How Tyrosine 15 Phosphorylation Inhibits the Activity of Cyclin-dependent Kinase 2-Cyclin A
2006; Elsevier BV; Volume: 282; Issue: 5 Linguagem: Inglês
10.1074/jbc.m609151200
ISSN1083-351X
AutoresJulie P. I. Welburn, Julie A. Tucker, Tim Johnson, Lisa M Lindert, Rhodri M. L. Morgan, Antony C. Willis, M.E.M. Noble, Jane Endicott,
Tópico(s)Ubiquitin and proteasome pathways
ResumoInhibition of cyclin-dependent kinase 1 (CDK1) activity by Tyr-15 phosphorylation directly regulates entry into mitosis and is an important element in the control of the unperturbed cell cycle. Active site phosphorylation of other members of the CDK family that regulate cell cycle progression instates checkpoints that are fundamental to eukaryotic cell cycle regulation. Kinetic and crystallographic analyses of CDK2-cyclin A complexes reveal that this inhibitory mechanism operates through steric blockade of peptide substrate binding and through the creation of an environment that favors a non-productive conformation of the terminal group of ATP. By contrast, tyrosine phosphorylation of CDK2 alters neither its Km for ATP nor its significant intrinsic ATPase activity. Tyr-15-phosphorylated CDK2 retains trace protein phosphorylation activity that should be considered in quantitative and qualitative cell cycle models. Inhibition of cyclin-dependent kinase 1 (CDK1) activity by Tyr-15 phosphorylation directly regulates entry into mitosis and is an important element in the control of the unperturbed cell cycle. Active site phosphorylation of other members of the CDK family that regulate cell cycle progression instates checkpoints that are fundamental to eukaryotic cell cycle regulation. Kinetic and crystallographic analyses of CDK2-cyclin A complexes reveal that this inhibitory mechanism operates through steric blockade of peptide substrate binding and through the creation of an environment that favors a non-productive conformation of the terminal group of ATP. By contrast, tyrosine phosphorylation of CDK2 alters neither its Km for ATP nor its significant intrinsic ATPase activity. Tyr-15-phosphorylated CDK2 retains trace protein phosphorylation activity that should be considered in quantitative and qualitative cell cycle models. The cyclin-dependent protein kinase (CDK) 4The abbreviations used are: CDK, cyclin-dependent kinase; AMPPNP, 5′-adenylylimido-diphosphate. family constitutes a highly conserved but functionally diverse family that is essential for many cellular regulatory processes, notably in the regulation of transcription and cell cycle progression (reviewed in Refs. 1Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1810) Google Scholar and 2Oelgeschlager T. J. Cell. Physiol. 2002; 190: 160-169Crossref PubMed Scopus (123) Google Scholar). For full activity, CDKs absolutely require association with an activating cyclin molecule, and they may also require phosphorylation of a conserved threonine residue that lies within the activation loop (Thr-160 in CDK2). Beyond these effects, CDKs are regulated by multiple mechanisms that include inhibitory phosphorylation of conserved residues within the active site (3Solomon M.J. Kaldis P. Results Probl. Cell Differ. 1998; 22: 79-109Crossref PubMed Scopus (58) Google Scholar) and binding of additional regulatory molecules (4Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5159) Google Scholar). Early studies in Schizosaccharomyces pombe demonstrated that Cdc2 (CDK1) Tyr-15 phosphorylation directly regulates entry into mitosis and is an important element in the control of the unperturbed cell cycle (5Gould K.L. Nurse P. Nature. 1989; 342: 39-45Crossref PubMed Scopus (855) Google Scholar, 6Morla A.O. Draetta G. Beach D. Wang J.Y. Cell. 1989; 58: 193-203Abstract Full Text PDF PubMed Scopus (306) Google Scholar, 7Krek W. Marks J. Schmitz N. Nigg E.A. Simanis V. J. Cell Sci. 1992; 102: 43-53PubMed Google Scholar). Subsequent studies showed that in higher vertebrates the adjacent threonine residue (Thr-14) is also a site of inhibitory phosphorylation, (8Krek W. Nigg E.A. EMBO J. 1991; 10: 305-316Crossref PubMed Scopus (314) Google Scholar, 9Norbury C. Blow J. 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Saccharomyces cerevisiae appears to be the exception in that phosphorylation of Tyr-19 is detectable (the residue equivalent to CDK1 Tyr-15) but its dephosphorylation is not a requirement for entry into mitosis, suggesting that other mechanisms must regulate the transition (12Amon A. Surana U. Muroff I. Nasmyth K. Nature. 1992; 355: 368-371Crossref PubMed Scopus (234) Google Scholar, 13Sorger P.K. Murray A.W. Nature. 1992; 355: 365-368Crossref PubMed Scopus (216) Google Scholar). Phosphorylation of CDK1 on Tyr-15 is catalyzed by members of the Wee1/Mik1 kinase family (14Featherstone C. Russell P. Nature. 1991; 349: 808-811Crossref PubMed Scopus (245) Google Scholar, 15Parker L.L. Piwnica-Worms H. Science. 1992; 257: 1955-1957Crossref PubMed Scopus (548) Google Scholar, 16Igarashi M. Nagata A. Jinno S. Suto K. Okayama H. Nature. 1991; 353: 80-83Crossref PubMed Scopus (148) Google Scholar, 17Lundgren K. Walworth N. Booher R. Dembski M. Kirschner M. Beach D. 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Other members of the human CDK family that regulate the cell cycle (CDKs 2, 4, and 6) all contain a tyrosine residue equivalent to CDK1 Tyr-15, and phosphoforms of these CDKs, phosphorylated on this tyrosine residue, have been detected in cultured cell lines (24Gu Y. Rosenblatt J. Morgan D.O. EMBO J. 1992; 11: 3995-4005Crossref PubMed Scopus (560) Google Scholar, 25Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (330) Google Scholar). Whether inhibitory phosphorylation is as crucial for their activities in the unperturbed cell cycle as it is for the activity of CDK1 is still unresolved. CDK2 is phosphorylated on Thr-14 and Tyr-15 during S and G2 phases and is dephosphorylated by Cdc25 (24Gu Y. Rosenblatt J. Morgan D.O. EMBO J. 1992; 11: 3995-4005Crossref PubMed Scopus (560) Google Scholar, 26Gabrielli B.G. Lee M.S. Walker D.H. Piwnica-Worms H. Maller J.L. J. Biol. Chem. 1992; 267: 18040-18046Abstract Full Text PDF PubMed Google Scholar, 27Sebastian B. 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As CDK2 is the major cell cycle regulator during these cell cycle transitions, it has been proposed that these effects are as a result of Cdc25A activity against CDK2. Indeed Cdc25A overexpression resulted in an increase in CDK2-cyclin A and cyclin E activity concomitant with CDK2 Tyr-15 dephosphorylation (30Hoffmann I. Draetta G. Karsenti E. EMBO J. 1994; 13: 4302-4310Crossref PubMed Scopus (424) Google Scholar). However, recent work showing that CDK1 can substitute for CDK2 to promote S-phase, as revealed by experiments in CDK2-/-p27-/- cells (31Aleem E. Kiyokawa H. Kaldis P. Nat. Cell Biol. 2005; 7: 831-836Crossref PubMed Scopus (318) Google Scholar), would suggest that further work is required to establish whether CDK2 and/or CDK1 is the essential Cdc25A target at G1/S. In contrast, the importance of CDK inhibition by phosphorylation in establishing cell cycle arrest in response to checkpoint activation is well documented. Active site phosphorylation of CDK1 (32Jin P. Gu Y. 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Nature. 2000; 408: 433-439Crossref PubMed Scopus (2645) Google Scholar, 39Donzelli M. Draetta G.F. EMBO Rep. 2003; 4: 671-677Crossref PubMed Scopus (351) Google Scholar, 40Bartek J. Lukas C. Lukas J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 792-804Crossref PubMed Scopus (603) Google Scholar). Exposure to transforming growth factor β results in a cell cycle arrest that is correlated with elevated levels of tyrosine-phosphorylated CDKs 4 and 6 (25Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (330) Google Scholar) and that may be mediated by down-regulation of Cdc25A. Genetic and biochemical studies to dissect the effect of tyrosine and threonine phosphorylation on CDK activity have focused on CDK1 because of its established role in controlling entry into M-phase. In mouse cells, expression of a CDK1 (Y15E) mutant produces a dominant negative phenotype (9Norbury C. Blow J. Nurse P. EMBO J. 1991; 10: 3321-3329Crossref PubMed Scopus (399) Google Scholar). Similarly chicken mutants (CDK1(T14E), CDK1(Y15E), CDK1(T14D, Y15F), and CDK1(T14A, Y15D)) were unable to rescue fission yeast cells lacking wild-type CDK1 function and caused a cdc phenotype upon overexpression (7Krek W. Marks J. Schmitz N. Nigg E.A. Simanis V. J. Cell Sci. 1992; 102: 43-53PubMed Google Scholar). These results suggest that negative charge at either position 14 or 15 is sufficient to inhibit kinase activity. Early biochemical studies suggested that Tyr-15-phosphoylated CDK1 is inactive (15Parker L.L. Piwnica-Worms H. Science. 1992; 257: 1955-1957Crossref PubMed Scopus (548) Google Scholar). Molecular models of CDK1 have suggested that a phosphate group located on Tyr-15 would present a steric and charge impediment to peptide substrate binding (41Marcote M.J. Knighton D.R. Basi G. Sowadski J.M. Brambilla P. Draetta G. Taylor S.S. Mol. Cell. Biol. 1993; 13: 5122-5131Crossref PubMed Scopus (43) Google Scholar, 42Endicott J.A. Nurse P. Johnson L.N. Protein Eng. 1994; 7: 243-253Crossref PubMed Scopus (26) Google Scholar). Biochemical characterization of suicide inhibitor labeling of Tyr-15-phosphorylated CDK1 has further suggested that phosphorylation of Tyr-15 might not inhibit ATP binding (43Atherton-Fessler S. Parker L.L. Geahlen R.L. Piwnica-Worms H. Mol. Cell. Biol. 1993; 13: 1675-1685Crossref PubMed Scopus (151) Google Scholar), an hypothesis supported by molecular modeling studies (44Bartova I. Otyepka M. Kriz Z. Koca J. Protein Sci. 2004; 13: 1449-1457Crossref PubMed Scopus (74) Google Scholar). Despite extensive x-ray crystallographic analysis of members of the CDK family, an atomic structure for a CDK in this phosphorylated state has not been described. In this report we present both the structure and the kinetic properties of CDK2-cyclin A phosphorylated on Tyr-15 and Thr-160. These results are discussed with respect to the molecular mechanism of CDK inhibitory phosphorylation and its physiological consequences. Purification of Phosphorylated CDK2-Cyclin A Complexes— T160pCDK2-cyclin A was purified as described (45Brown N.R. Noble M.E. Endicott J.A. Johnson L.N. Nat. Cell Biol. 1999; 1: 438-443Crossref PubMed Scopus (480) Google Scholar). Y15pT160pCDK2-cyclin A was purified by one of two methods. In the first method (Method 1), a modified pGEX-6P-1 vector (Amersham Biosciences) that independently expresses human CDK2 and the human Wee1 kinase catalytic domain (46McGowan C.H. Russell P. EMBO J. 1993; 12: 75-85Crossref PubMed Scopus (390) Google Scholar) as glutathione S-transferase fusion proteins (pGEX-6P-CDK2-Wee1) was transformed into Escherichia coli strain B834(DE3)/LysS. Notably, only GST-CDK2 contains a cleavage site for PreScission protease. GST-CDK2 and GST-Wee1 fusion proteins were purified together by affinity chromatography, and then GST-CDK2 was phosphorylated in vitro on Thr-160 by GST-CIV1 as described (47Brown N.R. Noble M.E. Lawrie A.M. Morris M.C. Tunnah P. Divita G. Johnson L.N. Endicott J.A. J. Biol. Chem. 1999; 274: 8746-8756Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Following a buffer exchange step, the mixture of GST-Y15pT160pCDK2, GST-CIV1, and GST-Wee1 was re-applied to a glutathione-Sepharose 4B column. Lysate from a culture of E. coli B834(DE3)/pLysS cells expressing a truncated version of human cyclin A (residues 174–432) (45Brown N.R. Noble M.E. Endicott J.A. Johnson L.N. Nat. Cell Biol. 1999; 1: 438-443Crossref PubMed Scopus (480) Google Scholar) was passed over the column. The binary Y15pT160pCDK2-cyclin A complex, GST-Wee1, and GST-CIV1 were eluted with glutathione; the mixture was incubated with PreScission protease and then subjected to gel filtration (Superdex 75 HR26/60; Amersham Biosciences) followed by a glutathione-Sepharose 4B column. The binary complex was recovered in the flow-through, and any remaining GST-CIV1 or GST-Wee1 was retained on the final column. In the second method (Method 2), pGEX-6P-CDK2-Wee1 and a pACYC-derived vector to express GST-CIV1 were cotransformed into E. coli strain B834(DE3). This expression system produced CDK2 phosphorylated on Tyr-15 and Thr-160. The Y15pT160pCDK2-cyclin A complex was subsequently prepared by a modified method to that described for preparation of T160pCDK2-cyclin A (45Brown N.R. Noble M.E. Endicott J.A. Johnson L.N. Nat. Cell Biol. 1999; 1: 438-443Crossref PubMed Scopus (480) Google Scholar). Briefly, Y15pT160pCDK2-cyclin A was purified by sequential affinity (glutathione-Sepharose 4B), size-exclusion, and then affinity chromatography steps as described (45Brown N.R. Noble M.E. Endicott J.A. Johnson L.N. Nat. Cell Biol. 1999; 1: 438-443Crossref PubMed Scopus (480) Google Scholar). The sample was then enriched for Y15pT160pCDK2 by exploiting a GST-Cdc25A affinity column. The Cdc25A construct used (GST-Cdc25AC430S (336–523)) encodes glutathione S-transferase fused to Cdc25A residues 336 to 523 and is a substrate-trapping mutant in which the catalytic cysteine (Cys-430) is mutated to serine. The construct was expressed in E. coli strain B834(DE3), and an S100 lysate was prepared and loaded onto a glutathione-Sepharose 4B column pre-equilibrated with HEPES-buffered saline. 10 mg of purified Y15pT160pCDK2-cyclin A was then applied to the GST-Cdc25AC430S(336–523) affinity column and washed to baseline with HEPES-buffered saline. Bound Y15pT160pCDK2-cyclin A was eluted with a gradient of dihydrogen ammonium phosphate (0–0.5 m). The eluted complex was buffer exchanged into HEPES-buffered saline and glycerol added to 50%. The inclusion of this additional affinity step in the purification procedure consistently enriched for Tyr-15-phosphorylated CDK2-cyclin A. Y15pT160pCDK2-cyclin A prepared by Method 2 was used for the ATPase assays and kinetic analysis. Analysis of Phosphorylated CDK2—The extinction coefficient (ϵ280) of T160pCDK2-cyclin A was determined by amino acid analysis. Subsequently, protein concentrations were determined from measured A280 values and an ϵ280 = 67420 m-1 cm-1. The extent of Tyr-15 phosphorylation was determined by N-terminal sequencing (described in supplemental information). Estimating the Extent of Phosphorylation of Tyr-15—The amount of amino acid liberated at each cycle of N-terminal sequencing was recorded for amino acid Gly-3 and for amino acids Gly-13 through Gly-19 in each of three separate sequencing runs of T160pCDK2-cyclin A (data presented in supplemental information for material prepared by Method 2, Table 1). Data for the different runs were placed on a common scale by dividing the amount of amino acid liberated at each cycle of a given run by the mean amount of amino acid liberated for cycles corresponding to Gly-3, Gly-13 through Thr-14, and Gly-16 through Tyr-19 of that run. The data were further normalized for cycle-to-cycle effects (principally expected to result from the relative efficiency of hydrolysis and derivatization of different amino acids) by dividing the scaled datum corresponding to a given cycle of a given run by the mean of the scaled data observed for that particular cycle in all runs. The variance of the resulting distribution of values, which should have a mean value of 1.0 for homogenous samples, is an estimate of the accuracy of the N-terminal sequencing protocol in determining the occupancy of the expected amino acid at any given sequence position. In this case, an accuracy of ± 0.11 was observed.TABLE 1Statistics of the datasets used and of the refined structuresY15pT160p CDK2-cyclinA-AMPPNPT160pCDK2-cyclinAaT160pCDK2-cyclin A and Y15pT160pCDK2-cyclin A crystals were grown in the presence of AMPPNP, cross-linked, and subsequently soaked in mother liquor supplemented with substrate peptide as described under "Experimental Procedures"Y15pT160pCDK2-cyclinAaT160pCDK2-cyclin A and Y15pT160pCDK2-cyclin A crystals were grown in the presence of AMPPNP, cross-linked, and subsequently soaked in mother liquor supplemented with substrate peptide as described under "Experimental Procedures"Cell dimensions (Å)a = 73.8, b = 133.2, c = 147.6a = 73.8, b = 133.2, c = 148.5a = 73.0, b = 133.6, c = 148.0Space groupP212121P212121P212121Maximum resolution (Å)2.33.23.2Observations490488223202207912Unique reflections, completeness (%)64652 (94.5)24761 (99.5)24165 (98.1)Rmerge (%)7.310.310.3Mean I/σ(I)4.22.62.6Highest resolution bin (Å):2.42-2.303.37-3.203.37-3.20Completeness (%)94.5100.0100.0Rmerge (%)bRmerge=∑h∑j|Ih,j-I¯h|∑h∑j|Ih,j| where Ih,j is the intensity of the jth observation of unique reflection h36.733.632.6Mean I/σ(I)1.92.31.9Statistics and model quality No. of protein atoms884090188856 No. of solvent molecules6584590 No. of peptide atoms0380 No. of AMPPNP atoms62062 Rfree (%)26.528.731.5 Rconv (%)cRconv=∑h‖Foh|-|Fch‖∑h|Foh| where Fo,h and Fc,h are the observed and calculated structure factor amplitudes for reflection h, k, l. Rfree is equivalent to Rconv but is calculated using a 5% disjoint set of reflections excluded from the least squares refinement stages20.123.123.9 Root mean square deviation on bonds (Å)0.018 Root mean square deviation on angles (°)1.724 Procheck Ramachandran core/allowed residues99.8%a T160pCDK2-cyclin A and Y15pT160pCDK2-cyclin A crystals were grown in the presence of AMPPNP, cross-linked, and subsequently soaked in mother liquor supplemented with substrate peptide as described under "Experimental Procedures"b Rmerge=∑h∑j|Ih,j-I¯h|∑h∑j|Ih,j| where Ih,j is the intensity of the jth observation of unique reflection hc Rconv=∑h‖Foh|-|Fch‖∑h|Foh| where Fo,h and Fc,h are the observed and calculated structure factor amplitudes for reflection h, k, l. Rfree is equivalent to Rconv but is calculated using a 5% disjoint set of reflections excluded from the least squares refinement stages Open table in a new tab A further four runs of N-terminal sequencing were carried out on the doubly phosphorylated sample, of which two yielded sequencing data over an amino acid range comparable with that sequenced in the singly phosphorylated samples. Run-to-run scaling was carried out as above, but the cycle-to-cycle scaling was performed using the scale factors determined for the singly phosphorylated sample. Correction by this method of the observed yields of unphosphorylated tyrosine at position 15 of the doubly phosphorylated samples yielded occupancies of 0.042 and 0.055, i.e. a mean of 0.049, with an S.E. of the mean of 0.077. ATPase Assay—The hydrolysis of ATP was monitored using a coupled spectrophotometric assay using material prepared by Method 2 as described previously (Refs. 48Prowse C.N. Lew J. J. Biol. Chem. 2001; 276: 99-103Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 49Stevenson L.M. Deal M.S. Hagopian J.C. Lew J. Biochemistry. 2002; 41: 8528-8534Crossref PubMed Scopus (38) Google Scholar and supplemental information). Assay of the Kinetics of Phosphorylation of Histone H1 by T160pCDK2-Cyclin A and Y15pT160pCDK2-Cyclin A— T160pCDK2-cyclin A (5.2 nm, prepared by Method 2) was incubated with histone H1 substrate at varying concentrations in the range 6.25–100 μm. The kinase reaction was started by the addition of [γ-32P]ATP/Mg2+ (600 cpm/pmol) in a final reaction volume of 10 or 20 μl containing 1 mm ATP, 10 mm MgCl2, 200 mm NaCl, 0.01% monothioglycerol, 100 mm Tris, pH 7.4. Triplicate reactions were allowed to proceed at 30 °C for 10 min before being terminated by the addition of 50% acetic acid. Phosphorylated histone was separated from free ATP by p81 paper cation exchange chromatography. Phosphate incorporation and hence the rates of reaction were determined by Cerenkov counting. The kinetic parameters from the data were determined by non-linear regression analysis. Y15pT160pCDK2-cyclin A was analyzed similarly except that a higher final enzyme concentration (1.6μm) and a higher substrate concentration range (up to 200μm) were used, reflecting the lower activity and higher Km of this sample. Analysis of the variation of initial rate with concentration of ATP for Y15pT160pCDK2-cyclin A suggested trace contamination of the sample with the more highly active T160pCDK2-cyclin A, particularly apparent at lower concentrations of substrate (Fig. 4). Regression analysis for this sample therefore used data from the higher concentration regime (50–200μm). Attempts to model the contribution of T160pCDK2-cyclin A to the kinetic data recorded from the Y15pT160pCDK2-cyclin A sample indicated a contamination of <1%, a value within the estimated accuracy of the amount indicated by N-terminal sequencing. Crystallization of Phosphorylated CDK2-Cyclin A Binary Complexes—Y15pT160pCDK2-cyclin A complex prepared by Method 1 in 40 mm HEPES, pH 7.4, 200 mm NaCl to 10 mg/ml and 5′-adenylylimido-diphosphate (AMPPNP) (Sigma) was added to an aliquot of the protein solution to a final concentration of 1 mm. Crystals were grown at 4 °C in 100 mm HEPES, pH 7.0, 5 mm dithiothreitol, 0.7 m KCl, and 1.20 m (NH4)2SO4. Crystals of T160pCDK2-cyclin A-AMPPNP were grown under very similar conditions as previously described (45Brown N.R. Noble M.E. Endicott J.A. Johnson L.N. Nat. Cell Biol. 1999; 1: 438-443Crossref PubMed Scopus (480) Google Scholar). A native Y15pT160pCDK2-cyclin A-AMPPNP dataset was collected from a crystal that, although soaked in a substrate peptide solution (sequence PKTPKKAKKL), had no bound peptide. After failing to bind peptide to these crystals despite repeated attempts, protein cross-linking was used to allow soaking at higher peptide concentrations. The crystals (T160pCDK2-cyclin A-AMPPNP and Y15pT160pCDK2-cyclin A-AMPPNP) were first cross-linked for 30–60 min at 4 °C by gentle vapor diffusion of glutaraldehyde added to the well solution (0.1%) and then soaked for 15 min in mother liquor supplemented with 20 mm substrate peptide (HHASPRK, previously shown to bind to T160pCDK2-cyclin A) (45Brown N.R. Noble M.E. Endicott J.A. Johnson L.N. Nat. Cell Biol. 1999; 1: 438-443Crossref PubMed Scopus (480) Google Scholar). For all data collection, crystals were briefly cryoprotected in 8 m sodium formate and flash frozen in liquid nitrogen. Data were integrated, scaled, and merged using the programs MOSFLM and SCALA. Statistics for the datasets are presented in Table 1. Structure Determination and Refinement—The structure of Y15pT160pCDK2-cyclin A-AMPPNP was solved by molecular replacement using the program Molrep via the CCP4 GUI (50Collaborative Computational Project 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The coordinates of a T160pCDK2-cyclin A complex (Protein Data Bank entry 1JST) (51Russo A.A. Jeffrey P.D. Pavletich N.P. Nat. Struct. Biol. 1996; 3: 696-700Crossref PubMed Scopus (505) Google Scholar) were used as the search model. Several cycles of rigid body refinement were initially pursued using REFMAC5. Subsequent refinement was then pursued using REFMAC in conjunction with manual rebuilding using the programs O (52Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and Coot (53Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60 (Pt. 1): 2126-2132Crossref PubMed Scopus (23628) Google Scholar). A detailed description of all steps in the structure determination and refinement procedure is provided in the supplemental information. Statistics for the final models are presented in Table 1. The Structure of CDK2-Cyclin A Phosphorylated on Tyr-15 and Thr-160—To provide a structural understanding of the role of Tyr-15 phosphorylation in regulating CDK activity we have determined the structure of a CDK2-cyclin A complex phosphorylated on Tyr-15 and Thr-160 (Y15pT160pCDK2-cyclin A) (Table 1) and characterized its catalytic activity (Table 2 and Fig. 4) A Cdc25A affinity column was found to be the most effective method to enrich for Y15pT160pCDK2-cyclin A, and exploiting this method preparations were conservatively estimated to contain 95.1% ± 7.8% CDK2 phosphorylated on Tyr-15 (supplemental Table S1). In our early CDK2/Wee1 coexpression studies we detected low levels of Thr-160 phosphorylation by SDS-PAGE gel shift assay and subsequent dephosphorylation by kinase-associated phosphate (KAP) (results not shown). Using more sensitive protein sequencing techniques we did not detect any phosphorylation on Thr-14 (supplemental Table S1). Taken together, these results suggest that the isolated Wee1 catalytic domain retains a degree of selectivity for Tyr-15 but that in this in vitro system Thr-160, and not Thr-14, is additionally susceptible to phosphorylation.TABLE 2Kinetic parameters for T160pCDK2-cyclin A and Y15pT160pCDK2-cyclin AParameterT160pCDK2-cyclin AY15pT160p CDK2-cyclin AFold change upon phosphorylationAverageS.D.nAverageS.D.nKinase activity (H1)34 Km (μm)281.978162.8 kcat (s–1)5.2(7)aPreviously reported values (54)0.40.080.0165 kcat/Km (μm–1 s–1)0.190.020.00100.0002190ATPase activity34 Km (μm)812072231.1 kcat (s–1)0.071 (0.2)aPreviously reported values (54)0.0090.0960.00731.3 kcat/Km (μm–1 s–1)0.000870.000240.00130.000141.5a Previously reported values (54Hagopian J.C. Kirtley M.P. Stevenson L.M. Gergis R.M. Russo A.A. Pavletich N.P. Parsons S.M. Lew J. J. Biol. Chem. 2001; 276: 275-280Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) Open table in a new tab The initial mFo - Fc maps calculated for the Y15pT160pCDK2-cyclin A complex co-crystallized with AMP-PNP included strong electron density for both phosphate groups attached to Tyr-15 and Thr-160 (Fig. 1). The Thr-160 phosphate group reorganizes the CDK2 activation segment to create the peptide substrate-binding site and, through interactions with the guanidinium groups of Arg-50 in the C-helix and Arg-126 and Arg-150 at the
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