p34~2: mutations of Thrl61 to nonphosphorylatable residues (except Glu) inactivate p34 ~2 protein kinase and interfere with cyclin binding Ducommun et al., 1991;Solomon et al., 1992;Krek et al., 1992a). Furthermore, there is a temporal correlation between the phosphorylation of Thrl61 in p34 ~2 and the time when its cyclin partners are synthesized (Krek and Nigg, 1991a), and, conversely, dephosphorylation of Thrl61 occurs concomitantly with p34 ~2 inactivation upon exit from mitosis (Lorca et al., 1992). Phosphorylation of a corresponding residue is believed to be required for the activity of other cdks (Gu et al., 1992;Kato et al., 1994), although this has not yet been directly demonstrated for all members of the family. Also, corresponding residues have been shown to be phosphorylated in cAMP-dependent protein kinase (PKA) and in MAP kinases, and the recent x-ray crystallographic analyses of PKA (Knighton et al., 1991a), cdk2 (De Bondt et al., 1993, and MAP-kinase (Zhang et al., 1994) provide plausible explanations for why attachment of a phosphate at this position may be critical for folding the various kinases in an active conformation (for reviews see Taylor et al., 1993;Morgan and De Bondt, 1994). Phosphorylation of this residue is considered to be essentially constitutive in the case of PKA (Taylor et al., 1993), but it clearly plays a key regulatory role in the activation of the MAP kinase cascade (reviewed in Pelech and Sanghera, 1992;Ahn et al., 1992;Nishida and Gotoh, 1993).
Recently, a kinase able to phosphorylate Thrl61 in p34 ~2 (and the corresponding residue in p33 cdu) has been identified and termed CAK. Originally, CAK was meant to stand for cdc2-activating kinase (Solomon et al., 1992); however, as the same kinase phosphorylates also p33 o~ and perhaps other cdks, at least in vitro, it seems appropriate to interpret the term CAK in a broader sense (i.e., cdk-activating kinase; see also Poon et al., 1993). CAK isolated from starfish and Xenopus (Fesquet et al., 1993;Poon et al., 1993;Solomon et al., 1993) was shown to contain a 40-kD catalytic subunit that had previously been isolated under the designation MO15 (Shuttleworth et al., 1990). Highly purified active CAK from starfish was found to contain a second polypeptide (Fesquet et al., 1993), but the identity of this potential regulatory subunit has not yet been elucidated. One of the key questions raised by the above findings is how the activity of CA/( is regulated during the somatic cell cycle. To address this issue, we have cloned a eDNA for a human homologue of Xenopus p40 M°lS, and we carded out a detailed analysis of p40U°t5/ CAK expression, activity, and subcellular localization in exponentially growing human cells. Our results have implications for both the temporal and the spatial regulation of cdk activities during the cell cycle.
A 198-bp partial human eDNA (originally designated as HsPK31), encoding a 66-amino acid polypeptide with 90.9% identity to the p40 u°15 protein kinase of Xenopus/aev/s (Shuttleworth et al., 1990), was originally isolated in the course of a PCR-based search for potential human homologues of the NIMA protein kinase, a cell cycle regulator in A~pergillus nidu/ans (Schultz and . To prepare a 32P-labeled probe for hybridization, the 19g-bp insert of the partial MOI5 eDNA was excised from the original HsPK31 plasmid by digestion with PstI and KPOI. The fragment was gel isolated and labeled with [~-32p]dATP using a random-primed DNA labeling kit (Boehringer Mannheim Biocbemicals, Indianapolis, IN). Inserts were excised from recombinant k phages by EcoRI digestion and subcloned into the EcoRI site of pBluescript II KS (Stratagene, La Jolla, CA). Plasmid DNAs were prepared using a Wizard Miniprep kit (Promega Corp., Madison, WI). Nested deletion templates for sequence analysis were generated using the Erase-A-Base kit (Promega Corp.). Double-stranded DNA sequencing was carried out for both strands by the dideoxynuclvotide method.
For expression studies, we have also constructed an NH2 terminally myc epitope-tagged version of p40 M°15 in the eukaryotic expression plasmid pRc/CMV (Invitrogen, San Diego, CA). The protein produced by this pinsmid contains the peptide MEQKLISEEDLNMNF fused in frame to the initiator methionine of p40MO15; thus, the anti-myc epitope mAb 9El0 (Evan et al., 1985) could be used for its detection. In vitro translation of p40 M°15 or myc epitope-tagged p40 M°'5 (constructed in pBluescript; Schmidt-Zachmann and , was performed by priming the coupled transcription-translation reticulocyte lysate system (Promega Corp.) with the corresponding Bluescript plasmids, as described by the manufacturer. using super medium (25 g bacto-tryptone, 15 g bacto-yeast extract, and 5 g NaCl/liter). Recombinant protein was purified under denaturing conditions as described by the manufacturer (QIAGEN Inc.). Poly-His-tagged MO15 protein was finally eluted in buffer E (8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 4.5) and stored at -20°C.
Rabbit antibodies against bacterially expressed MOI5 protein were produced as follows: intramuscular injections were carried out every 4 wk with 280 t~g of purified recombinant MO15 protein in buffer E emulsified in Freund's adjuvant. Complete adjuvant was used for the first injection, incomplete adjuvant for all subsequent injections. The polyhistidine-tagged MO15 fusion protein was also used to raise mAbs in mouse, as described previously (Lukas et al., 1992). The mAb MO-I.I used throughout this study is an IgG2b.

AJ~inity Purification of the Rabbit Polyclonal Antibody and Preparation of lmmunoglobulin Beads
For all experiments involving rabbit (R30) polyclonal anti-MOl5 antibodies, these were affinity purified as described by Harlow and Lane (1988). Briefly, purified recombinant MO15 protein was subjected to polyacrylamide gel electrophoresis, blotted onto a nitrocellulose membrane, and a nitrocellulose strip containing the MO15 protein was excised. After incubation with R30 anti-MOl5 serum, this strip was extensively washed with PBS. Immunoglobulins were eluted with 0.1 M glycine, pH 2.5, neutralized with an equal volume of I M Tris-HCl, pH 8.0, and stored at 4°C. For coupling of antibodies to a solid support, affinity-purified rabbit immunoglobulins or hybridoma supernatants were incubated for 2 h at 4°C with protein A or protein G-Sepharose, respectively, equilibrated in 0.5 M Tris-HC1, pH 7.5. Immunoglobulin-protein A/G-Sepharose beads were washed with PBS and stored at 4°C.
Kinase assays were carried out in a total volume of 50 t~l of the above assay buffer, supplemented with 4 mM ATP, 10 t~Ci of [3,-32p]ATP (Amersham Corp., Arlington Heights, IL), and 400 t~g/ml of purified GST-cdk2 protein, as a substrate. After 30 min at 32°C, the reaction was stopped with 50 t~l of 3 × gel sample buffer. Phosphorylation of GST-cdk2 protein was analyzed by SDS-PAGE and autoradiography.

Miscellaneous Techniques
HeLa cells were synchronized either by centrifugal elutriation (Draetta and Beach, 1988;Golsteyn et al., 1994) or using drug arrest-release protocols (Krek and Nigg, 1991a). The procedures for immunoblotting, indirect immunofluorescence microscopy, and enucleation have also been described previously Nigg, 1991a, 1991b;Krek et al., 1992b). Onedimensional peptide mapping with V8 protease was carried out according to Cleveland (1983), and two-dimensional tryptic phosphopeptide mapping and phosphoamino acid analysis was conducted according to Boyle et al. (1991).

Cloning of a Human MO15 cDNA and Identification of p40 M°15 in Different 7))pes of Human Cells
By screening a human placenta library with a PCR fragment isolated previously (originally termed HsPK31; Schultz and , 13 overlapping cDNAs coding for human p40 M°15 were isolated. The nucleotide sequence of the longest cDNA (1,304 bp) and the corresponding translation product are shown in Fig. 1. The predicted human MO15 protein is composed of 346 amino acids, has an Mr of 39,123, and displays 86% identity with p40 M°15 from Xenopus laevis ( Fig. 1). A histidine-tagged fragment encompassing the COOH-terminal 214 amino acids of p40 M°15 was expressed in E. coli, and the purified fusion protein was used to raise both polyclonal (rabbit) and monoclonal (mouse) antibodies. The specificity of these antibodies is illustrated in Fig. 2. As determined by immunoblotting, the R30 rabbit antibodies detected a single 40-kD protein in whole-cell extracts prepared from either human cancer-derived cell lines (HeLa and UMSCC2; Fig. 2, lanes 3 and 4) or cultures of nontransformed fibroblasts (IMR90 and WI38; Fig. 2, lanes 5 and 6). Interestingly, when comparable amounts of total protein were loaded in each lane (see the Coomassie blue staining profiles shown in Fig. 2, lanes 7-10), both cancer-derived cell lines expressed three to five times higher levels of p40 M°15 than the nontransformed cell types (Fig. 2, lanes 3 and 4 vs lanes 5 and 6). The immunoreactive 40-kD protein comigrated exactly with the product obtained after in vitro translation of MO15 eDNA-derived RNA in a reticulocyte lysate (Fig. 2, lane 2). As expected, translation of a myc-GGG CGGGG TTGGAGGCTTTAAGGTAG C TTTAAATTCG TG TTG TCCTGGG AGCTCG CCCTT TTCGG CTC-G AG TCGGGCTTTACGGCGC CGGATGG C TCTGGACG TGAAGTCTCGGGCAAAG TGG ACAACATTTTACTACTGAGGGAAATAGCCAAAAAGGCAAATAATGG AAAAATAG TAA ACATTAAGTAAATGCTGTAGAAGTGAGTTTGTAAATATTCTACACATGTAAAATATGTAA AACTATG GG TTA~'FI'±'I'ATTAAATG TATTTTAAAAAAAAAAAAA tagged MO15 RNA yielded a slightly larger product of 42 kD (lane/). Several mAbs, e.g., MO-I.1 (Fig. 2, lane 1/), were also found to be highly specific for the 40-kD MO15 protein.

Expression of p40 uo'J Is Invariant throughout the Somatic Cell Cycle
To determine the steady-state levels of p40 M°~5 during the cell cycle, HeLa cells were synchronized by centrifugal elutriation (Fig. 3). Aliquots of each sample, containing equal amounts of total protein, were then analyzed by immunoblotting using antibodies specific for p40 uo'5. For control, the same samples were also probed with anti-cyclin A and anti-PSTAIRE antibodies (a convenient tool to detect p34 ~a and other cdk family members; Yamashita et al., 1991), and a population of exponentially growing cells was  3, lanes I and 2), but it accumulated as cells progressed towards S and G2 phases (Fig. 3, lanes 3-6); in contrast, the PSTAIRE-positive signal (Fig. 3, bottom panel) was essentially invariant throughout the cell cycle, except for the appearance of a slower migrating PSTAIRE protein in S-and G2/M-phase cells, which is characteristic of tyrosine-phosphorylated p34 ~'2 (Solomon et al., 1992).

p4Ouol~-associated CAK Activity is Constant throughout the Cell Cycle
To assay for CAK activity associated with p40 M°lS, the protein was immunoprecipitated from asynchronous HeLa cells, using either mAb MO-I.1 or the R30 rabbit anti-MO15 antibody. After extensive washing, the immunoprecipitates were incubated with [3,-3~P]ATP and bacterially expressed GST-cdk2 as a substrate, as described by Poon et al. (1993). As shown in Fig. 4 A, GST-cdk2 was readily phosphorylated when incubated with either type of MO15 immunoprecipitate (Fig. 4, A, lanes 2 and 3), but not when exposed to mock immunoprecipitates carried out with protein G-or protein A-Sepharose beads, respectively (Fig. 4, A, lanes I and 4). Tryptic phosphopeptide mapping and phosphoaminoacid analyses showed that GST-cdk2 was phosphorylated exclusively on threonine and on a single tryptic peptide (Fig. 4 B). These results are in full agreement with previous data showing that phosphorylation of GST-cdk2 by Xenopus CAK occurs exclusively on threonine 160 (Poon et al., 1993). Using the above assay, we next investigated p40 M°lSassociated CAK activity in HeLa cells that had been synchronized at various stages of the cell cycle. Cells were either arrested in mitosis using nocodazole, or at the G1/S boundary using aphidicoline, as described previously (Krek and Nigg, 1991a). Then, they were released from these blocks for various lengths of time, and p40 M°15 was immunoprecipitated. For each sample, the amount of p40 M°~5 recovered was determined by immunoblotting, and p40M°lS-associated CAK activity was assayed as described above. In parallel, aliquots of cells were used for FACS ® analyses to determine the proportions of cells at different stages of the cell cycle. In a first experiment, cells were released for comparatively short time intervals from a nocodazole-induced block in prometaphase (Fig. 5 A). Under these conditions, cells inactivate mitotic cdc2 kinase through destruction of B-type cyclins and complete mitosis; indeed, as assessed by microscopic monitoring of cell spreading to the substratum, . For control, mock immunoprecipitations were carried out using protein G beads (lane 1) or protein A beads (lane 4). Washed immune complexes were then assayed for kinase activity using GST-edk2 as a substrate, as described previously (Poon et al., 1993). (B) Phosphorylated GST-cdk2 protein (obtained as described in A) was subjected to two-dimensional tryptic phosphopeptide mapping and phosphoamino acid analysis (Boyle et al., 1991). O marks the site of sample application, and the inset shows the result of the phosphoamino acid analysis (S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine).  Figure 5. p40U°~5-associated CAK activity is constant throughout the cell cycle. (A) HeLa cells were arrested in prometaphase with nocodazole. Before release from the nocodazole block (0) or at the indicated time intervals after the release, p40 u°t5 was immunoprecipitated from equal numbers of cells, using mAb MO-I.1. Each sample was assayed for CAK activity, using GST-cdk2 as a substrate (upper part), and recovery of p40 u°15 was controlled by immunoblotting using the R30 antibody (lower part ). (B) HeLa cells were arrested either in prometaphase with nocodazole, or at the G1/S boundary with aphidicolin, exactly as described previously (Krek and Nigg, 1991a). Before release from these blocks, or at the indicated time intervals after the release, p40 u°~5 was immunoprecipirated and its recovery and associated CAK activity were determined as described above (A). In parallel, aliquots of each sample were subjected to FACS ® analyses. The specific CAK activities (i.e., phosphate incorporation into GST-cdk2, relative to the amounts p40 M°~ protein present in the immune complexes) were then determined by densitometric scanning of the data, and results from two independent experiments are shown in the form of a histogram. Below, the results of the FACS ® analyses are summarized: numbers to the left refer to experiment 1 (hatched bars), numbers to the fight to experiment 2 (open bars).
daughter cells reentered G1 phase by "~4-6 h (data not shown). Fig. 5 A illustrates that the level of p40 M°15, as well as its associated CAK activity, remained virtually constant through mitosis and reentry into the subsequent G1 phase. Fig. 5, B and C, summarize the results obtained when similar drug-arrest/release experiments, using aphidicolin and nocodazole, respectively, were carried out over the entire cell cycle. Again, the specific activity of p40M°lS-associated CAK was constant at all stages of the cell cycle. Yet, previous studies based on a virtually identical experimental approach had revealed striking cell cycle-dependent changes in the ac-  (D) p40 M°lS, p34, and p32 were subjected to one-dimensional tivity of p34 cd¢2 (Krek and Nigg, 1991a). We are forced to conclude from these results that progression through the somatic cell cycle is not accompanied by any major changes in p40Mols-associated CAK activity, at least as assayed in immunoprecipitates (see Discussion). In this context, we note also that p40 M°15 is a phosphoprotein in vivo (Tassan, J.-P., and E. A. Nigg, unpublished results). Yet, CAK activity associated with p40 M°~5 immunoprecipitates was unaffected by the presence or absence of phosphatase inhibitors in lysis and washing buffers (data not shown).

Idenfffication of Two Candidate Complex Partners of p40 M°~
In starfish and Xenopus eggs, p40 M°~5 appears to associate with other proteins to form active CAK (Fesquet et al., 1993;Poon et al., 1993). To determine whether p40 M°t5 forms complexes with other proteins in somatic cells, a gel filtration experiment was carried out (Fig. 6). HeLa cells were lysed in the presence of NP-40 detergent, and proteins were size fractionated on a Superose 6 column. Then, each fraction was subjected to immunoprecipitation with anti-MO15 antibodies, and immunoprecipitates were probed by immunoblotting for the abundance of p40 M°15 (Fig. 6, upper panel). In parallel, they were assayed for CAK activity, using GST-cdk2 as a substrate (Fig. 6, lower panel). This experiment shows that the bulk of p40 u°ls did not occur as a monomer but, instead, migrated in complexes with an apparent molecular mass of "~60-120 kD. Interestingly, the CAK activity of each fraction paralleled the abundance of p40 M°tS, indicating that, at least under the lysis conditions used here, the bulk of the p40M°~5-associated kinase was recovered in an active state.
To search for cellular proteins that might specifically associate with p40 M°tS, immunoprecipitation experiments were carried out. As shown in Fig. 7 A, both polyclonal (lane 3) and monoclonal (lane 4) anti-MO15 antibodies specifically precipitated two polypeptides with molecular masses of 34 and 32 kD, in addition to p40 Mo~s. A third protein of ,o57 kD was also precipitated, particularly when using the rabbit anti-MO15 antibodies (Fig. 7 A, lane 3; arrowhead); however, recovery of this 57-kD protein was variable (Fig. 7, A vs B), and its identity has not been further investigated. Importantly, none of these proteins was precipitated by protein A-or protein G-Sepharose (Fig. 7 A, lanes 2 and 5). Also, when immune complexes were boiled and then subjected to a second immunoprecipitation with anti-MO15 antibodies, only p40 M°~5 was recovered (Fig. 7 B, lane 2 vs lane 1). This indicates that none of the coprecipitating proteins were recognized directly by anti-MO15 antibodies.
As indicated by peptide mapping with V8 protease, the 34and 32-kD proteins appear to be structurally distinct from each other, as well as from p40 M°15 (Fig. 7 D). Also, the 34/32-kD proteins were not recognized by anti-PSTAIRE antibodies, indicating that they are not identical with p34 ~d~2 or p33 °du (data not shown), a conclusion supported further by microsequence analysis (Tassan, J.-P., S. Frutiger, G. J. Hughes, and E. A. Nigg, unpublished results). Complexes peptide mapping (Cleveland, 1983) using increasing amounts of V8 protease as indicated. Digestion products were analyzed by electrophoresis on a 15% SDS-polyacrylamide gel.
Tassan et al. Cell Cycle Regulation of Human p4OMO15/CAK by Coomassie blue (Fig. 7 B, lane 3), indicating that all three proteins coimmunoprecipitate in similarly large amounts and may, thus, form a near-stoichiometric complex.
To determine whether p40 ~°~s might undergo cell cycle-dependent complex formation, immunoprecipitation experiments were carried out from HeLa cells that had been synchronized by drug-arrest/release protocols and pulse labeled with [35S]methionine/cysteine at different stages of the cell cycle (Krek and Nigg, 1991a). As shown in Fig. 8, no significant cell cycle-dependent changes could be detected in either the accumulated levels of p40 ~°15 or its association with the 34/32-kD proteins. Figure 8. Association of p40 M°~5 with p34/p32 is constant during the cell cycle. Immunoprecipitations with rabbit R30 anti-MO15 antibodies were carried out from HeLa cells that had been labeled for 4 h with [ssS]methionine/cysteine (100 #Ci/ml). Lysates were prepared from cells representing progressive stages of the cell cycle (Krek and Nigg, 1991a). Lanes 2 and 3, Exponentially growing cells (lane 2 shows a protein A-Sepharose mock immunoprecipitation for control); lane 4, cells predominantly in G1 phase (5 h after a release from a nocodazole block in prometaphase); lane 5, cells at G1/S phase (arrested with aphidicolin); lane 6, cells in S and G2 phase (5 h after release from the aphidicolin block); lane 7, cells in M phase (arrested with nocodazole). The contribution of G1, S, and G2/M phase cells to each population was determined by FACS ® analysis (using duplicate plates), and the results are indicated below each lane. between t>40 M°t5 and the 34/32-kD polypeptides could be detected, not only in HeLa cells, but also in UMSCC2 carcinoma ceils, and in two different types of nonimmortalized human fibroblasts (Fig. 7 C). Remarkably, p40 M°~5 and its putative 34/32-kD complex partners could be stained readily

Human p40 M°15 Protein Localizes to the Cell Nucleus
Indirect immunofluorescence microscopy was used to examined the subcellular localization of p40 M°~s (Fig. 9). After incubation with R30 anti-MO15 antibodies, interphase HeLa ceils displayed a strong staining of nuclei, with negligible staining of the cytoplasm (Fig. 9, a, f, and g). Uniform results were obtained for all cells in exponentially growing cultures, indicating that p40 M°ts does not undergo any significant redistributions during interphase of the cell cycle. In mitotic cells, p40 M°~5 was diffusely distributed, with no evidence for a specific association with either condensed chromosomes or spindle microtubules (Fig. 9, c and f ) . Virtually identical results were obtained when analyzing nonimmortalized IMR90 human fibroblasts (Fig. 9 h) or when using monoclonal antibodies for detection of p40 M°l~ (Fig.  9 f). Also, results were indistinguishable regardless of whether ceils were fixed and permeabilized using paraformaldehyde detergent (Fig. 9, a, g, and h) or organic solvents (Fig. 9 f). Finally, when HeLa were transfected with a cDNA encoding a myc epitope-tagged version of p40 M°~5, anti-myc epitope antibodies also stained the nuclei of transfected ceils, indicating that the exogenously expressed MO15 protein localized to the same compartment as endogenous p40 M°15 (Fig. 9 i). Figure 9. Immunofluorescent localization of p40 M°15. Endogenous p40 ~°t5 was visualized by indirect immunofluorescence microscopy, using either R30 antibodies (a, c, f, and g) or mAb MO-I.1 (h). Both HeLa (a-e, f, and h) and IMR90 cells (g) were examined. Fixation-permeabilization was achieved using either paraformaldehyde-Trit on X-100 (a-e and g-i) or methanol/acetone (f), with virtually identical results. Finally, HeLa cells were transiently transfected with apCMV plasmid coding for a myc-epitopetagged p40 M°lS, and the subcellular localization of the ectopically expressed protein was visualized using the antimyc tag mAb 9El0 (i; arrowheads indicate untransfected ceils). Note the exclusive nuclear localization of p40 M°15 in interphase cells (a vs b; phase contrast). In mitotic cells (c-e), p40 M°15 was diffusely distributed and showed no obvious association with condensed chromosomes (d; DNA staining with Hoechst 33258; e; phase contrast). Bar in a, 10/~m. Exponentially growing HeLa ceils were enucleated as described previously for chicken DU249 cells (Krek et al., 1992b;Hermekes et al., 1993). Nuclear (N) and cytoplasmic (C) fractions, as well as total cellular lysates (T), were then analyzed by immunoblotting using antibodies against p40 M°~5. In parallel, appropriate antibodies were used to monitor the partitioning of lamin B2 (a marker for the nucleus) and tubulin (a marker for the cytoplasm).
To corroborate these immunocytochemical results by independent biochemical evidence, enucleation experiments were carried out. HeLa ceils were grown on round coverslips and then fractionated by centrifugation in the presence of cytochalasin D (Krek et al., 1992b;Hennekes et al., 1993). This procedure yields nuclei with some contamination by cytoplasm, and cytoplasts with a minimal contamination by intact cells (Gordon et al., 1981;Banerle and Baltimore, 1988). After separation of nuclei and cytoplasts, the partitioning of p40 M°ls was examined by immunoblotting. For control, the distributions of a nuclear marker (lamin Bz) and a cytoplasmic marker (tubulin) were assayed in parallel.
As shown in Fig. 10 (upper panel), p40 M°lS was recovered almost exclusively from the nuclear fraction; the minimal amount of p40 M°~s detectable in cytoplasts (9%) can readily be attributed to contamination by intact cells because a similar proportion (7 %) of nuclear lamin was also detectable in the cytoplasts (Fig. 10, middle panel). Conversely, tubulin was present predominantly in the cytoplasts, as expected ( Fig. 10, lower panel). Based on the results of both immunocytochemistry and biochemical analysis of fractionated ceils, we thus conclude that at all interphase stages of the cell cycle p40 M°~5 is a predominantly nuclear protein.

Discussion
The p34 ~2 protein kinase and structurally related cdks play a pivotal role in promoting cell cycle progression in all eukaryotes. In addition to cell cycle-dependent changes in the subunit composition of cdk/cyclin complexes, the reversible phosphorylation of p34 ~2 and the other cdks has been recognized as a major mechanism for regulating their activities (for references see Introduction). Since the phosphorylation state of cdks appears to be controlled by external signals and/or internal checkpoint controls, it is important to identify the kinases and phosphatases acting on cdks, and to determine how their activities are integrated with intracellular signal transduction pathways. Important progress in this field has been made recently with the identification of p40 M°t~ as the catalytic subunit of CAK, the enzyme responsible for phosphorylating p34 ~2 on Thrl61 (Fesquet et al., 1993;Pooh et al., 1993;Solomon et al., 1993). This enzyme is also able to phosphorylate a corresponding residue in p33 ~u and possibly in other cdks, but its precise target range has not yet been determined (for reviews see Draetta, 1993;Solomon, 1994).
In this study, we describe a molecular characterization and cell cycle analysis of human p40M°tVCAK. We have cloned, from a human placenta library, a cDNA coding for a 40-kD protein that displays 86% sequence identity to p40 M°~5 from Xenopus laevis. Independently, partial cDNAs for human and mouse p40 M°t5 have been isolated in other laboratories (Ershler et al., 1993;Canoe et al., 1993). Using a CAK assay described by Pooh et al. (1993), we further show that antibodies raised against bacterially expressed human p40 Mot5 readily immunoprecipitate CAK activity from cultured human cells. From these results, we conclude that we have isolated a functional homologue of the Xenopus MOI5 gene.
Highly specific poly-and monoclonal antibodies were then used to study the expression and subcellular localization of t>40 u°~5, as well as the activity and subunit composition of CAK, during the human somatic cell cycle.
Perhaps the most unexpected result of the present study is that no cell cycle-dependent changes could be detected in either the expression of MO15 protein or its associated CAK activity. Previous studies had failed to reveal any significant variations in CAK activity during the early embryonic development ofXenopus laevis (Solomon et al., 1992;Poon et al., 1993). However, because cell growth is uncoupled from cell division in these early cleavage embryos, the embryonic cell cycles lack bonafide G1 and G2 phases, and at least some checlq~int controls are not operational (for review see Murray and Hunt, 1993). It was clearly important, therefore, to study the expression and regulation of CAK activity during progression through a somatic cell cycle. On the basis of the results obtained here and results obtained independently by Pooh and colleagues (1994), it is difficult to escape the conclusion that CAK activity is constant not only in early embryonic cell cycles, but also in exponentially growing somatic cells. This conclusion, however, is subject to one important caveat: it is virtually impossible to exclude the possibility that p40 M°t5 might be regulated within living cells by rapidly reversible posttranslational modifications, or by labile interactions with other proteins. Such modifications or weakly interacting proteins might be lost during immunoprecipitation of p40 M°tS, and changes in CAK activity might thus go undetected in standard in vitro assays. In spite of this caveat, however, the available evidence suggests that the phosphorylation state of Thrl61 in p34 ~2 (and of the corresponding residues in other cdks) may be regulated primarily, and perhaps exclusively, at the level of the substrate and/or the corresponding phosphatase(s). This implies that cdks are likely to be phosphorylated by a constitutively active CAK as soon as they become available in a suitable conformation.
We emphasize that our study focused on CAK activity in exponentially growing cells. We have not described results on CAK expression or activity in Go arrested cells, but Poon et al. (1994) recently found that p40 M°~s is absent from serum-starved mouse 3T3 cells. This result is not unexpected, and it falls in line with the observation that cdk-cyclin complexes, the targets of CAK, are themselves absent from Go-arrested cells (e.g., Lee et al., 1988;Matsushime et al., 1994). Also, we emphasize that the results described here do not exclude that CAK may be responsive to external growth regulatory signals, or to internal checkpoint controls monl-toting the quality of cell cycle progression. However, they do strongly indicate that CAK activity is not subject to regulation as part of a program intrinsic to every cell cycle.
Immunoffuorescence microscopy, as well as biochemical fractionation experiments, unequivocally demonstrate that p40 uo~ is nuclear throughout interphase of the cell cycle. This result also constitutes a surprise. Although several potential targets of CAK do occur within the nucleus, e.g., the complexes between cdks and cyclin A (Pines and Hunter, 1991b;Girard et al., 1991;Pagano et al., 1992;Zindy et al., 1992;Maridor et al., 1993), cyclin D1 (Baldin et al., 1993), or cyclin E (Lehner, C. E, personal communication), the virtually exclusive nuclear localization of p40 u°~5 is difficult to reconcile with the cytoplasmic localization of some of the purported CAK substrates, notably the p34~cVcyclin B complexes (Pines and Hunter, 1991b;Gallant and Nigg, 1992;Ookata et al:, 1992). These complexes are presumed to be phosphorylated in the cytoplasm, before they translocate to the nucleus at the onset of mitosis. To resolve this apparent paradox, one has to argue that either cytoplasmic cdk/cyclin complexes are substrates for CAKs containing as yet unidentified catalytic subunits distinct from p40 M°'5, or, alternatively, that some of the protein complexes must cross the nuclear envelope. Considering that nucleocytoplasmic interactions are much more dynamic than suggested by the analyses of steady-state protein distributions (for discussion see , it is possible that p40~°~VCAK or p34~2/cyclin B complexes, or both, might shuttle between nucleus and cytoplasm (Schmidt-Zachmann et al., 1993). Furthermore, considering that p34 ~c2 occurs in excess over cyclins, it is attractive to speculate that p34 cdc2 protein might be phosphorylated by CAK within the nucleus, and then be captured by a B-type cyclin after its diffusion to the cytoplasm. It appears doubtful that free monomeric p34 ca2 could efficiently be phosphorylated by CAK (Solomon, 1994), but one could invoke a model in which a chaperonelike protein might bind to nuclear p34 ¢ac2, thereby conferring a conformation appropriate for the phosphorylation of Thrl61. The fact that a GST-cdk2 fusion protein can readily serve as a substrate for CAK (Poon et al., 1993, and this study) illustrates that cdk phosphorylation can occur in the absence of cyclin binding. Perhaps the GST fusion moiety confers to cdk2 a conformation that would normally result from the binding of a cyclin or a (hypothetical) chaperone.
Previous work strongly suggests that the starfish oocyte MO15 protein may form a stoichiometric complex with a second, somewhat larger polypeptide (Fesquet et al., 1993). Although no information has yet been reported on the molecular identity of this putative regulatory subunit, there is additional evidence to support the idea that CA/( may be a multiprotein complex. Gel filtration of starfish and Xenopus oocyte extracts revealed two complexes containing MO15 protein, only one of which, migrating at 180-220 kD, displayed the bulk of the CAK activity (Solomon et al., 1992;Poon et al., 1993). Also, bacterially expressed Xenopus MO15 protein was reported to be inactive unless it was incubated with cellular extracts, suggesting that either binding of another protein or a posttranslational modification of p40 M°" was required for kinase activation (Poon et al., 1993). Given the structural similarity between p40 M°~5 and cdks (Shuttleworth et al., 1990), it is tempting to speculate that p40 M°t5 might require a cyclin-like partner. It is per-haps significant, therefore, that the closest relative of p40 u°15 in S. cerevisiae, the product of the KIN28 gene, has recently been shown to associate with a cyclin-like protein (Valay et al., 1993). However, as yet, there is no direct evidence for a functional homology between the KIN28 and the MO15 gene products.
In this study, we provide evidence for the existence of a CAK multiprotein complex in somatic human cells. As determined by fractionation on a Superose 6 column, both p40 M°15 protein and CAK activity eluted as a broad peak with an apparent molecular mass of ,~60-120 kD. In direct support of a multiprotein CAK complex, immunoprecipitation of p40 M°15 from human cells allowed the identification of two potential complex partners. Using two different antibody preparations, two proteins of 34 and 32 kD were found to coimmunoprecipitate invariably with p40 M°~5 and CAK activity from all cell types analyzed and at all stages of the cell cycle. Binding of these proteins to p40 M°~5 appeared to be of high affinity because the complexes remained intact in the presence of mixed detergents (1% NP-40, 0.5 % deoxycholate, 0.1% SDS; see Fig. 7), as well as in high ionic strength solutions (2 M NaCI; Tassan, J.-P., and E. A. Nigg, unpublished results). Furthermore, all three coimmunoprecipitated proteins could readily be visualized in similar quantities by Coomassie blue staining, indicating that they may form a heterotrimeric complex. Alternatively, it is possible that our antibodies immunoprecipitate a mixture of several complexes; one such complex might then contain p40 M°~5 and only the 34-kD protein, another p40 M°~s associated only with the 32-kD protein. Whatever the precise stoichiometry of these complexes, we note that human CAK is abundant in somatic cells, unlike CAK in Xenopus eggs : on the basis of partial purification experiments, we estimate that p40 M°t5 and its two putative complex partners may each be present at ~1-5 x 1@ copies per HeLa cell, and at only three to five times lower levels in nontransformed human fibroblasts.
At present, we have no definitive information on the molecular identity of the 34-and 32-kD proteins. Preliminary results from microsequence analyses did not reveal any significant similarities to protein sequences presently available in databases, but they confirm that the two proteins are distinct from each other and from p40 ~°t5 (Tassan, J.-P., S. Frutiger, G. J. Hughes, and E. A. Nigg, unpublished resuits). Also, we note that the two proteins identified here differ in molecular weight from the single putative MO15 complex partner purified from starfish oocytes (Fesquet et al., 1993). Clearly, the molecular cloning of these putative p40 Mo15 partners will be indispensable for a better understanding of CAK function in different cell types.