Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV
2004; Springer Nature; Volume: 23; Issue: 19 Linguagem: Inglês
10.1038/sj.emboj.7600375
ISSN1460-2075
AutoresChristine Koch, Roger Agyei, Sarah Galicia, Pavel Metalnikov, Ed O’Donnell, Andrei Starostine, Michael Weinfeld, Daniel Durocher,
Tópico(s)PARP inhibition in cancer therapy
ResumoArticle23 September 2004free access Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV Christine Anne Koch Corresponding Author Christine Anne Koch Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Radiation Medicine Program, Princess Margaret Hospital (UHN), Toronto, ON, Canada Department of Radiation Oncology, University of Toronto, Toronto, Canada Search for more papers by this author Roger Agyei Roger Agyei Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Sarah Galicia Sarah Galicia Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Pavel Metalnikov Pavel Metalnikov Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Paul O'Donnell Paul O'Donnell Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Andrei Starostine Andrei Starostine Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Michael Weinfeld Michael Weinfeld Cross Cancer Institute, Edmonton, Alberta, Canada Search for more papers by this author Daniel Durocher Corresponding Author Daniel Durocher Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada Search for more papers by this author Christine Anne Koch Corresponding Author Christine Anne Koch Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Radiation Medicine Program, Princess Margaret Hospital (UHN), Toronto, ON, Canada Department of Radiation Oncology, University of Toronto, Toronto, Canada Search for more papers by this author Roger Agyei Roger Agyei Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Sarah Galicia Sarah Galicia Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Pavel Metalnikov Pavel Metalnikov Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Paul O'Donnell Paul O'Donnell Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Andrei Starostine Andrei Starostine Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Search for more papers by this author Michael Weinfeld Michael Weinfeld Cross Cancer Institute, Edmonton, Alberta, Canada Search for more papers by this author Daniel Durocher Corresponding Author Daniel Durocher Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada Search for more papers by this author Author Information Christine Anne Koch 1,2,3, Roger Agyei1, Sarah Galicia1, Pavel Metalnikov1, Paul O'Donnell1, Andrei Starostine1, Michael Weinfeld4 and Daniel Durocher 1,5 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada 2Radiation Medicine Program, Princess Margaret Hospital (UHN), Toronto, ON, Canada 3Department of Radiation Oncology, University of Toronto, Toronto, Canada 4Cross Cancer Institute, Edmonton, Alberta, Canada 5Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada *Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Room 1073, 600 University Avenue, Toronto, Canada ON M5G 1X5. Tel.: +1 416 586 4800 x2544; Fax: +1 416 586 8869; E-mail: [email protected] Medicine Program, Princess Margaret Hospital (UHN), 610 University Avenue, 5th floor Toronto, Canada ON M5G 2M9 The EMBO Journal (2004)23:3874-3885https://doi.org/10.1038/sj.emboj.7600375 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4–DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5′-OH and 3′-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA–Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4–PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation. Introduction DNA double-strand breaks (DSBs) pose a major threat to cell survival and genome stability. If left unrepaired, DSBs can result in the loss of genetic material or cell death. Moreover, mutations or gross genetic aberrations can occur if DSB repair (DSBR) is inaccurate (Mills et al, 2003). Great efforts are therefore made by the cell to prevent, detect, signal and repair DSBs. In vertebrates, a major DSBR pathway is nonhomologous end joining (NHEJ). NHEJ is active throughout the cell cycle, although it predominates during G0/G1 (Lieber et al, 2003). In addition, NHEJ plays a key role in the processing and repair of the developmentally regulated DSBs that occur during V(D)J recombination, the process by which T-cell receptor and antibody diversity is established (Gellert, 2002). NHEJ has several known components: the DNA-PK holoenzyme (composed of the DNA end-binding complex Ku70/Ku80 and the DNA-PKcs serine/threonine kinase; Smith and Jackson, 1999), the Artemis nuclease and the Xrcc4–DNA ligase IV complex (Lieber et al, 2003). Mammalian cells deficient in these NHEJ components share phenotypes that include sensitivity to ionizing radiation (IR) and impaired V(D)J recombination, underscoring their critical role in DSBR (Lieber et al, 2003). Most models of mammalian NHEJ do not take into account the requirement for DNA end processing prior to ligation. Indeed, most sources of DNA damage either produce nonligatable chemical modifications or generate protein–DNA adducts at DNA termini that must require processing prior to DNA ligation. For example, IR produces nonligatable 5′-OH and 3′-phosphate DNA termini among other types of ends (Coquerelle et al, 1973). Therefore, the recruitment and regulation of specific DNA end processors is critical for NHEJ depending on the type of terminal groups present at DSBs. The Artemis protein was recently shown to possess overhang endonucleolytic activity and is perhaps the best characterized example of a DNA end processor associated with NHEJ (Lieber et al, 2003). Artemis forms a complex with DNA-PKcs and its interaction with, along with its phosphorylation by, DNA-PKcs is required to convert Artemis from an exonuclease to an endonuclease (Ma et al, 2002). These findings link DSB detection by DNA-PK to DNA end processing by Artemis. However, Artemis cannot process every type of nonligatable ends and therefore other types of end-processing enzymes must act during NHEJ. The mammalian polynucleotide kinase (PNK) enzyme is a good candidate for an NHEJ DNA end processor as it contains both 5′-DNA kinase and 3′-DNA phosphatase activities that could specifically act during the repair of 5′-OH and 3′-phosphate-modified DNA termini (Karimi-Busheri et al, 1999). Interestingly, recent studies from Chappell and colleagues, using an in vitro NHEJ system, identified PNK as a key factor for the processing of DSBs with 5′-OH DNA termini in vitro (Chappell et al, 2002). In addition to its potential role in NHEJ, PNK has a well-established role in single-strand break repair (SSBR), where it interacts with a multi-protein complex that includes the protein Xrcc1 (Whitehouse et al, 2001). In metazoans, from Drosophila to humans, PNK orthologs also harbor a putative FHA domain N-terminal to their catalytic domains. The FHA domain is found in a number of proteins of diverse functions, where it acts as a phosphothreonine-binding module (Durocher and Jackson, 2002). The presence of an FHA domain on PNK therefore suggests that PNK engages in phosphorylation-dependent interactions and indicates that the assembly of the SSBR or DSBR machineries may be in part controlled by phosphorylation. In this study, we sought to identify phosphorylation-dependent PNK-interacting proteins, identify the kinase(s) responsible for these interactions, as well as characterize the functional importance of these interactions in DNA repair. Here, we show that PNK physically interacts with Xrcc4 in a phosphorylation- and FHA-dependent manner. Disruption of the Xrcc4–PNK interaction is associated with increased sensitivity to IR, concomitant with a diminished efficiency of DNA end joining in vitro. This study therefore identifies the mechanism by which PNK participates in NHEJ and uncovers a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation. Results PNK interacts with Xrcc4 in an FHA-dependent manner To identify proteins that specifically interact with the PNK FHA domain, we purified the PNK FHA domain as a GST fusion protein (PNKFHA) and separately purified a mutated version of the PNK FHA domain substituting a conserved arginine residue (Arg35) to alanine (PNKFHA-R35A), a mutation shown in other FHA domains to be essential for phosphothreonine binding (Durocher and Jackson, 2002). Both purified fusion proteins were immobilized on glutathione–sepharose beads and incubated with whole-cell extracts (WCEs) from human HEK293T cells. We excised a total of 18 bands that displayed selective interaction with the PNKFHA domain and prepared them for tandem mass spectrometry. As a control to ensure identification of proteins retrieved in a PNKFHA-dependent fashion, corresponding gel slices in the PNKFHA-R35A lane were also excised and analyzed by mass spectrometry. As shown in Table I, the PNKFHA protein, in contrast to PNKFHA-R35A, selectively retrieved SSBR components known to interact with PNK (Xrcc1, DNA ligase III and PARP; Whitehouse et al, 2001), indicating that PNK may interact with Xrcc1 in an FHA domain-dependent manner, a result recently confirmed by the Caldecott group (Loizou et al, 2004; see Table I). We also identified a number of novel PNK interactors such as NHEJ components (Xrcc4, the Ku70/Ku80 heterodimer and DNA-PKcs), as well as other regulators of DNA repair and chromatin remodeling such as components of the FACT complex (Table I). The interaction between PNK and NHEJ components was of particular interest, given the recent report by Chappell et al (2002) that suggests a role for PNK during NHEJ. The physical and functional association of PNK with NHEJ components was therefore further investigated. Table 1. Summary of MS data Banda Protein Peptides identified Accession numberb Functionc a Xrcc4 10 gi∣12408647 NHEJ HnRNP H1 5 gi∣5764101 RNA metabolism b AdoHcyase 9 gi∣6094223 Metabolism c DKFZp451l037 6 gi∣34364737 Unknown TRIM26 3 gi∣4508005 Unknown NASP 3 gi∣27262628 Binds histone H1 d Ku70 30 gi∣4503841 NHEJ e Nohitswith>2 peptides f Ku80 33 gi∣10863945 NHEJ Xrcc1 8 gi∣40226177 SSBR SSRP1 3 gi∣4507241 Part of FACT complex; DNA repair g MCM5 19 gi∣1232079 DNA replication Xrcc1 10 gi∣5454172 SSBR i MCM3 22 gi∣1552242 DNA replication DNA ligase III 25 gi∣7710126 SSBR j Gemin 4 11 gi∣10503982 Protein of 'Gem bodies' PITSLRE 8 gi∣3978441 Protein kinase DNA ligase III 8 gi∣7710126 SSBR k PARP 10 gi∣190167 SSBR DDB1 3 gi∣13435359 NER (UV-binding protein) HnRNP U 3 gi∣14141161 Matrix-associated protein l NOPP140 3 gi∣12804871 Nucleolar protein m FACT140 10 gi∣6005757 Chromatin remodeling NRD-C 6 gi∣29840826 Protease NASP 4 gi∣27262628 Binds histone H1 n KIAA1035 7 gi∣20521740 Unknown TCOF1 3 gi∣15079955 Treacher–Collins syndrome o TCOF1 7 gi∣4507411 Treacher-Collins syndrome VprBP 3 gi∣7662316 Binds HIV-1 Vpr p E2-230K 12 gi∣12698013 Ubiquitin conjugation TCOF1 3 gi∣4507411 Treacher–Collins syndrome q TCOF1 17 gi∣1778432 Treacher–Collins syndrome RBAF600 4 gi∣4557447 Rb-associated factor CHD1 7 gi∣24416002 Chromatin remodeling r DNA-PKcs 11 gi∣13654237 NHEJ RBAF600 13 gi∣24416002 Rb-associated factor ATRX 8 gi∣20336207 Alpha-thalassemia/mental retardation syndrome/chromatin remodeling s RBAF600 56 gi∣24416002 Rb-associated factor a Refer to Figure 1A. b Accession number retrieved by the Mascot search engine. c NHEJ: nonhomologous end joining; SSBR: single-strand break repair; NER: nucleotide excision repair. We first sought to determine whether the NHEJ components identified by mass spectrometry interacted with full-length PNK in an FHA-dependent manner. Full-length, epitope-tagged, PNK or PNKR35A were expressed in human HEK293T cells and tested for their ability to physically interact with NHEJ proteins in co-immunoprecipitation experiments. As shown in Figure 1B, PNK co-immunoprecipitates with Xrcc4 and DNA ligase IV, and these interactions are dependent on the phosphothreonine-binding activity of the FHA domain, as PNKR35A fails to interact with either protein. However, the potential interaction between PNK and the DNA-PK holoenzyme was not confirmed by co-immunoprecipitation and therefore was not investigated further. The PNK–DNA ligase IV interaction is also detected when the direction of the co-immunoprecipitation is reversed (Figure 1C) and, importantly, the interaction between PNK and the Xrcc4-DNA ligase IV complex is not bridged by DNA since the interaction is resistant to 50 μg/ml of the DNA intercalating agent ethidium bromide (Figure 1D). Finally, co-immunoprecipitation experiments using anti-PNK antibodies confirmed that the interaction between endogenous Xrcc4 and PNK occurs in vivo (Figure 1E). These results indicate that PNK interacts with the Xrcc4–DNA ligase IV complex in human cells and that their association is dependent on a functional PNK FHA domain. Figure 1.PNK physically interacts with Xrcc4 in an FHA-dependent manner. (A) Recombinant GST-PNKFHA and -PNKFHA-R35A proteins were incubated with HEK293T WCEs, the interacting proteins were resolved on SDS–PAGE, stained with GelCode, and prepared for mass spectrometry. The identities of the proteins found in gel slices are found in Table I. (B) WCE from HEK293T cells transfected with V5-tagged PNK, PNKR35A or empty vector (V) were immunoprecipitated with an anti-V5 antibody. The precipitates or WCEs were separated on SDS–PAGE, and probed to detect PNK, Xrcc4, DNA ligase IV, DNA-PKcs and Ku70/Ku80 as indicated. (C) WCEs from HEK293T cells transfected with DNA ligase IV, V5-tagged PNK or empty vector were immunoprecipitated with anti-DNA ligase IV antibody, followed by anti-V5 immunoblotting (top panel). WCEs were also probed with anti-DNA ligase IV antibody (middle panel) and anti-V5 antibody (bottom panel). We note that, for the Xrcc4–PNK interaction, the immunoprecipitation cannot be reversed for technical reasons. (D) WCEs from (A) treated with or without ethidium bromide were immunoprecipitated with anti-V5 antibody and probed with anti-Xrcc4 and DNA ligase IV antibodies. (E) HEK293T WCEs were immunoprecipitated with anti-PNK antibody, followed by immunoblotting with anti-Xrcc4 antibody or anti-PNK antibody. (F) Recombinant GST, PNKFHA, or PNKFHA-R35A were mixed in PD assays with HEK293T WCEs treated with or without λ protein phosphatase (λp'tase) as indicated. As a control, 2.5% of input WCE was loaded. The samples were resolved on SDS–PAGE and probed with anti-Xrcc4 antibody. Download figure Download PowerPoint Since FHA domains are phosphothreonine recognition modules, we examined if protein phosphorylation was required for the PNK–Xrcc4 interaction. We used the PNKFHA pull-down (PD) assay used in Figure 1A, as a means to rapidly monitor the PNKFHA–Xrcc4 interaction. As shown in Figure 1F, PNKFHA efficiently retrieves Xrcc4 from WCEs and the interaction is essentially abolished by the Arg35Ala mutation. Furthermore, λ protein phosphatase treatment of cell extracts prior to incubation with PNKFHA completely abolishes the interaction between Xrcc4 and PNKFHA. These results support the notion that the interaction is phosphorylation-dependent and indicate that the PNK FHA domain is both sufficient and essential to promote an interaction with Xrcc4. Threonine 233 (Thr233) of Xrcc4 is required for binding to PNK Next, we sought to determine the region of Xrcc4 required for its association with PNK. A series of epitope-tagged Xrcc4 constructs were generated (Figure 2A), expressed in HEK293T cells and tested for their ability to interact with PNKFHA in PD assays. As shown in Figure 2B, PNKFHA efficiently retrieves Xrcc4 and Xrcc4250Δ from WCEs, but is unable to interact with Xrcc4213Δ or Xrcc4179Δ. From these observations, we conclude that the PNK–Xrcc4 interaction requires a region of Xrcc4 encompassing amino-acid residues 213–250. Figure 2.The Xrcc4 Thr233 residue is required for interaction with PNK. (A) Schematic representation of Xrcc4 proteins analyzed for interaction with PNK: wild-type Xrcc4; Xrcc4250Δ (residues 1–250); Xrcc4 213Δ (residues 1–213); Xrcc4 179Δ (residues 1–179). Also indicated is the DNA-ligase IV Xrcc4-interacting region (residues 179–213), and the portion of Xrcc4 important for mediating homodimerization (residues 115–204). (B) WCEs from HEK293T cells transfected with V5-tagged Xrcc4 constructs (or empty vector as control) were incubated with recombinant GST-PNKFHA in PD assays. PNKFHA-interacting proteins were resolved by SDS–PAGE, and detected with an anti-V5 antibody (top panel). As a control, 20 μg of WCE was analyzed by immunoblotting (bottom panel). (C) Conserved threonine residues of Xrcc4 were mutated to alanine, and the resulting mutants were expressed in HEK293T cells. PD assays were carried out with PNKFHA, and Xrcc4 was detected by anti-V5 immunoblotting (top panel). In the bottom panel, 20 μg of WCE was immunoblotted with anti-V5 antibody to control for protein expression. (D) CHO Xrcc4-deficient (XR-1) cell lines stably transfected with Xrcc4WT, Xrcc4T233A, Xrcc4250Δ, Xrcc4250ΔT233A, or an empty vector were treated with or without 10 Gy of X-rays and assessed for their ability to interact with PNKFHA in PD assays and detected by immunoblotting (IB) with anti-V5 antibody (top panel). The blot was stripped and immunoblotted with anti-DNA ligase IV antibody (middle panel). Examination of WCEs (bottom panel) by anti-V5 immunoblotting revealed similar expression levels between the cell lines. (E) WCEs from XR-1 lines transfected with Xrcc4WT, Xrcc4T233A or a control vector were immunoprecipitated (IPs) with the anti-V5 antibody and probed for the presence of DNA ligase IV by anti-DNA ligase IV immunoblotting. DNA ligase IV was expressed at similar levels in both cell lines, as revealed by anti-DNA ligase IV immunoblotting from WCEs. DNA-ligase IV is indicated by the top arrow, and the bottom arrow represents a faster migrating nonspecific (NS) band. Download figure Download PowerPoint Since FHA domains are phosphothreonine-binding modules, we next assessed whether conserved threonine residues on Xrcc4 are required for the Xrcc4–PNK interaction. Five highly conserved threonine residues in Xrcc4 were mutated to alanine (Thr233, 264, 282, 306 and 321; see Supplementary Figure 1A). Of these, Thr321 is known to be phosphorylated by DNA-PK (Yu et al, 2003), making it a particularly interesting candidate. The resulting Xrcc4 mutants were expressed in HEK293T cells and assessed for binding to PNKFHA in PD assays (Figure 2C). We found that only the Thr233Ala mutation resulted in the impairment of the Xrcc4–PNKFHA interaction. Notably, the location of this residue in Xrcc4 is in agreement with the regional mapping data presented in Figure 2B, which located the PNK-binding site between amino-acid residues 213 and 250, and suggests that the PNK FHA domain recognizes an epitope encompassing Thr233 on Xrcc4. Since Xrcc4 is oligomeric in nature (dimeric or tetrameric; Junop et al, 2000; Lee et al, 2000), we sought to ensure that multimerization with endogenous Xrcc4 present in HEK293T extracts did not enable some Xrcc4 mutants to interact with the PNK FHA domain. We therefore generated stable cell lines expressing V5-epitope-tagged Xrcc4, Xrcc4250Δ, Xrcc4T233A and Xrcc4250ΔT233A in the Xrcc4-deficient CHO cell line XR-1, and tested their ability to be bound by PNKFHA in PD assays (Figure 2D). Again, we confirmed that the Xrcc4 Thr233 residue is critical for association with the PNK FHA domain, both in the context of full-length Xrcc4 and Xrcc4250Δ proteins (Figure 2D). Interestingly, the association between Xrcc4 and PNK is not modulated by DNA damage as the interaction is observed at similar levels after 10 Gy of X-rays (Figure 2D). In addition, the binding of DNA ligase IV to PNKFHA also depends on the Xrcc4 Thr233 residue (Figure 2D). This observation raises the possibility that DNA ligase IV interacts with PNK via a bridging interaction with Xrcc4. To rule out the possibility that the loss of the DNA ligase IV–PNK interaction in cells expressing Xrcc4T233A was due to a disruption of the Xrcc4–DNA ligase IV complex, Xrcc4 or Xrcc4T233A proteins were immunoprecipitated from XR-1 stable transfectants and examined for association with DNA ligase IV (Figure 2E). We found that DNA ligase IV interacts equally well with wild-type Xrcc4 or with Xrcc4T233A. As DNA ligase IV stability is dependent on its association with Xrcc4 (Bryans et al, 1999), we also note that Xrcc4T233A stabilizes endogenous DNA ligase IV to the same extent as wild-type Xrcc4. Collectively, these results are consistent with the formation of a tripartite complex comprising PNK, Xrcc4 and DNA ligase IV and that the PNK–Xrcc4 interaction requires Thr233 on Xrcc4. PNK FHA domain displays a novel phosphothreonine-binding mode The results presented above suggest that Thr233 may be phosphorylated and bound by the PNK FHA domain. We thus tested if peptides derived from Xrcc4 and containing the sequence surrounding Thr233 could interact with PNKFHA or PNKFHA-R35A proteins in peptide PD assays. Biotinylated, Thr233-phosphorylated and unphosphorylated Xrcc4-derived peptides (designated as T233P and T233 peptides, respectively; Figure 3A) were employed in peptide-binding assays to assess whether the interaction is phosphorylation-dependent. Both peptides were coupled to streptavidin magnetic beads and incubated with PNK and PNKR35A. As shown in Figure 3B, only the phosphorylated peptide (T233P) is able to bind appreciably to PNK. Conversely, when PNK and PNKR35A were compared for their ability to bind to T233P, only PNK bound efficiently (Figure 3B). These results indicate that the FHA domain of PNK is required to directly interact with an epitope on Xrcc4 surrounding the phosphorylated threonine at position 233. Figure 3.The PNK FHA domain binds to phosphorylated Thr233. (A) Sequence (in single-letter amino acids) of the biotinylated unphosphorylated (T233) and phosphorylated (T233P) peptides from Xrcc4 corresponding to amino acids 229–238. Two C-terminal lysines and two N-terminal glycine residues were also added. (B) The immobilized T233P peptide was mixed with recombinant GST fusion proteins (PNK or PNKR35A proteins), and peptide binding was detected by anti-GST immunoblotting (top panel). In the bottom panel, PNK was incubated with the T233 or T233P coupled peptides, and association was assessed by anti-GST immunoblotting. Input amounts of the recombinant proteins (for both the upper and lower panels) are indicated in the middle panel, and have also been detected by probing with anti-GST antibody. Download figure Download PowerPoint The PNK FHA domain is part of a newly identified class of FHA domains comprising the FHA domain of aprataxin (Caldecott, 2003). Interestingly, iterative BLAST searches with the FHA domain of PNK identify a third member of this subfamily of FHA domains (MGC47799, Supplementary Figure 2). In order to test whether this FHA domain subfamily may display a unique mode of phosphopeptide recognition, we characterized the binding properties of the PNK FHA domain in more detail. First, we determined the dissociation constant for the interaction between purified PNKFHA and the T233P peptide using isothermal titration calorimetry (ITC; Figure 4A) and fluorescence polarization (FP), using a fluorescein-labeled version of the T233P peptide (Figure 4B). We obtained a KD of 4.1 μM using ITC and 4.2 μM by FP. A similar dissociation constant is also obtained when full-length recombinant PNK is used in ITC experiments (KD of 2.5 μM; Mark Glover, personal communication). The low micromolar affinity of the T233P peptide–PNKFHA interaction is within the range of dissociation constants calculated for other FHA-peptide-binding interactions (Durocher and Jackson, 2002). Figure 4.The PNK FHA domain demonstrates unique phosphopeptide-binding selectivity. (A) Peptide-binding affinity determined by ITC. A representative ITC trace is shown outlining the binding of the T233P phosphopeptide to recombinant PNKFHA. (B) PNKFHA binding to fluorescein-labeled 7816 peptide was quantitated by FP. The calculated dissociation constant is indicated. (C) PNKFHA domain binding to a filter array of peptides. The PNKFHA domain was detected by immunoblotting with anti-GST antibody. The resulting consensus binding motif is indicated on the right column. 'X' indicates no dominant selection, and letters enclosed in square brackets are specifically counter-selected. The left row indicates the amino acid that was substituted in the Xrcc4 peptide sequence and the top row indicates to which amino acid the residue was substituted for. (D, E) PNKFHA binding to 7814 (D) or 7815 peptides (E) was quantitated by FP. A dissociation constant could not be calculated for the 7815 peptide. Download figure Download PowerPoint To further characterize the binding specificity of the PNK FHA domain, we synthesized a peptide array based on the sequence encompassing Thr233 of Xrcc4 on a cellulose membrane, using a SPOT peptide synthesizer. We tested the ability of PNKFHA to interact with the peptide array using Southwestern blotting. As shown in Figure 4C, substitution of residues located C-terminal to the phosphothreonine does not, in large part, affect binding to PNKFHA. However, substitution at the -2 or -3 positions relative to pThr can only be tolerated by a few amino acids (Arg/Asp/Cys/Glu at -2, and Asn/Asp/Cys/Trp at -3), indicating that the binding selectivity of the PNK FHA domain resides N-terminal to the phosphothreonine residue. This result was unexpected given that every FHA domain characterized to date possesses major binding selectivity determinants C-terminal to the phosphothreonine (Durocher and Jackson, 2002). To confirm this unique binding selectivity, we generated two additional fluorescein-labeled peptides (Figure 4D and E), designed to either disrupt or maintain PNKFHA binding, and examined the interactions using FP. As predicted from the peptide array data, the peptide containing alanine substitutions at the -2 and -3 positions abolished PNKFHA binding (Figure 4E). In contrast, substitution of five out of nine amino acids in the native Xrcc4 sequence for residues expected to maintain binding did in fact augment PNKFHA-binding affinity relative to the wild-type peptide (KD of 2.3 versus 4.2 μM, respectively; Figure 4B and D). These results are consistent with the notion that positions N-terminal to the phosphothreonine site, in particular the -2 and -3 positions, are important for determining PNKFHA-binding specificity. Therefore, among FHA domains, PNKFHA recognizes phosphopeptides with a unique mode of binding selectivity. Xrcc4 Thr233 is a CK2 phosphorylation site The amino-acid sequence surrounding Thr233 (Asp–Glu–Ser–Thr233–Asp–Glu–Glu) is highly acidic and suggests that Thr233 may be a target for acidophilic kinases. Indeed, analysis of the Xrcc4 sequence in Scansite (Yaffe et al, 2001) reveals that the sequence encompassing Thr233 conforms to a consensus CK2 phosphorylation site (along with Ser232). We therefore tested if the Xrcc4-derived peptide containing Thr233 (T233 peptide, and see above) could act as a substrate for CK2. As shown in Figure 5A, the T233 peptide is efficiently phosphorylated by recombinant CK2. As a control, we also assessed if the phosphorylated T233P peptide could act as a substrate for CK2. Figure 5A shows that the T233P peptide is a much poorer substrate for recombinant CK2 relative to the T233 peptide. The residual phosphorylation of the phosphopeptide may be explained by some phosphorylation at the serine equivalent to Ser232 on Xrcc4, which also conforms to a CK2 consensus phosphorylation site. Figure 5.The Thr233 residue of Xrcc4 is phosphorylated by CK2. (A) The T233 and T233P peptides were used in CK2 protein kinase assays. The histogram values represent the corrected means of at least two indep
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