Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange
2006; Springer Nature; Volume: 25; Issue: 13 Linguagem: Inglês
10.1038/sj.emboj.7601209
ISSN1460-2075
AutoresAntony W. Oliver, Angela Paul, K. Boxall, S. Elaine Barrie, G. Wynne Aherne, Michelle D. Garrett, Sibylle Mittnacht, Laurence H. Pearl,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle22 June 2006free access Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange Antony W Oliver Corresponding Author Antony W Oliver Cancer Research UK DNA Repair Enzymes Group, Section of Structural Biology, The Institute of Cancer Research, Chelsea, London, UK Search for more papers by this author Angela Paul Angela Paul Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, Chelsea, London, UK Search for more papers by this author Katherine J Boxall Katherine J Boxall Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK Search for more papers by this author S Elaine Barrie S Elaine Barrie Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK Search for more papers by this author G Wynne Aherne G Wynne Aherne Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK Search for more papers by this author Michelle D Garrett Michelle D Garrett Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK Search for more papers by this author Sibylle Mittnacht Sibylle Mittnacht Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, Chelsea, London, UK Search for more papers by this author Laurence H Pearl Corresponding Author Laurence H Pearl Cancer Research UK DNA Repair Enzymes Group, Section of Structural Biology, The Institute of Cancer Research, Chelsea, London, UK Search for more papers by this author Antony W Oliver Corresponding Author Antony W Oliver Cancer Research UK DNA Repair Enzymes Group, Section of Structural Biology, The Institute of Cancer Research, Chelsea, London, UK Search for more papers by this author Angela Paul Angela Paul Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, Chelsea, London, UK Search for more papers by this author Katherine J Boxall Katherine J Boxall Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK Search for more papers by this author S Elaine Barrie S Elaine Barrie Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK Search for more papers by this author G Wynne Aherne G Wynne Aherne Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK Search for more papers by this author Michelle D Garrett Michelle D Garrett Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK Search for more papers by this author Sibylle Mittnacht Sibylle Mittnacht Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, Chelsea, London, UK Search for more papers by this author Laurence H Pearl Corresponding Author Laurence H Pearl Cancer Research UK DNA Repair Enzymes Group, Section of Structural Biology, The Institute of Cancer Research, Chelsea, London, UK Search for more papers by this author Author Information Antony W Oliver 1, Angela Paul2, Katherine J Boxall3, S Elaine Barrie3, G Wynne Aherne3, Michelle D Garrett3, Sibylle Mittnacht2 and Laurence H Pearl 1 1Cancer Research UK DNA Repair Enzymes Group, Section of Structural Biology, The Institute of Cancer Research, Chelsea, London, UK 2Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, Chelsea, London, UK 3Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK *Corresponding authors: Cancer Research UK DNA Repair Enzymes Group, The Institute of Cancer Research, 237 Fulham Road, Chelsea, London SW3 6JB, UK. Tel.: +44 20 7153 5571; Fax: +44 20 6153 5457; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2006)25:3179-3190https://doi.org/10.1038/sj.emboj.7601209 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine–glutamine/threonine–glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation. Introduction DNA double-strand breaks, generated by ionising radiation, genotoxic chemicals or collapsed replication forks, are the most serious type of DNA damage with which the eukaryotic cell must contend. Survival depends on a coordinated response whereby the DNA lesion is repaired, and the progress of the cell cycle is halted to allow that repair to occur. Key to this response is the activation of a signalling network, dependent on the phosphatidylinositol kinase-like kinases (PIKKs), ATM and ATR, which are primary sensors of DNA damage (McGowan and Russell, 2004; Lavin et al, 2005) (Figure 1A). ATM/ATR driven signals modulate transcription through stabilisation of p53, and directly regulate cell cycle progression by promoting the degradation of the Cdc25 phosphatases that activate Cdk2/CyclinE complexes (Falck et al, 2001). An immediate downstream target of the ATM branch of the network is checkpoint kinase 2 (Chk2) (Cds1), a Ser/Thr kinase consisting of an N-terminal serine–glutamine/threonine–glutamine cluster domain (SCD), a middle phosphothreonine-binding FHA domain (Li et al, 2002) and a C-terminal catalytic domain (Figure 1B). Activated Chk2 in turn phosphorylates a range of downstream targets including Cdc25A and Cdc25C, BRCA1 and p53 (Bartek and Lukas, 2003). Chk2 activation is a multistep process, initiated by ATM, which phosphorylates Chk2 on Thr68 in a segment of the protein upstream of the FHA domain (Ahn et al, 2000; Matsuoka et al, 2000; Melchionna et al, 2000). Once phosphorylated, the pThr68 segment of Chk2 can bind in trans to the FHA domain of another molecule promoting dimerisation and trans-activating phosphorylation of Thr383 and Thr387 in the activation segment or ‘T-loop’ of the catalytic domain (Ahn et al, 2002; Xu et al, 2002). However, the mechanism by which dimerisation promotes T-loop phosphorylation is not understood. Figure 1.Chk2 in the DNA damage signalling network. (A) Simplified diagram of the ATM/ATR modulated signalling pathway. The PIKKs ATM and ATR are activated by DNA damage; ATM principally by DSB in DNA and ATR by UV damage and its associated lesions. Activated ATM phosphorylates the ‘transducer’ kinases Chk1 and Chk2, which in turn phosphorylate the CDC25 phosphatases promoting their degradation. Subsequent downstream events lead to cell cycle arrest, facilitating DNA repair processes. ATR similarly promotes CDC25 degradation, but only via Chk1 activity. The ‘effector’ protein p53, in undamaged cells, is rapidly degraded due to Mdm2-mediated ubiquitylation targeting the protein to the proteasome. Upon DNA damage p53 is phosphorylated (ATM, ATR, Chk2) inhibiting its interaction with Mdm2, hence stabilising the protein and allowing it to function as a transcription factor. Phosphorylation of Mdm2 by ATM also prevents the protein from shuttling p53 from the nucleus to the cytoplasm for degradation. (B) Schematic representation of the functional domains of Chk2. SCD=serine–glutamine/threonine–glutamine cluster domain, FHA=Forkhead-associated domain and KINASE=kinase domain. Predicted amino-acid ranges for each domain are indicated (from UniProt entry CHK2_HUMAN), along with phosphorylation sites (T69, T383, T387 and S516). The approximate amino-acid ranges for each protein/proteolysis product are also shown. (C) SDS–PAGE of selected gel filtration fractions. M=molecular size marker, with sizes indicated in kilodaltons (kDa). (D) MALDI-TOF analysis of proteolysis products. Molecular masses determined for each peak are shown. In (B–D), the full-length protein (amino acids 210–531) is labelled with an asterisk (*) and the proteolysis products are labelled (1) and (2) respectively. Download figure Download PowerPoint The role played by Chk2 in mediating the cellular response to DNA damage gives it an important function as a tumour suppressor (McGowan, 2002). Truncations and missense mutations of Chk2 have been identified in a wide range of tumours, particularly breast (reviewed in Bartek and Lukas, 2003). Although Chk2 is formally a tumour suppressor, there is a growing interest in its inhibition in the treatment of cancer, both to radio-sensitise tumour cells already defective in other damage checkpoints (e.g. p53−/−), and to inhibit radiation induced apoptosis in sensitive collateral tissues (Bartek and Lukas, 2003; Collins and Garrett, 2005; Pommier et al, 2005). Towards understanding the molecular mechanism for Chk2 activation and to facilitate development of specific inhibitors, we have now determined the crystal structure of a catalytic domain construct of human Chk2 in complex with Mg-ADP, and with the ATP-competitive kinase inhibitor debromohymenialdisine (DBQ) (Meijer et al, 2000; Sharma and Tepe, 2004). The structure of the Chk2 kinase domain reveals an unusual dimeric arrangement involving exchange of T-loops, and suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation. Results Chk2 kinase domain constructs As attempts to crystallise full-length Chk2 were unsuccessful, we sought smaller constructs that would encapsulate the catalytic domain. Initial trials, based on sequence threading predictions, used an N-terminally His6-tagged construct running from Ser210 to the natural C-terminus at Leu543, but these were also unsuccessful. During purification of this protein, two proteolysis products were observed in SDS–PAGE. Western blotting with an anti-His6 antibody confirmed the retention of the N-terminal tag indicating that these products were C-terminally truncated (Figure 1C). The masses of the truncated proteins were determined using MALDI-TOF mass spectrometry (Figure 1D). The mass for the larger of the two bands equated to a protein containing amino acids 210–528, while the smaller equated to amino acids 210–512. A new recombinant construct based on the larger of these (210–531) was constructed as a protease-cleavable N-terminal GST fusion (see Materials and methods), which expressed at high levels, could be fully released from the fusion protein, and was readily purified to homogeneity. Crystal structure of the binary Chk2 kinase domain/ADP complex The purified Chk2(210-531) protein (CHK2-KD) crystallised as a binary complex with Mg-ADP. The structure was solved by molecular replacement using the structure of PKA as a search model (see Materials and methods), then rebuilt and refined at 2.25 Å. Statistics for the structure determination and refinement are given in Table I. Clear interpretable electron density is seen for residues 210–504 of the molecule, except for the loop comprised of residues 255–268 (connecting strand β3 to helix αC), which appears to be poorly ordered in these crystals. Similarly, at the C-terminus residues 505–531 have no discernable electron density and are also considered to be disordered. As expected, CHK2-KD has the two-lobe architecture of the canonical kinase fold, with the nucleotide-binding site residing between the two lobes (Figure 2). The N-lobe is folded around a five-stranded antiparallel β-sheet and contains the αC helix and the glycine-rich loop (residues 227–232 in Chk2), which is fully ordered in the Mg–ADP complex. The C-lobe is predominantly helical and contains the highly conserved DFG and APE motifs, and the intervening T-loop (residues 368–394); the hinge region comprised of residues 303–321 links the two lobes. Figure 2.Structure of Chk2 kinase domain. Secondary structure cartoon of the binary complex of CHK2-KD with ADP. The glycine-rich loop is coloured pink, the DFG and APE motifs red, and the T-loop yellow. Secondary structure elements corresponding to β-strands are coloured blue and α-helices cyan. ADP is shown as a ‘stick’ model. The catalytic residue Asp347, conserved residues Lys249 and Glu373, and autophosphorylation sites Thr383 and Thr387 are also highlighted. Molecular images in this and subsequent figures were generated using PyMOL (www.pymol.org). Download figure Download PowerPoint Table 1. Data collection and refinement statistics CHK2-KD/MgADP CHK2-KD/DBQ Data collection Space group P3221 P3221 Cell dimensions a, b, c (Å) 90.87, 90.87, 92.91 88.65, 88.65, 88.90 α, β, γ (deg) 90, 90, 120 90, 90, 120 Resolution (Å) 40.83–2.25 (2.31–2.25)a 39.65–2.70 (2.85–2.70) Rmerge 0.076 (0.474) 0.067 (0.533) Mn I/σI 16.5 (3.2) 16.0 (2.8) Completeness (%) 100 (100) 98.7 (100) Redundancy 5.5 (5.6) 4.3 (4.3) Refinement Resolution (Å) 2.25 2.70 No. of reflections 20 374 10 709 Rwork/Rfree 0.20/0.24 0.21/0.27 No. of atoms Protein 2271 2153 Ligand/ion 1(Cl−), 2(Mg2+), 4(NO3−), 27(ADP) 1(Mg2+), 4(NO3−), 18 (DBQ) Water 156 44 B-factors Protein 46.6 55.0 Ligand/ion 43.5(Cl−), 48.1(Mg2+), 40.0(NO3−), 42.3(ADP) 65.8(Mg2+), 74.0(NO3−), 48.6(DBQ) Water 47.6 63.2 R.m.s. deviations Bond lengths (Å) 0.02 0.02 Bond angles (deg) 1.73 2.23 a Highest resolution shell is shown in parenthesis. T-loop exchange The most immediately striking feature of the CHK2-KD crystal structure is the presence of an ordered T-loop or activation segment, projecting from the body of the kinase to interact with a second Chk2 molecule, forming a two-fold symmetric dimer interaction (Figure 3A and B). As well as its unusual location, the structure of the T-loop itself departs from the norm significantly, consisting of an antiparallel pair of close-packed amphipathic α-helices (377–386, 392–402) connected by an extended but well-ordered loop. The residues at the tip of the loop (Pro388, Thr389 and Tyr390) and the beginning of the second helix (Ala392, Pro393, Leu396 and Val397) pack into a hydrophobic recess on the dimer-related Chk2 lined by residues from the α-helices 405–423 and 435–442, and the loop from 466 to 476 (Figure 3C). Additionally, Glu394 (from the conserved APE motif) on the second helix of the projecting T-loop makes a bidentate ion-pair/hydrogen bond interaction with Arg474 (Figure 3D). The first helix of the projecting T-loop, however, makes no interactions with the dimer-related Chk2 molecule and has significantly higher temperature factors than the second helix. Figure 3.Chk2 T-loop conformation and exchange. (A) T-loop exchanged dimer. One molecule of the dimer is shown as a molecular surface representation (coloured cyan), and the second as a secondary structure cartoon (yellow). (B) As in (A) but rotated 90° around the indicated axis. (C) Molecular details of T-loop exchange. Residues Pro388, Thr389 and Tyr390 comprising the tip of the exchanged loop (coloured yellow), along with residues Ala392, Pro393, Leu396 and Val397 forming the start of a helix (red) pack into a hydrophobic recess in a dimer-related CHK2-KD molecule (cyan). This recess is lined by two helices (residues 405–423 and 435–442) and the loop comprised of residues 466–476 (blue). (D) APE motif: Glu394 of the APE motif (coloured red) is hydrogen-bonded to the canonically conserved arginine (Arg474) despite coming from an exchanged T-loop (yellow). Helices and loops comprising the hydrophobic recess are coloured blue, as in (C). Tyr390 is also shown, as a guide for orientating the figure. (E) Chk2/Chk1 comparison. Despite the exchange of T-loops between Chk2 molecules, the orientation, position and conformation of the C-terminal part of this loop (coloured red) is very similar to that of Chk1 (PDB: 1ia8). Download figure Download PowerPoint CHK2-KD is in an ‘active’ state Comparison of the CHK2-KD structure with other known protein structures using DALI (Holm and Sander, 1993) (www.ebi.ac.uk/dali), highlighted the Ser/Thr protein kinases PKA and Chk1 as the most similar, with root-mean-squared deviations of 1.64 and 1.75 Å between 205 and 204 Cα positions, respectively (Krissinel and Henrick, 2004) (PDBs: 1cdk and 1ia8). The CHK2-KD structure is judged to be in an ‘active’ conformation, both by comparison to the PKA and Chk1 structures (also in active conformations (Bossemeyer et al, 1993; Chen et al, 2000)) and through several features conserved throughout protein kinases (Johnson et al, 1996): the essential catalytic residues Lys249, Asp368 and Asp347 (equivalent to Lys72, Asp166 and Asp184 in PKA, respectively) are conserved both in identity and spatial arrangement, the conserved lysine–glutamate salt-bridge (Lys249—Glu273) is maintained, the nucleotide binding site is open and accessible, and the relative orientation of the two lobes with respect to each other is consistent with an active state. Most significantly, the T-loop is fully ordered, with the APE motif and loop tip properly engaged in interactions with the body of the C-lobe, albeit in the dimer-related Chk2 molecule (Figure 3E). The orientation, position and conformation of this C-terminal part of the T-loop in Chk2 is extremely similar to that of PKA and Chk1. However, the helical conformation of the N-terminal side of the Chk2 T-loop is radically different to that of PKA/Chk1, and indeed to all other kinases we are aware of, for which ‘active’ structures have been described. Nucleotide and inhibitor binding Clear electron density is visible for the ADP molecule located in the nucleotide-binding site of CHK2-KD and for the glycine-rich loop, which is fully ordered in the complex and contacts the phosphate groups of the bound ADP (Figure 4A and C). The phosphates additionally receive hydrogen bonds from the side chains of Lys249, Asp368 and Asn352, and are coordinated by one of the two magnesium ions located in the catalytic site. The phenylalanine residue (Phe369) of the adjacent DFG motif is in a ‘flipped out’ conformation. The adenine ring of ADP makes hydrogen bonds to the backbone groups of hinge residues Glu302 and Met304, while the oxygen of the ribose moiety make hydrogen bonds to the side chain of Glu308, and the backbone of Glu351. Figure 4.Nucleotide and drug binding to Chk2 kinase domain. (A) Electron density for ADP bound to CHK2-KD, contoured at a level of 4.5σ from an Fo−Fc omit map. (B) Electron density for the inhibitor DBQ bound to CHK2-KD, contoured at a level of 3σ from an Fo−Fc omit map. (C) LIGPLOT diagram (Wallace et al, 1995) detailing the interactions made between ADP and CHK2-KD. (D) LIGPLOT diagram detailing the interactions made between DBQ and CHK2-KD. In (A) and (B), residues involved in hydrogen bonds to the bound ligand are shown in ‘stick’ representation. Water molecules are shown as grey spheres, and magnesium ions as green spheres. Secondary structure elements corresponding to β-sheets are coloured blue, and α-helices cyan. In (C) and (D), green dotted lines represent hydrogen-bonds (<3.5 Å) whereas brown ‘sunbursts’ represent hydrophobic-type interactions between the protein and ligand. Download figure Download PowerPoint While there is growing interest in Chk2 as a therapeutic target in cancer (Bartek and Lukas, 2003; Collins and Garrett, 2005; Pommier et al, 2005), few highly potent and specific inhibitors have been described. Hymenialdisine is a natural product found in marine sponges, which is an ATP-competitive inhibitor of several protein kinases (Meijer et al, 2000) and derivatives of this are showing promise as Chk2 inhibitors (Sharma and Tepe, 2004). Therefore, as a possible starting point for developing specific Chk2 inhibitors, we have determined the structure of a binary complex between CHK2-KD and DBQ at 2.7 Å resolution; a compound with an IC50 of 3.5 μM (Curman et al, 2001). As expected from biochemical studies, the compound occupies the nucleotide-binding pocket, making hydrogen bonds to several key residues (Figure 4B and D). Principally, two hydrogen bonds are made to the backbone of hinge residues Glu302 and Met304, mimicking the interactions made by the adenine group of bound ADP, a binding-mode equivalent to that reported for other kinase/DBQ complexes (Meijer et al, 2000; Lougheed et al, 2004). Hydrogen bonds are also made between the amino group of the dihydroimidazolone moiety and the side-chains of Glu308 and Asn352, and with the backbone oxygen of Asn352, similar to the contacts made by the ribose and phosphate groups of bound ADP. An additional hydrogen bond is made between the N4H position of DBQ and the side-chain of Glu308. In contrast to other kinase/DBQ complexes, however, no polar interactions, direct or water-mediated, are made to either Asp368 of the DFG motif, or its conserved salt-bridge partner Lys249. Furthermore, no interactions are made between DBQ and the glycine-rich loop, which is disordered in the drug complex. One effect of the extensive hydrogen-bonding network involving Glu308, Asn352 and DBQ is to change the orientation of the dihydroimidazolone moiety with respect to the rest of the DBQ molecule, compared to the conformation in other kinases, a difference that might be exploited in drug development. Chk2 dimerisation and activation The current model for Chk2 activation in response to DNA damage involves two distinct phases of phosphorylation. Initially, Chk2 is primed for activation by phosphorylation of Thr68 within its SCD, principally by the ATM kinase (Matsuoka et al, 2000; Melchionna et al, 2000). Thr68-phosphorylation of Chk2 converts it to a state where it is competent to autophosphorylate two residues in its T-loop, Thr383 and Thr387 (Lee and Chung, 2001). Phosphorylation of Thr68 had been proposed to activate Chk2 by promoting allosteric changes in the kinase domain that facilitate autophosphorylation (Bartek et al, 2001). However, a body of subsequent data have shown that the main effect of Thr68 phosphorylation is to promote homo-dimerisation of Chk2 via interaction of the phospho-Thr68 segment of one molecule with the FHA domain of the other (Ahn et al, 2002; Xu et al, 2002). Once dimerised, one Chk2 molecule can phosphorylate the T-loop in the other Chk2 molecule, to give a fully active species which may then be able to cis-phosphorylate at Thr68 and Ser516 (Schwarz et al, 2003), although the significance of this latter phosphorylation is unknown. The activated conformation and dimeric interactions of CHK2-KD in the crystal structure suggests that it is (or closely resembles) the trans-phosphorylation-competent state engendered by Thr68 phosphorylation. In support of this idea we first sought to determine that the Chk2 kinase domain does indeed have an inherent propensity to dimerise and that the observed dimer is not merely the result of crystallisation. We therefore incubated CHK2-KD in dilute solution (0.1 mg/ml, compared to 20 mg/ml used for crystallisation) in the presence of increasing concentrations of a lysine-specific crosslinking reagent, and analysed the result on SDS–PAGE as previously described (Dajani et al, 2001). At all concentrations of the crosslinker, we observed a consistent crosslinked dimer band, but with no significant higher bands that would indicate nonspecific aggregation of the protein at this concentration, suggesting that CHK2-KD does exist in a monomer–dimer equilibrium in solution (Figure 5A). Although this does not prove that the T-loop exchanged dimer in the crystals is that which forms in solution, the surface area buried by formation of this interface (1344 Å2 per monomer, 2688 Å2 in total) is in the range typically found for functional interactions (Ponstingl et al, 2000) and significantly larger than any other intermolecular contact observed in the crystals. The equilibrium for the isolated kinase domain clearly favours the monomer over the dimer at this low protein concentration (8 μM). However in the intact protein, tethering of two Chk2 molecules via pThr68–FHA domain interactions would massively increase the effective concentration of the associated kinase domains, shifting the equilibrium significantly towards the dimerised state. Figure 5.Dimerisation, Auto- and substrate-phosphorylation of CHK2-KD. (A) SDS–PAGE analysis of CHK2-KD crosslinking. (B) SDS–PAGE analysis of GST-CHK2-KD crosslinking. In each case, a fixed concentration of protein was incubated with increasing concentrations of the homo-bifunctional crosslinker BS3. Mw=molecular mass marker, O=protein not exposed to crosslinking agent, D=dimeric species, M=monomeric species. (C) Autophosphorylation assay. Fixed concentrations of GST-CHK2-KD or CHK2-KD were incubated with γ-32P-labelled ATP for increasing periods of time (as indicated). Samples were analysed by SDS–PAGE, visualised by phosphorimager and autoradiography. (D) Coomassie-stained SDS–PAGE gel of (C). (E) Autophosphorylation specifically occurs within the kinase domain of Chk2. Autophosphorylated samples of GST-CHK2-KD (Lane 1) and CHK2-KD (3) were incubated with rhinovirus 3C-protease (Lanes 2 and 4, respectively) then analysed by SDS–PAGE. Incorporation of γ-32P-labelled ATP was visualised by autoradiography. (F) Coomassie-stained SDS–PAGE gel of (E). (G) DELFIA assay. Samples of CHK2-FL, GST-CHK2-KD and CHK2-KD at varying concentrations were incubated, in the presence of ATP, with a substrate peptide (corresponding to the sequence flanking the Ser216 phosphorylation site of CDC25C). Phosphorylation was detected by incubation with a polyclonal anti-CDC25C(Ser216) antibody, followed by a Europium-labelled secondary antibody. A fluorescent plate reader was then used to quantitate the level of peptide phosphorylation (as Europium counts). (H) Trans-phosphorylation assay. Increasing amounts of CHK2-FL were incubated with a fixed concentration of CHK2-KD (as indicated) in the presence of γ-32P-labelled ATP. Samples were analysed by SDS–PAGE, then visualised by phosphorimager and autoradiography. (I) Coomassie-stained SDS–PAGE gel of (H). Download figure Download PowerPoint We next asked whether the dimeric arrangement of the kinase domains was architecturally consistent with the proposed activation mechanism in which the phosphorylated Thr68 segments of each Chk2 protomer could bind to the FHA domain of the other and promote kinase domain association. The visible C-terminus of the crystal structure of the Chk2 FHA domain (Asp207) is only separated by a couple of residues from the visible N-terminus of the CHK2-KD structure (Ser210), so that the juxtaposition of the two domains is closely constrained, and the FHA domain and N-terminal lobe of the kinase, are likely to be in contact. With this constraint, we constructed a structure-based model for a Chk2 FHA-KD segment dimer. Taking the pThr residue bound to the FHA domain structure to represent pThr68, 20 residues separate the C-terminal end of the phosphopeptide (equivalent to Chk2 residue 72) bound to the FHA domain on one chain, and the visible N-terminus of the FHA domain (Pro92) on the other (Figure 6). The distance between residues 72 and 92 (∼50–70 Å) could be spanned comfortably by this amount of polypeptide chain, so that the model is geometrically reasonable. While this is of course a hypothetical model, that it fits comfortably within the architectural constraints of Thr68-driven dimerisation is reassuring, and further supports the idea that the kinase-domain dimer in the crystals is biologically authentic. Figure 6.Model of the trans-activating Chk2 dimer. Schematic model of a full-length Chk2 dimer. The X-ray structure of the Chk2 FHA domain and its associated phospho-peptide (PDB: 1GXC), along with the CHK2 kinase domain reported here, were used to build a theoretical model for the full-length Chk2 molecule. Predictive modelling of the FHA/Kinase domain junction is facilitated due to the short (2 amino acid) distance between the reported visible termini in each structure (Asp207 and Ser210 respectively), which suggests a spatially constrained arrangement. The position of the FHA-bound phosphothreonine is taken to represent Thr68. The visible C-terminus of the bound peptide therefore equates to Tyr72. The distance between this residue and the visible N-terminus of a dimer-related FHA molecule (Pro92) is estimated to be between 50 and 70 Å. A connecting polypeptide chain of 30 amino acids would be therefore be sufficient to connect the two termini. See Figure 1 for SCD, FHA and KINASE abbreviations. N and C termini for each polypeptide chain are coloured blue and red, respectively. Download figure Download PowerPoint We then sought to determine whether dimerisation per se had any effect on the ability of Chk2 to autopho
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