THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 42, Issue of October 20, pp. 32578 –32584, 2000 Printed in U.S.A.

The Effects of Changing the Site of Activating Phosphorylation in CDK2 from Threonine to Serine* Received for publication, April 14, 2000, and in revised form, July 20, 2000 Published, JBC Papers in Press, August 7, 2000, DOI 10.1074/jbc.M003212200

Philipp Kaldis‡, Aiyang Cheng, and Mark J. Solomon From the Yale University School of Medicine, Department of Molecular Biophysics and Biochemistry, New Haven, Connecticut 06520-8114

Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue. This essential phosphorylation is carried out by the CDK-activating kinase (CAK). Here we examine the effects of replacing this threonine residue in human CDK2 by serine. We found that cyclin A bound equally well to wild-type CDK2 (CDK2Thr-160) or to the mutant CDK2 (CDK2Ser-160). In the absence of activating phosphorylation, CDK2Ser-160-cyclin A complexes were more active than wild-type CDK2Thr-160-cyclin A complexes. In contrast, following activating phosphorylation, CDK2Ser-160-cyclin A complexes were less active than phosphorylated CDK2Thr-160-cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2Ser-160. The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity. Interestingly, the CDK2Ser-160 mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II. CDK2Ser-160 was efficiently phosphorylated by CAKs, both human p40MO15(CDK7)-cyclin H and budding yeast Cak1p. In fact, the kcat values for phosphorylation of CDK2Ser-160 were significantly higher than for phosphorylation of CDK2Thr-160, indicating that CDK2Ser-160 is actually phosphorylated more efficiently than wild-type CDK2. In contrast, dephosphorylation proceeded more slowly with CDK2Ser-160 than with wild-type CDK2, either in HeLa cell extract or by purified PP2C␤. Combined with the more efficient phosphorylation of CDK2Ser-160 by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins.

Cyclin-dependent kinases (CDKs),1 a subfamily of protein kinases, promote cell cycle progression. In mammals, nine different CDKs (CDK1 to CDK9; CDK1 is better known as CDC2) have been identified (for a review, see Ref. 1). The activity of

* This work was supported by National Institutes of Health Grant GM47830 (to M. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: NCI-Frederick Cancer Research and Development Center, Regulation of Cell Growth Laboratory, Bldg. 560, W. 7th St., Frederick, MD 21702-1201. Tel.: 301846-1988; Fax: 301-846-1666; E-mail: [email protected]. 1 The abbreviations used are: CDK, cyclin-dependent kinase; GST, glutathione S-transferase; DTT, dithiothreitol; CAK, CDK-activating kinase; PAGE, polyacrylamide gel electrophoresis; CTD, C-terminal domain.

CDKs is regulated on various levels including binding of proteins (cyclins, inhibitors, and assembly factors), protein degradation, transcriptional control, localization, and multiple phosphorylations (2–5). This leads to timely regulation of CDK activity, allowing progression from one cell cycle phase to the next. CDKs are regulated by both inhibitory and activating phosphorylations. Threonine 14 and tyrosine 15 (in human CDK2) were identified as inhibitory phosphorylation sites that are phosphorylated by WEE1-like kinases and dephosphorylated by the CDC25 dual specificity phosphatases. For full activation, the cell cycle CDKs need to bind a cyclin and to be phosphorylated on a conserved threonine residue, located in the T-loop (Thr-160 in CDK2). Activating phosphorylation is essential for CDK activity in vitro (6, 7) and for growth of yeast cells (8, 9). Activating phosphorylation is carried out by the CDK-activating kinase (CAK; for a review, see Ref. 10), an enzyme that, except in budding yeast, is composed of a catalytic subunit, p40MO15 (see Refs. 11–14; also called CDK7); a regulatory subunit, cyclin H (15, 16); and an assembly factor, MAT1 (17–19). In vitro, MO15 phosphorylates CDC2 (11, 12), CDK2 (11–13, 15, 20, 21), CDK3 (22), CDK4 (23–25), and CDK6 (21, 26, 27). Budding yeast CAK, Cak1p, is very different from MO15type CAKs. Cak1p is active as a monomer and is only distantly related to CDKs (28 –30). Cak1p is an essential gene product that phosphorylates and activates Cdc28p (the major CDK in budding yeast, a homolog of CDC2) in vivo (28, 29). In vitro, Cak1p can phosphorylate Cdc28p (28 –31), CDC2 (32), CDK2 (21, 28 –30), CDK6 (21), and Kin28p (33). Activating phosphorylation is reversed by type 2C protein phosphatases (34). Specifically, budding yeast Ptc2p and Ptc3p dephosphorylate Cdc28p in vivo. Deletion of the corresponding genes partially rescues a cak1 temperature-sensitive mutant at restrictive temperature, and overexpression renders a cak1 mutant inviable at semirestrictive temperature (34), indicating that Ptc2p and Ptc3p are the physiological phosphatases that dephosphorylate the activating threonine in Cdc28p. PP2Cs are also responsible for dephosphorylating human CDK2 in HeLa cell extracts (34). Human CDK2 was identified by complementation of cdc2 mutants in Schizosaccharomyces pombe (35) and cdc28 mutants in Saccharomyces cerevisiae (36, 37), indicating that CDK2 can function in all phases of the yeast cell cycle and that it is a substrate for Cak1p. In human cells, CDK2 binds to cyclins A and E and promotes entry into and progression through S phase. The substrate specificity of CDKs is defined by at least two factors: (i) the intrinsic kinase specificity and (ii) a docking site in the cyclin subunit. Most CDKs phosphorylate serines and threonines within the general consensus sequence (S/T)PX(K/R), although there are strong differences among the various CDKs (38 – 41), and, in addition, many CDKs have not

32578

This paper is available on line at http://www.jbc.org

Effects of T160S Mutation in CDK2 been tested in such assays. Some CDK substrates such as Rb, p27, and CDC25 require docking to the cyclin subunit for efficient phosphorylation (42– 46), whereas other substrates are independent of docking. On a structural level, the understanding of substrate specificity has been helped by a recent co-crystal structure of a substrate peptide with CDK2-cyclin A3 (47). The basic residue at the P⫹3-position (with respect to the phosphorylation site) interacts with the phosphate on threonine 160, explaining both the requirement for phosphorylation on threonine 160 and the preference for a basic residue at the P⫹3-position of the substrate (47). It is curious that all cell cycle CDKs contain a threonine, and never a serine, at the site of activating phosphorylation. Replacement of Thr-160 by serine leads to serine-phosphorylated CDK2 in cell lines and indicates that this molecule is generally active (48). In this study, we investigated the detailed biochemical effects of replacing the activating threonine in human CDK2 by a serine residue. We found that this mutant bound cyclin A like wild-type CDK2 and displayed elevated activity in the absence of CAK phosphorylation. Following phosphorylation, CDK2Ser-160 had a normal affinity for ATP and histone H1 but was compromised in phosphorylation of Rb, the CTD peptide, and a synthetic peptide substrate GST-KSPRK. Furthermore, CDK2Ser-160 was more efficiently phosphorylated by CAKs and less efficiently dephosphorylated by PP2C than wildtype CDK2. EXPERIMENTAL PROCEDURES

Mutagenesis and Constructs—QuikChange mutagenesis (Stratagene, La Jolla, CA) was performed to introduce the T160S mutation into the GST-CDK2 sequence with the following primers (altered codons are underlined): T160S, 5⬘-GTT CGT ACT TAC TCC CAT GAG GTG GTG-3⬘ and 5⬘-CAC CAC CTC ATG GGA GTA AGT ACG AAC-3⬘. Human PP2C␤ (AJ005801) was amplified from HeLa cDNA with the following primers: 5⬘-GC CCC ATG GGT GCA TTT TTG GAT AAA CG-3⬘ (NcoI) and 5⬘-CCC CTC GAG TAT TTT TTC ACC ACT CAT CTT TG-3⬘ (XhoI). The PCR product was digested with NcoI–XhoI, cloned into pET28a, and sequenced. For in vitro transcription and translation, an NcoI–BamHI fragment containing cyclin A173– 432 was removed from GST-cyclin A173– 432 (PKB257; Ref. 34) and cloned into the ⌬13Tb vector (49) to create PKB382. Cyclin A173– 432 was transcribed and translated in vitro using the TNT coupled reticulocyte lysate system (Promega) according to the manufacturer’s instructions with 1 ␮Ci of [35S]methionine (PerkinElmer Life Sciences)/␮l of reaction volume. Protein Expression—GST-CDK2, GST-Rb605–928 (21), p40MO15-cyclin H (50), human PP2C␤ (34), GST-KSPRX substrates (40), and GSTcyclin A173– 432 (34) were purified as described. Plasmids for expression of GST-CDK2, GST-CDK2T160A, and GST-CDK2T160E in E. coli were kind gifts of J. Wade Harper. For expression of GST-Cak1p in insect cells, a BamHI (5⬘)–EcoRI (3⬘) fragment containing CAK1 (28) was cloned into pEG[KG] (51). From there, a SacI (5⬘)–EcoRI (3⬘) fragment encompassing GST-CAK1 was transferred to pBacPAK8 (CLONTECH). The virus was generated as described (52). Cyclin A Binding—GST-CDK2 (0, 0.05, 0.1, or 0.2 ␮g) was incubated for 2 h at room temperature with GST-Cak1p (0, 16.3, 32.6, or 65.1 ng) in the presence of either 5 mM ATP (“CAK-phosphorylated”) or 18 mM EDTA (“unphosphorylated”) in a total volume of 28.6 ␮l in buffer A (80 mM ␤-glycerophosphate (pH 7.3), 20 mM EGTA, 15 mM MgCl2, 10 mM DTT, 1 mg/ml ovalbumin, and 10⫻ protease inhibitors (1⫻ protease inhibitors corresponds to 10 ␮g/ml each of leupeptin, chymostatin, and pepstatin (Chemicon, Temecula, CA)). To each sample, 5 ␮l of in vitro translated, 35S-labeled cyclin A173– 432 was added, and incubation was continued for 1 h at room temperature. Each sample was diluted with 250 ␮l of buffer A containing 1% Nonidet P-40 followed by the addition of 25 ␮l of glutathione-agarose beads (Sigma; G4510). After rotation for 2 h at 4 °C, beads were washed five times with 400 ␮l of buffer A containing 1% Nonidet P-40, twice with 300 ␮l of buffer A, and resuspended in 20 ␮l of SDS-PAGE sample buffer. For the time course experiment (Fig. 1B), CDK2 and cyclin A were incubated for the indicated times and diluted with 475 ␮l of buffer A containing 1% Nonidet P-40 and 25 ␮l of glutathione-agarose beads. After rotation for 10 min at 4 °C, beads were treated as above. Proteins were electrophoresed in

32579

10% SDS-polyacrylamide gels followed by phosphor imaging and autoradiography. Kinetics of CDK2 Phosphorylation by CAKs—19 ng (0.273 pmol) of GST-Cak1p or 30.6 ng (0.4 pmol) of p40MO15-cyclin H was incubated with 5 ␮Ci of [␥-32P]ATP, 30 ␮M ATP, 20 mM MgCl2, and 6, 12.1, 24.2, 48.3, 96.7, 193.3, 386.6, 773.2, or 1546.4 nM GST-CDK2 in 16 ␮l of buffer A. Assays using GST-Cak1p were done in the absence of cyclin, whereas those using p40MO15-cyclin H contained 1.54 ␮g (1750 nM) of GST-cyclin A173– 432. After incubation for 30 min at room temperature, reactions were stopped by adding 7 ␮l of 5⫻ SDS-PAGE sample buffer. Preparation of CAK-phosphorylated CDK2—8.9 ␮g (0.15 nmol) of GST-CDK2 was incubated for 3 h at room temperature with 2.9 ␮g (41.5 pmol) of GST-Cak1p in the presence of 5 mM ATP in 100 ␮l of buffer B (100 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mg/ml ovalbumin, 10 mM DTT, 1⫻ protease inhibitors). Samples were desalted in Vivaspin 6-ml concentrators (30-kDa molecular mass cut-off, Vivascience Ltd., Lincoln, United Kingdom) and diluted to a final volume of 440 ␮l of 100 mM HEPES (pH 8), 1 mM DTT. Aliquots of this preparation are referred to here as CAK-phosphorylated CDK2. Activity of CDK2—Unphosphorylated or CAK-phosphorylated GSTCDK2 (0.1 ␮g) was incubated for 30 min at room temperature with GST-cyclin A173– 432 (0.01, 0.05, 0.1, or 1 ␮g) in 10 ␮l of buffer B. Samples were then added to 6 ␮l of histone H1 mix (1.5 ␮Ci of [␥-32P]ATP, 1.5 ␮g of histone H1 (Roche Molecular Biochemicals catalog no. 1004875; dissolved in 20 mM Tris (pH 7.4), 200 mM NaCl, 1 mM DTT, 1⫻ protease inhibitors)), 6 ␮l of CTD mix (3 ␮Ci of [␥-32P]ATP, 4 ␮g of CTD4 peptide ((YSPTSPS)4 dissolved in buffer B)), or 6 ␮l of Rb mix (1.5 ␮Ci of [␥-32P]ATP, 5 ␮l of GST-Rb605–928 (21) bound to glutathioneagarose beads in buffer B). All assays using the CTD peptide or unphosphorylated CDK2 contained 0.625 ␮M ATP, unless stated otherwise. All other assays contained 375 ␮M ATP. After incubation for 15 min at room temperature, the reactions were terminated by the addition of 7 ␮l of 5⫻ SDS-PAGE sample buffer. For the determination of substrate specificity using the GST-KSPRX substrates (Fig. 6A), 0.005 ␮g (0.08 pmol) of CAK-phosphorylated GSTCDK2 with 0.0064 ␮g (0.1 pmol) of GST-cyclin A173– 432 or 0.01 ␮g (0.17 pmol) of GST-CDK2Ser-160 with 0.0128 ␮g (0.21 pmol) of GST-cyclin A173– 432 was incubated with 50 ␮M substrate in 10 ␮l of buffer B. 6 ␮l of mix (5 ␮Ci of [␥-32P]ATP, 400 ␮M ATP, 20 mM MgCl2 in buffer B) was added. After incubation for 15 min at room temperature, reactions were stopped by adding 7 ␮l of 5⫻ SDS-PAGE sample buffer. To compare the activities of unphosphorylated and CAK-phosphorylated CDK2, 2-fold serial dilutions were prepared starting from 3.2 ␮g (53.3 pmol) of unphosphorylated GST-CDK2 with 3.2 ␮g (58.2 pmol) of GST-cyclin A173– 432 or from 0.1 ␮g (1.7 pmol) of CAK-phosphorylated CDK2 with 0.11 ␮g (2 pmol) of GST-cyclin A173– 432 in buffer B. 10 ␮l of each complex was added to 6 ␮l of histone H1 mix (8 ␮Ci of [␥-32P]ATP, 100 ␮M ATP, 1.5 ␮g of histone H1 in 20 mM Tris (pH 7.4), 200 mM NaCl, 1 mM DTT, 1⫻ protease inhibitors). After incubation for 15 min at room temperature, reactions were stopped by adding 7 ␮l of 5⫻ SDS-PAGE sample buffer. Km Determinations of CDK2 for ATP, Histone H1, and GST-KSPRK— 0.1 ␮g (1.7 pmol) of CAK-phosphorylated GST-CDK2 or 0.2 ␮g (3.3 pmol) of GST-CDK2Ser-160 was mixed with 0.12 ␮g (2.1 pmol) or 0.23 ␮g (4.1 pmol) of GST-cyclin A173– 432 in the presence of 15 ␮g (0.71 nmol) of histone H1 in 8 ␮l of buffer B. 8 ␮l of mix in buffer B was added containing 2.5, 5, 10, 20, 40, 80, 160, 320, or 640 ␮M ATP at a specific activity of 5 ␮Ci of [␥-32P]ATP/nmol. After incubation for 15 min, reactions were terminated by adding 7 ␮l of 5⫻ SDS-PAGE sample buffer. The data shown in Fig. 5A represent the averages of six independent sets of experiments. The same amounts of CDK2-cyclin A as above were mixed with 0.6, 1.2, 2.3, 4.7, 9.3, 18.6, 37.2, 74.4, and 148.8 ␮M histone H1 in 13 ␮l of buffer B. 3 ␮l of mix (8 ␮Ci of [␥-32P]ATP, 0.5 mM ATP, 20 mM MgCl2 in buffer B) was added. Reactions were terminated after 15 min at room temperature by adding 7 ␮l of SDS-PAGE sample buffer. The data shown in Fig. 5B represent the averages of 10 independent sets of experiments. 0.0025 ␮g (0.04 pmol) of CAK-phosphorylated GST-CDK2 with 0.0064 ␮g (0.1 pmol) of GST-cyclin A173– 432 or 0.015 ␮g (0.25 pmol) of GST-CDK2Ser-160 with 0.02 ␮g (0.32 pmol) of GST-cyclin A173– 432 was mixed with 11.2, 22.5, 44.9, 89.9, 179.75, 359.5, 719, 1438, and 2875.9 ␮M GST-KSPRK (Ref. 40; a kind gift of Jennifer Holmes) in 12 ␮l of buffer A. 4 ␮l of mix (5 ␮Ci of [␥-32P]ATP, 100 ␮M ATP, 20 mM MgCl2 in buffer A) was added. Reactions were terminated after incubation for 15 min at room temperature and treated as described above. The data shown in Fig. 6B represent the averages of four independent sets of experiments. Dephosphorylation of CDK2—Preparation of 32P-labeled substrates

32580

Effects of T160S Mutation in CDK2

FIG. 2. Activity of unphosphorylated CDK2-cyclin A. 0.1 ␮g of wild-type CDK2Thr-160 (Thr160, lanes 1– 4), mutant CDK2Ser-160 (Ser160, lanes 5– 8), mutant CDK2Ala-160 (Ala160, lanes 9 –12), and mutant CDK2Glu-160 (Glu160, lanes 13–16) was bound to cyclin A, and the activity toward histone H1 (A), CTD4 peptide (B), and GSTRb605–928 (C) was determined. The following GST-cyclin A173– 432 concentrations were used: 1 ␮g (lanes 1, 5, 9, and 13), 0.1 ␮g (lanes 2, 6, 10, and 14), 0.05 ␮g (lanes 3, 7, 11, and 15), and 0.01 ␮g (lanes 4, 8, 12, and 16). Kinase assays were analyzed by autoradiography following SDS-PAGE.

FIG. 1. Cyclin A binding to wild-type and mutant CDK2. Cyclin A was translated in vitro and labeled with [35S]methionine. A, after mixing with buffer (column 1), unphosphorylated wild-type CDK2Thr-160 (column 2), CAK-phosphorylated CDK2Thr-160 (column 3), unphosphorylated mutant CDK2Ser-160 (column 4), or CAK-phosphorylated CDK2Ser-160 (column 5), CDK2 was pulled down via its GST tag, and bound cyclin A was measured by phosphorimaging following SDSPAGE. Each column represents the average of three different CDK2 concentrations. Immunoblotting confirmed that identical amounts of CDK2 were precipitated (data not shown). B, time course of cyclin A binding to unphosphorylated wild-type CDK2Thr-160 (E) and unphosphorylated mutant CDK2Ser-160 (f). Each time point corresponds to the average of three independent measurements. and dephosphorylation reactions were done as described previously (34). RESULTS

CDK2Ser-160 has been shown previously to function normally, at least to a first approximation, following transfection into cell lines (48). However, in preliminary experiments we observed clear differences between CDK2Ser-160 and wild-type CDK2. We explored the causes of these observations. Since cyclin binding is an essential step in the activation of CDKs, we first compared the ability of cyclin A to bind to CDK2Ser-160 and to wild-type CDK2 (CDK2Thr-160). In vitro translated, radiolabeled cyclin A was incubated for 1 h with identical amounts of CDK2Ser-160 or CDK2Thr-160 that were unphosphorylated or prephosphorylated by Cak1p (see below). The CDK2 was precipitated, and the amount of bound cyclin A was determined by SDS-PAGE. Both unphosphorylated and prephosphorylated CDK2Ser-160 bound similar amounts of cyclin A as the corresponding forms of CDK2Thr-160 (Fig. 1A, lanes 2–5). Phosphorylation of either CDK2 protein stimulated the binding to cyclin A as has been reported previously for wild-type CDK2 (53, 54). We next examined cyclin binding after short incubation times to determine whether there was a kinetic difference in the ability of CDK2Thr-160 and CDK2Ser-160 to bind cyclin (Fig. 1B). Since activating phosphorylation strengthens the cyclinCDK2 interaction (Fig. 1A), we performed this experiment with unphosphorylated CDK2, reasoning that a kinetic effect would be more apparent. Cyclin binding occurred very quickly with half-maximal binding at 18 s for CDK2Thr-160 and at 13 s for

CDK2Ser-160. CDK2Thr-160 bound approximately 15% more cyclin A than CDK2Ser-160 (Fig. 1B), even when the time course was extended to 4 h (data not shown). We do not know if these differences are significant. Nevertheless, they were small and should not affect the outcome of the following experiments, which involve longer incubation times in the presence of excess cyclin. We tested the effects of threonine 160 mutations on the low but detectable kinase activity of unphosphorylated CDK2-cyclin A complexes (55, 56). Detection of this low activity required the use of a higher specific activity of radiolabeled ATP than was used in other experiments. In addition to wild-type CDK2 and CDK2Ser-160, we also tested a nonphosphorylatable mutant (threonine 160 replaced by alanine; Ala160) and a mutant designed to mimic constitutive phosphorylation (threonine 160 replaced by glutamic acid; Glu160). CDK2Ala-160 displayed lower activity than CDK2Thr-160 toward all substrates tested (histone H1 (Fig. 2A), CTD peptide (Fig. 2B), and Rb (Fig. 2C)), whereas CDK2Glu-160 displayed higher activity (Fig. 2, compare lanes 13–16 with lanes 1– 4). Interestingly, CDK2Ser-160 was more active than CDK2Thr-160, similar to CDK2Glu-160 (compare lanes 5– 8 with lanes 1– 4 and lanes 13–16). We next compared the abilities of two CAKs, budding yeast Cak1p (Fig. 3A) and human p40MO15-cyclin H (Fig. 3B), to phosphorylate CDK2Thr-160 and CDK2Ser-160. The assays were performed over a range of CDK2 concentrations so that we could derive the kinetic parameters Km (substrate concentration that yields half-maximal velocity) and kcat (maximal velocity at saturating substrate concentrations divided by the enzyme concentration). The Km(CDK2) for Cak1p was approximately 4-fold higher for CDK2Ser-160 than for CDKThr-160 (Table I). However, the Km(CDK2) for MO15 was very similar for both substrates. Interestingly, the kcat of each CAK using CDK2Ser-160 was approximately 2.5 times as high as when using CDK2Thr-160. We compared the activities of phosphorylated CDK2-cyclin A complexes toward histone H1 (Fig. 4A, upper panel), the CTD peptide (Fig. 4A, middle panel), and Rb (Fig. 4A, lower panel). The activities toward histone H1 were similar for CDK2Thr-160 and CDK2Ser-160 (Fig. 4A, upper panel). However, the activities of CDK2Ser-160 toward the CTD peptide and Rb were substantially lower than those of CDK2Thr-160 (Fig. 4A, middle and lower panels), suggesting that phosphorylation of the activating threonine selectively affects phosphorylation of different substrates. Since phosphorylation of the CTD peptide was car-

Effects of T160S Mutation in CDK2

FIG. 3. CAK phosphorylation of CDK2. Phosphorylation of wildtype CDK2Thr-160 (E) and mutant CDK2Ser-160 (f) by budding yeast Cak1p (A) and p40MO15-cyclin H (B) is shown. The following CDK2 concentrations were used: 6, 12.1, 24.2, 48.3, 96.7, 193.3, 386.6, 773.2, and 1546.4 nM. Assays using GST-Cak1p were done in the absence of cyclin, and those using p40MO15-cyclin H contained 1.54 ␮g (1750 nM) GST-cyclin A173– 432. The concentration of ATP was 30 ␮M. The data shown represent averages of six independent experiments in A and of four in B. The y axis corresponds to the reaction velocity (M䡠min⫺1) divided by the concentration of CAK (M). TABLE I Kinetic parameters for phosphorylation of CDK2 by Cak1p and MO15 p40 (CDK7) Km and kcat values for Cak1p and p40MO15(CDK7) using CDK2Thr-160 and CDK2Ser-160 as substrates were determined by standard MichaelisMenten analysis as shown in Fig. 3. Enzyme

Km(Thr-160) nM

kcat(Thr-160) min

⫺1

Km(Ser-160)

kcat(Ser-160)

nM

min⫺1

Cak1p

246

0.055

1270

0.222

p40MO15(CDK7)

297

0.022

365

0.100

ried out at a low ATP concentration, we repeated the phosphorylation of the CTD peptide under both low and high ATP concentrations and obtained essentially identical results (Fig. 4B). These findings indicate that the reduced phosphorylation of the CTD peptide by CDK2Ser-160 is not due to a lower affinity of CDK2Ser-160 for ATP relative to wild-type CDK2. We further analyzed the mutant CDK2Ser-160 by performing a kinetic analysis. The Km(ATP) for CDK2Ser-160 was 34% higher than for wild-type CDK2Thr-160 (Fig. 5A and Table II), and the Km(histone H1) was increased by 92% (Fig. 5B and Table II). These are modest differences considering the high concentrations of ATP and histone H1 used in Fig. 4; these differences cannot explain the lower activity of CDK2Ser-160 toward the CTD peptide and Rb (Fig. 3A). Interestingly, the kcat of CDK2Ser-160 toward histone H1 was only 33% that of wild-type CDK2Thr-160 (Fig. 5B, Table II). This low kcat does not reflect lower levels of phosphorylation by CAK, since CDK2Ser-160 is actually phosphorylated more efficiently than CDK2Thr-160 (see Fig. 3).

32581

FIG. 4. Phosphorylation of histone H1, Rb, and the CTD peptide by wild-type and mutant CDK2. A, CAK-phosphorylated wildtype CDK2Thr-160 (lanes 1– 4) and mutant CDK2Ser-160 (lanes 5– 8) with cyclin A was used to phosphorylate histone H1 (upper panel), the CTD4 peptide (middle panel), and GST-Rb605–928 (bottom panel). The following concentrations of cyclin A were used: 1 ␮g (lanes 1 and 5), 0.1 ␮g (lanes 2 and 6), 0.05 ␮g (lanes 3 and 7), and 0.01 ␮g (lanes 4 and 8). B, the CTD assay from A was repeated at the same ATP concentration (375 nM) and at a higher ATP concentration (375 ␮M). CDK2 was prephosphorylated by Cak1p at a concentration of 150 nM CDK2, ensuring that both CDK2Ser-160 and CDK2Thr-160 were phosphorylated to similar levels (see Fig. 3A).

Because of the striking effects of the T160S mutation on the phosphorylation of the CTD peptide and Rb by phosphorylated CDK2 (Fig. 4), we performed a kinetic analysis to determine whether this effect was due to an increase in Km, a decrease in kcat, or a combination of the two. We could not use Rb or the CTD peptide for this analysis. At high concentrations, Rb binds to CDK2-cyclin A and inhibits its activity (data not shown). The CTD peptide was phosphorylated too weakly at high ATP concentrations and at lower CTD peptide concentrations (data not shown). Therefore, we turned to a systematic panel of CDK substrates in which a substrate peptide (KSPRK) was fused to the C terminus of glutathione S-transferase (GST-KSPRK; Ref. 40). 19 such substrates, containing all possible amino acids except for isoleucine at the P⫹3-position with respect to the serine, were analyzed. The initial assays were carried out at low (below the Km) concentrations of substrates, yielding phosphorylation efficiencies relative to the phosphorylation of the KSPRK substrate by CDK2Thr-160, which was defined as 100%. CDK2Ser-160 phosphorylated the wild-type substrate, KSPRK, much more poorly than did CDK2Thr-160 (16.7 ⫾ 3%; Fig. 6A). This situation resembles our observations using the CTD peptide and Rb as substrates (see Fig. 4). Both CDK2Thr-160 and CDK2Ser-160 were highly sensitive to replacement at the P⫹3position, as has been observed previously for wild-type CDK2 (40). Interestingly, CDK2Ser-160 phosphorylated KSPRP about as well as KSPRK (19.6 ⫾ 13.1 versus 16.7 ⫾ 3%) and better than KSPRR (8.9 ⫾ 6.1%). In contrast, CDK2Thr-160 phosphorylated KSPRK much better than KSPRP and KSPRR (100% versus 16.6 ⫾ 9.1 and 17.6 ⫾ 5.3%, respectively). No clear pattern emerged from the other minor differences among the substrates. We performed a kinetic analysis of the GSTKSPRK substrate and found not only that the Km was lower for CDK2Thr-160 (661 versus 1815 ␮M) but also that the kcat was much higher (Fig. 6B; 1.48 versus 0.17 min⫺1). Thus, kcat/Km is 24-fold as high for CDK2Thr-160 as for CDK2Ser-160, explaining why KSPRK is not as good a substrate for CDK2Ser-160. The stimulation of CDK2 upon activating phosphorylation

32582

Effects of T160S Mutation in CDK2

FIG. 5. Kinetic analysis of CDK2 using histone H1 as a substrate. CAK-phosphorylated wild-type CDK2Thr-160 (E) and mutant CDK2Ser-160 (f) with cyclin A was used to phosphorylate histone H1 over a range of ATP (A) and histone H1 (B) concentrations. In A, the concentration of histone H1 was 45 ␮M. In B, the concentration of ATP was 500 ␮M. The y axis corresponds to the reaction velocity (M䡠min⫺1) divided by the concentration of CDK2 (M). TABLE II Kinetic parameters for phosphorylation of histone H1 by CDK2Thr-160 and CDK2Ser-160 Km values for CDK2Thr-160 and CDK2Ser-160 in complex with cyclin A using histone H1 as a substrate were determined by standard Michaelis-Menten analysis as shown in Fig. 5. Enzyme

Km(ATP)

kcat(ATP)

Km(histone H1)

kcat(histone H1)

␮M

min⫺1

␮M

min⫺1

CDK2Thr-160

33.6

3.9

4.8

2.1

CDK2Ser-160

45.0

1.7

9.2

0.7

was analyzed using dilution series of CDK2Thr-160 and CDK2Ser-160 to phosphorylate histone H1 (Fig. 7, A and B). As observed in Fig. 2, CDK2Thr-160 was less active than CDK2Ser160 when unphosphorylated (Fig. 7A) but more active than CDK2Ser-160 following phosphorylation by CAK (Fig. 7B). Specific activities calculated from the slopes in Fig. 7, A and B, indicated that wild-type CDK2Thr-160 was activated approximately 46.1-fold by CAK phosphorylation (Fig. 7C, compare columns 1 and 2), whereas mutant CDK2Ser-160 was stimulated only 9.8-fold (Fig. 7C, compare columns 3 and 4). Thus, the dynamic range of activation of mutant CDK2Ser-160 upon CAK phosphorylation was only 21% as much as for wild-type CDK2Thr-160, providing less room for regulation by phosphorylation. Dephosphorylation of the activating threonine in CDKs is carried out by type 2C phosphatases (34). Since PP2C has been reported to display a 20-fold preference for a phosphothreonine peptide substrate compared with an equivalent phosphoserine substrate (57), we tested if CDK2Ser-160 could serve as a substrate for PP2C. Mutant and wild-type CDK2 proteins were phosphorylated in vitro and added to a HeLa cell extract.

FIG. 6. Determination of the peptide substrate specificity of CDK2Ser-160. CAK-phosphorylated wild-type CDK2Thr-160 (open bars/ circles) and mutant CDK2Ser-160 (filled bars/squares) with cyclin A was used to phosphorylate 50 ␮M GST-KSPRX substrate (with the indicated amino acid in the P⫹3-position) in the presence of 400 ␮M ATP (A) and the indicated concentration of GST-KSPRK in the presence of 100 ␮M ATP (B). Each point represents the average of four independent measurements. The y axis corresponds to the reaction velocity (M䡠min⫺1) divided by the concentration of CDK2 (M).

Although both CDK2Thr-160 and CDK2Ser-160 were dephosphorylated in a Mg2⫹-dependent (Fig. 8A) and cyclin-inhibitable fashion (data not shown), mutant CDK2Ser-160 was dephosphorylated more slowly than wild-type CDK2Thr-160. To obtain quantitative data, the linear phase of this experiment was repeated three times (Fig. 8B). Mutant CDK2Ser-160 was dephosphorylated approximately 25% as rapidly as wild-type CDK2Thr-160 by a HeLa cell extract. A similar value (⬃17%) was obtained using purified recombinant human PP2C␤ (Fig. 8C), indicating that the slower dephosphorylation of CDK2Ser-160 was a direct effect and not due to other factors in the cell extract. DISCUSSION

Superficially, CDK2 seems to function well with a serine residue in place of threonine 160 (our results; Ref. 48). However, closer examination revealed effects on its activity, its phosphorylation by CAK, and on its dephosphorylation by PP2C. These results have implications for the functions of the activating threonine. Site-directed mutagenesis is widely used to elucidate the functions of proteins. Replacement of a threonine residue by serine is considered to be a “conservative” mutation that should

Effects of T160S Mutation in CDK2

FIG. 7. Quantitative effects of activating phosphorylation of CDK2Thr-160 and CDK2Ser-160. Dilutions of cyclin-bound unphosphorylated CDK2Thr-160 (A; E) or CDK2Ser-160 (A; f) and of CAK-phosphorylated CDK2Thr-160 (B; E) or CDK2Ser-160 (B; f) were used to phosphorylate histone H1. Activities were calculated from the linear fits in A and B and are depicted in C relative to the activity of phosphorylated CDK2Thr-160. Activation upon CAK phosphorylation was approximately 46.1-fold for CDK2Thr-160 and 9.8-fold for CDK2Ser-160.

mimic the functions of the replaced threonine. The serine residue can be phosphorylated and dephosphorylated like the threonine residue. Nevertheless, every mutation has effects, even if they are subtle. Our results demonstrate that the serine replacement of threonine 160 in human CDK2 displays a variety of defects that can only be detected by detailed quantitative analysis. Our results suggest that CDK2Ser-160 displays no general defects in catalytic activity, since the kinetic parameters for ATP and histone H1 were similar to those of wild-type CDK2 (Table II). Surprisingly, phosphorylated CDK2Ser-160 was compromised specifically in phosphorylating a CTD peptide, Rb (Fig. 4A), and the GST-KSPRK model substrate (Fig. 6). However, unphosphorylated CDK2Ser-160-cyclin A complexes phosphorylated the CTD peptide and Rb much better than did CDK2Thr-160-cyclin A (Fig. 2). Rb is one of the substrates that requires a docking site located on the cyclin subunit (RXL; Refs. 44 – 47 and 58), whereas phosphorylation of histone H1, presumably of the CTD peptide, and of GST-KSPRK are independent of the docking site. These results suggest that the T160S mutation selectively affects the phosphorylation of different substrates.

32583

FIG. 8. Dephosphorylation of CDK2. CAK-phosphorylated wildtype CDK2Thr-160 (lanes 1–3) and CDK2Ser-160 (lanes 4 – 6) were subjected to dephosphorylation by 2.5 ␮g of HeLa cell extract (A) in the presence (lanes 2 and 5) or absence (lanes 1, 3, 4, and 6) of 5 mM Mg2⫹. Samples were analyzed by autoradiography (AR) and immunoblotting (IB) with antibodies against CDK2 following SDS-PAGE. B, quantitation of three sets of experiments as in A considering only data from the linear phases of the reactions. C, dephosphorylation of 32P-labeled GSTCDK2Thr-160 and GST-CDK2Ser-160 by recombinant human PP2C␤. 50 ng of substrate was incubated for 20 min at room temperature with the indicated amounts of human PP2C␤. After termination of the reaction and separation by SDS-PAGE, the data were analyzed by phosphorimaging.

A recent report of the crystal structure of CDK2-cyclin A3 with a substrate peptide (47) indicates that the phosphate group on threonine 160 makes direct contact with the P⫹3 residue of the substrate peptide. Although the absence of a methyl side group on serine appears unlikely to have a significant effect on the distance between the substrate phosphorylation site and the phosphate on CDK2, it could have subtle but important effects on the orientation of this side chain. For instance, the added methyl group of threonine might constrain the movement of the side chain. This constraint might place the phosphate in an optimal orientation for efficient substrate binding. With a serine, however, the side chain might be less constrained and might spend less time in the optimal position. In contrast, the weak substrate phosphorylation by unphosphorylated CDK2Thr-160 might reflect a low time-averaged occupancy of the most favorable orientation in the absence of phosphorylation. By allowing greater motion, serine 160 might spend a greater fraction of time in a favorable orientation for substrate phosphorylation. Thus, greater motion of serine 160 might account for the paradoxical observation that CDK2Ser-160

32584

Effects of T160S Mutation in CDK2

has higher activity than CDK2Thr-160 when unphosphorylated but lower activity when phosphorylated. Such speculative ideas are, of course, difficult to test in the absence of crystal structures of phosphorylated and unphosphorylated CDK2Ser-160. Interestingly, threonine is also found as the catalytic residue in all protease subunits of the proteasome. Although replacement with serine compromises activity, no structural explanation of this surprising effect is apparent (59). A previous in vivo study confirms some of the predictions of our analysis. Mutation of threonine 160 to serine in CDK2 led to a ⬎20-fold increase of 32P labeling in vivo (48), supporting our finding that CDK2Ser-160 is phosphorylated more efficiently by CAK (Fig. 3) and dephosphorylated less efficiently by PP2C (Fig. 8). In addition, Gu et al. (48) found that the specific activity of CDK2Ser-160 was only 50% that of CDK2Thr-160, consistent with the reduced activity we see in vitro (Fig. 7C). Therefore, our findings reflect differences that can be observed in an in vivo situation. No phenotype was observed when CDK2Ser-160 was overexpressed up to 50-fold (48), excluding the possibility of a dramatic effect of CDK2Ser-160 function in vivo. Examination of more subtle effects will require replacing both copies of CDK2Thr-160 with CDK2Ser-160. Threonine is used as the site of activating phosphorylation in all known cell cycle CDKs from all species. Our results suggest the following possible explanations for this conservation: (i) CDK2Thr-160 has a broader substrate utilization than CDK2Ser-160; (ii) CDK2Thr-160 displays a greater dynamic range of activity upon phosphorylation than CDK2Ser-160; and (iii) the slower phosphorylation of CDK2Thr-160 by CAK (24-fold) combined with its faster dephosphorylation by PP2Cs (⬃4-fold) shifts the equilibrium toward unphosphorylated monomeric CDK2, which would prevent an immediate activation of CDK2 after cyclin binding. All of these changes could hinder the precise control of CDK2Ser-160 activity during the cell cycle. Acknowledgments—We thank Adrienne Natrillo for technical support. For discussion, support, and comments on the manuscript, we thank Janet Burton, Denis Ostapenko, Karen Ross, and Vasiliki Tsakraklides. GST-CDK2 (wild type, T160A, and T160E) clones were kindly provided by Wade Harper, p40MO15-cyclin H proteins were provided by Alicia Russo and Nikola Pavletich, and GST-KSPRX substrates were a kind gift of Jennifer Holmes. REFERENCES 1. Morgan, D. O. (1997) Annu. Rev. Cell Dev. Biol. 13, 261–291 2. Pines, J. (1995) Biochem. J. 308, 697–711 3. King, R. W., Deshaies, R. J., Peters, J.-M., and Kirschner, M. W. (1996) Science 274, 1652–1659 4. Sherr, C. J., and Roberts, J. M. (1995) Genes Dev. 9, 1149 –1163 5. Solomon, M. J., and Kaldis, P. (1998) in Results and Problems in Cell Differentiation (Pagano, M., ed) Vol. 22, pp. 79 –109, Springer, Heidelberg 6. Desai, D., Gu, Y., and Morgan, D. O. (1992) Mol. Biol. Cell 3, 571–582 7. Solomon, M. J., Lee, T., and Kirschner, M. W. (1992) Mol. Biol. Cell 3, 13–27 8. Gould, K. L., Moreno, S., Owen, D. J., Sazer, S., and Nurse, P. (1991) EMBO J. 10, 3297–3309 9. Cismowski, M. J., Laff, G. M., Solomon, M. J., and Reed, S. I. (1995) Mol. Cell. Biol. 15, 2983–2992 10. Kaldis, P. (1999) Cell. Mol. Life Sci. 55, 284 –296 11. Solomon, M. J., Harper, J. W., and Shuttleworth, J. (1993) EMBO J. 12, 3133–3142 12. Fesquet, D., Labbe´, J.-C., Derancourt, J., Capony, J.-P., Galas, S., Girard, F., Lorca, T., Shuttleworth, J., Dore´e, M., and Cavadore, J.-C. (1993) EMBO J. 12, 3111–3121 13. Poon, R. Y. C., Yamashita, K., Adamczewski, J. P., Hunt, T., and Shuttleworth, J. (1993) EMBO J. 12, 3123–3132 14. Tassan, J.-P., Schultz, S. J., Bartek, J., and Nigg, E. A. (1994) J. Cell Biol. 127, 467– 478

15. Fisher, R. P., and Morgan, D. O. (1994) Cell 78, 713–724 16. Ma¨kela¨, T. P., Tassan, J.-P., Nigg, E. A., Frutiger, S., Hughes, G. J., and Weinberg, R. A. (1994) Nature 371, 254 –257 17. Devault, A., Martinez, A.-M., Fesquet, D., Labbe´, J.-C., Morin, N., Tassan, J.-P., Nigg, E. A., Cavadore, J.-C., and Dore´e, M. (1995) EMBO J. 14, 5027–5036 18. Fisher, R. P., Jin, P., Chamberlin, H. M., and Morgan, D. O. (1995) Cell 83, 47–57 19. Tassan, J.-P., Jaquenoud, M., Fry, A. M., Frutiger, S., Hughes, G. J., and Nigg, E. A. (1995) EMBO J. 14, 5608 –5617 20. Poon, R. Y. C., Yamashita, K., Howell, M., Ershler, M. A., Belyavsky, A., and Hunt, T. (1994) J. Cell Sci. 107, 2789 –2799 21. Kaldis, P., Russo, A. A., Chou, H. S., Pavletich, N. P., and Solomon, M. J. (1998) Mol. Biol. Cell 9, 2545–2560 22. Labbe´, J.-C., Martinez, A.-M., Fesquet, D., Capony, J.-P., Darbon, J.-M., Derancourt, J., Devault, A., Morin, N., Cavadore, J.-C., and Dore´e, M. (1994) EMBO J. 13, 5155–5164 23. Kato, J.-Y., Matsuoka, M., Storm, D. K., and Sherr, C. J. (1994) Mol. Cell. Biol. 14, 2713–2721 24. Matsuoka, M., Kato, J.-y., Fisher, R. P., Morgan, D. O., and Sherr, C. J. (1994) Mol. Cell. Biol. 14, 7265–7275 25. Blain, S. W., Montalvo, E., and Massague´, J. (1997) J. Biol. Chem. 272, 25863–25872 26. Aprelikova, O., Xiong, Y., and Liu, E. T. (1995) J. Biol. Chem. 270, 18195–18197 27. Iavarone, A., and Massague´, J. (1997) Nature 387, 417– 422 28. Kaldis, P., Sutton, A., and Solomon, M. J. (1996) Cell 86, 553–564 29. Thuret, J.-Y., Valay, J.-G., Faye, G., and Mann, C. (1996) Cell 86, 565–576 30. Espinoza, F. H., Farrell, A., Erdjument-Bromage, H., Tempst, P., and Morgan, D. O. (1996) Science 273, 1714 –1717 31. Espinoza, F. H., Farrell, A., Nourse, J. L., Chamberlin, H. M., Gileadi, O., and Morgan, D. O. (1998) Mol. Cell. Biol. 18, 6365– 6373 32. Egan, E. A., and Solomon, M. J. (1998) Mol. Cell. Biol. 18, 3659 –3667 33. Kimmelman, J., Kaldis, P., Hengartner, C. J., Laff, G. M., Koh, S. S., Young, R. A., and Solomon, M. J. (1999) Mol. Cell. Biol. 19, 4774 – 4787 34. Cheng, A., Ross, K. E., Kaldis, P., and Solomon, M. J. (1999) Genes Dev. 13, 2946 –2957 35. Lee, M. G., and Nurse, P. (1987) Nature 327, 31–35 36. Elledge, S. J., and Spottswood, M. R. (1991) EMBO J. 10, 2653–2659 37. Ninomiya-Tsuji, J., Nomoto, S., Yasuda, H., Reed, S. I., and Matsumoto, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9006 –9010 38. Songyang, Z., Blechner, S., Hoagland, N., Hoekstra, M. F., Piwnica-Worms, H., and Cantley, L. C. (1994) Curr. Biol. 4, 973–982 39. Higashi, H., Suzuki-Takahashi, I., Taya, Y., Segawa, K., Nishimura, S., and Kitagawa, M. (1995) Biochem. Biophys. Res. Commun. 216, 520 –525 40. Holmes, J. K., and Solomon, M. J. (1996) J. Biol. Chem. 271, 25240 –25246 41. Kitagawa, M., Higashi, H., Jung, H.-K., Suzuki-Takahashi, I., Ikeda, M., Tamai, K., Kato, J., Nishimura, S., and Taya, Y. (1996) EMBO J. 15, 7060 –7069 42. Zhu, L., Harlow, E., and Dynlacht, B. D. (1995) Genes Dev. 9, 1740 –1752 43. Adams, P. D., Sellers, W. R., Sharma, S. K., Wu, A. D., Nalin, C. M., and Kaelin, W. G. (1996) Mol. Cell. Biol. 16, 6623– 6633 44. Chen, J., Saha, P., Kornbluth, S., Dynlacht, B. D., and Dutta, A. (1996) Mol. Cell. Biol. 16, 4673– 4682 45. Schulman, B. A., Lindstrom, D. L., and Harlow, E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10453–10458 46. Adams, P. D., Li, X., Sellers, W. R., Baker, K. B., Leng, X., Harper, J. W., Taya, Y., and Kaelin, W. G. (1999) Mol. Cell. Biol. 19, 1068 –1080 47. Brown, N. R., Noble, M. E. M., Endicott, J. A., and Johnson, L. N. (1999) Nat. Cell Biol. 1, 438 – 443 48. Gu, Y., Rosenblatt, J., and Morgan, D. O. (1992) EMBO J. 11, 3995– 4005 49. Gautier, J., Solomon, M. J., Booher, R. N., Bazan, J. F., and Kirschner, M. W. (1991) Cell 67, 197–211 50. Russo, A. A., Jeffrey, P. D., and Pavletich, N. P. (1996) Nat. Struct. Biol. 3, 696 –700 51. Mitchell, D. A., Marshall, T. K., and Deschenes, R. J. (1993) Yeast 9, 715–723 52. Enke, D. A., Kaldis, P., Holmes, J. K., and Solomon, M. J. (1999) J. Biol. Chem. 274, 1949 –1956 53. Desai, D., Wessling, H. C., Fisher, R. P., and Morgan, D. O. (1995) Mol. Cell. Biol. 15, 345–350 54. Ross, K. E., Kaldis, P., and Solomon, M. J. (2000) Mol. Biol. Cell 11, 1597–1609 55. Connell-Crowley, L., Solomon, M. J., Wei, N., and Harper, J. W. (1993) Mol. Biol. Cell 4, 79 –92 56. Jeffrey, P. D., Russo, A. A., Polyak, K., Gibbs, E., Hurwitz, J., Massague´, J., and Pavletich, N. P. (1995) Nature 376, 313–320 57. Donella Deana, A., McGowan, C. H., Cohen, P., Marchiori, F., Meyer, H. E., and Pinna, L. A. (1990) Biochim. Biophys. Acta 1051, 199 –202 58. Zhu, L., Enders, G., Lees, J. A., Beijersbergen, R. L., Bernards, R., and Harlow, E. (1995) EMBO J. 14, 1904 –1913 59. Kisselev, A. F., Songyang, Z., and Goldberg, A. L. (2000) J. Biol. Chem. 275, 14831–14837