PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Use of Quantitative Mass Spectrometric Analysis to Elucidate the Mechanisms of Phospho-priming and Auto-activation of the Checkpoint Kinase Rad53 in Vivo

The cell cycle checkpoint kinases play central roles in the genome maintenance of eukaryotes. Activation of the yeast checkpoint kinase Rad53 involves Rad9 or Mrc1 adaptor-mediated phospho-priming by Mec1 kinase, followed by auto-activating phosphorylation within its activation loop. However, the mechanisms by which these adaptors regulate priming phosphorylation of specific sites and how this then leads to Rad53 activation remain poorly understood. Here we used quantitative mass spectrometry to delineate the stepwise phosphorylation events in the activation of endogenous Rad53 in response to S phase alkylation DNA damage, and we show that the two Rad9 and Mrc1 adaptors, the four N-terminal Mec1-target TQ sites of Rad53 (Rad53-SCD1), and Rad53-FHA2 coordinate intimately for optimal priming phosphorylation to support substantial Rad53 auto-activation. Rad9 or Mrc1 alone can mediate surprisingly similar Mec1 target site phosphorylation patterns of Rad53, including previously undetected tri- and tetraphosphorylation of Rad53-SCD1. Reducing the number of TQ motifs turns the SCD1 into a proportionally poorer Mec1 target, which then requires the presence of both Mrc1 and Rad9 for sufficient priming and auto-activation. The phosphothreonine-interacting Rad53-FHA domains, particularly FHA2, regulate phospho-priming by interacting with the checkpoint mediators but do not seem to play a major role in the phospho-SCD1-dependent auto-activation step. Finally, mutation of all four SCD1 TQ motifs greatly reduces Rad53 activation but does not eliminate it, and residual Rad53 activity in this mutant is dependent on Rad9 but not Mrc1. Altogether, our results provide a paradigm for how phosphorylation site clusters and checkpoint mediators can be involved in the regulation of signaling relay in protein kinase cascades in vivo and elucidate an SCD1-independent Rad53 auto-activation mechanism through the Rad9 pathway. The work also demonstrates the power of mass spectrometry for in-depth analyses of molecular mechanisms in cellular signaling in vivo.Eukaryotic cells are most vulnerable to exogenous DNA-damaging agents during the S phase of the cell cycle, when unprogrammed DNA lesions interfere with the tightly choreographed DNA replication process. DNA damage during this phase leads to the activation of two overlapping checkpoint pathways in Saccharomyces cerevisiae, the DNA replication checkpoint and the intra-S-phase DNA damage checkpoint (1, 2). Phospho-priming for auto-activation of the central checkpoint kinase Rad53 by the upstream kinase Mec1/Tel1 depends on Mrc1 as an adaptor in the DNA replication checkpoint pathway and Rad9 as an adaptor in the DNA damage checkpoint pathway (3–10). Rad53, a well-accepted model system for studying the function and regulation of Chk2-like kinases, contains two forkhead-associated (FHA)¹ domains (FHA1 and -2) and two SQ/TQ cluster domains (SCD1 and -2) enriched in Mec1/Tel1-target phosphorylation sites (11–13).Mrc1 normally is a replisome component that functionally couples DNA Pol ε with Cdc45 and MCM helicase during replication fork progression (14, 15). As the replication forks are stalled by replication stress, the recruited checkpoint sensor kinase Mec1 phosphorylates the SCD of Mrc1, which abolishes its N-terminal interaction with Pol ε and enables Mrc1 to recruit Rad53 and promote Rad53 phosphorylation by Mec1 as an initial step in the activation of Rad53 in the Mrc1 branch (6, 14, 16). Alanine substitution of all Mec1 target sites of Mrc1 (designated the mrc1-AQ allele) has been shown to selectively disable its checkpoint function for Rad53 activation without affecting its DNA replication functions (4). In response to DNA damage, Rad9 is able to associate with damaged chromatin via its BRCT and Tudor domains, which tether it to Ser129-phosphorylated histone H2A (γH2A) and Lys79-methylated histone H3, respectively (17, 18). Alternatively, the recruitment of Rad9 onto damaged DNA could also be facilitated by its phosphorylation by CDK1, which enables the specific interaction of Rad9 with Dpb11, allowing the formation of the ternary complex of Dpb11, Mec1, and Rad9 (19, 20). Similar to Mrc1, Mec1 activates the adaptor function of Rad9 by phosphorylation of its SCD, which then binds to the Rad53-FHA domains to promote Rad53 phosphorylation by Mec1 (3, 5, 10).Beyond serving as scaffolds to recruit Rad53, Mrc1 and Rad9 have been shown to promote Rad53 phosphorylation by Mec1 in a dose-dependent manner in vitro (3, 16), underlining their adaptor role to enhance the enzyme–substrate (Mec1–Rad53) interaction. However, how they can specifically regulate the priming phosphorylation at specific sites and how this then leads to Rad53 activation remains poorly understood. Finally, hyperphosphorylated Rad9 has also been shown to catalyze the auto-phosphorylation of recombinant Rad53 (21), but it remains to be examined whether and how this occurs in vivo.The activation of SCD-FHA containing kinases such as human Chk2 and fission yeast Cds1 has been suggested to involve a two-step phosphorylation process: first, SCD phosphorylation by an ATM/ATR-like kinase leads to intermolecular binding to the FHA domain of another Chk2/Cds1 monomer, which then results in dimerization/oligomerization-dependent auto-phosphorylation within the kinase activation loop (22–26). In addition to the characteristic N-terminal SCD-FHA module of Chk2-like kinases, Rad53 contains another SCD2-FHA2 module C-terminal to its kinase domain. Similar to its orthologues, Rad53 activation has been proposed to depend on SCD1 phosphorylation (but not SCD2 phosphorylation) and partially redundant functions of the two FHA domains (9, 27–29). However, although Rad53-FHA1 can interact with SCD1 in a phospho-threonine (pT)-dependent manner in vitro (9, 28), it appears to be required for Rad53 activation only in G2/M-arrested cells (27, 29). In contrast, the FHA2 domain, which seems to be more important overall for Rad53 activation, does not appreciably bind phospho-SCD1 peptides in vitro (27, 28). Thus, the mechanisms by which Mrc1, Rad9, SCD1 phosphorylation, and FHA domains interact during checkpoint-dependent Rad53 priming and auto-activation remain to be elucidated.Quantitative mass spectrometric analysis has revolutionized the functional analysis of cellular signaling pathways, including site-specific phosphorylation events of key signaling molecules (30–33), but an important caveat is that MS studies often involve protein tags or nonphysiological expression levels that can interfere with normal protein functions. For example, the integration of a triple HA tag into the endogenous RAD53 gene locus has been shown to reduce Rad53 protein levels, resulting in significantly altered checkpoint activity (34). In this study we used quantitative MS analyses to dissect the stepwise phosphorylation events of endogenous, untagged Rad53 in response to MMS-induced alkylation DNA damage and replication stress during the S phase. Together with functional analyses, our results delineate how the two Mec1 adaptors Rad9 and Mrc1 can coordinate with the four SCD1 priming sites (T5, T8, T12, and T15) to regulate the phospho-priming of Rad53 by Mec1. In addition, an SCD1-priming independent Rad53 auto-activation mechanism and the specific roles of the FHA domains during Rad53 hyperphosphorylation are also elucidated in this work.     

Two Regions of Simian Virus 40 T Antigen Determine Cooperativity of Double-Hexamer Assembly on the Viral Origin of DNA Replication and Promote Hexamer Interactions during Bidirectional Origin DNA Unwinding

Phosphorylation of simian virus 40 large tumor (T) antigen on threonine 124 is essential for viral DNA replication. A mutant T antigen (T124A), in which this threonine was replaced by alanine, has helicase activity, assembles double hexamers on viral-origin DNA, and locally distorts the origin DNA structure, but it cannot catalyze origin DNA unwinding. A class of T-antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has remarkably similar properties, although these proteins are phosphorylated on threonine 124, as we show here. By comparing the DNA binding properties of the T124A and class 4 mutant proteins with those of the wild type, we demonstrate that mutant double hexamers bind to viral origin DNA with reduced cooperativity. We report that T124A T-antigen subunits impair the ability of double hexamers containing the wild-type protein to unwind viral origin DNA, suggesting that interactions between hexamers are also required for unwinding. Moreover, the T124A and class 4 mutant T antigens display dominant-negative inhibition of the viral DNA replication activity of the wild-type protein. We propose that interactions between hexamers, mediated through the DNA binding domain and the N-terminal phosphorylated region of T antigen, play a role in double-hexamer assembly and origin DNA unwinding. We speculate that one surface of the DNA binding domain in each subunit of one hexamer may form a docking site that can interact with each subunit in the other hexamer, either directly with the N-terminal phosphorylated region or with another region that is regulated by phosphorylation.

The initiation of simian virus 40 (SV40) DNA replication by the viral T antigen is a complex series of events that begins when T antigen binds specifically to a palindromic arrangement of four GAGGC pentanucleotide sequences in the minimal origin of viral DNA replication (recently reviewed in references 1, 2, 3, 22, and 48). In the presence of Mg-ATP, T antigen assembles cooperatively on the two halves of the palindrome as a double hexamer (10, 11, 13, 24, 30, 38, 51, 53). The DNA conformation flanking the T-antigen binding sites is locally distorted upon hexamer assembly (reference 7 and references therein). One pair of pentanucleotides is sufficient to direct double-hexamer assembly and local distortion of the origin DNA but not to initiate DNA replication (25). ATP hydrolysis by T-antigen hexamers then catalyzes bidirectional unwinding of the parental DNA (reference 53 and references therein). A mutant origin with a single nucleotide insertion in the center of the palindromic T-antigen binding site prevents cooperative interactions between hexamers and cannot support bidirectional origin unwinding (8, 51), suggesting that both processes require interactions between T-antigen hexamers. After assembly of the two replication forks, bidirectional replication is carried out by 10 cellular proteins and T antigen, which remains at the forks as the only essential helicase (reviewed in references 3, 22, and 48).The phosphorylation state of SV40 T antigen governs its ability to initiate viral DNA replication (reviewed in references 15 to 17 and 39). T antigen contains two clusters of phosphorylation sites located at the N and C termini (40, 41). Phosphorylation of T antigen on threonine 124 in the N-terminal cluster was shown to be essential for viral DNA replication in monkey cells and in vitro (5, 14, 32–36, 44). Efforts to define what step in viral DNA replication requires modification of threonine 124 revealed that Mg-ATP-induced hexamer formation of T antigen in solution and DNA helicase activity of T antigen did not require phosphorylation at this site (33, 36). Origin DNA binding of T antigen lacking the modification at residue 124 was weaker than that of the modified T antigen (33, 34, 36, 44), but the reduction in binding was modest under the conditions used for SV40 DNA replication in vitro (36). Moreover, a mutant T antigen containing alanine in place of the phosphorylated threonine (T124A) assembled as a double hexamer on the viral origin and altered the conformation of the early palindrome and AT-rich sequences flanking the T-antigen binding sites in the viral origin in the same manner as the wild-type protein, except that higher concentrations were required (36). However, even at an elevated concentration, these mutant double hexamers were unable to unwind closed circular duplex DNA containing the viral origin (33, 36), suggesting that the defect in unwinding was responsible for the inability of T124A T antigen to replicate SV40 DNA. One possible explanation for the unwinding defect of the mutant T antigen, despite its helicase activity, was that some essential interaction between the two hexamers during bidirectional unwinding depended upon phosphorylation of threonine 124. Electron micrographs of SV40 DNA unwinding intermediates, which showed two single-stranded DNA loops protruding between two hexamers of T antigen, provided support for this explanation, implying that a double hexamer pulled the parental duplex DNA into the protein complex and spooled the single-stranded DNA out (53). Furthermore, double-hexamer formation significantly enhanced the helicase activity of T antigen (47, 47a).Most of the T antigen isolated from mammalian cells is in a hyperphosphorylated form, containing multiple phosphoserines, as well as two phosphothreonines, and supports SV40 DNA replication in vitro poorly but can be stimulated by treatment with alkaline phosphatase or protein phosphatase 2A (19, 28, 37, 42, 49, 50). Hyperphosphorylated T antigen is unable to unwind duplex closed circular duplex DNA harboring the viral origin (4, 6, 51). Dephosphorylation of serines 120 and 123 restores its ability to unwind origin DNA (14, 43, 51). Studies of double-hexamer assembly on the origin indicate that phosphorylation of T antigen on serines 120 and 123 also impairs the cooperativity of double-hexamer assembly (14, 51). These results demonstrate that hyperphosphorylation of T antigen interferes with interactions between hexamers that are required for origin unwinding and raise the question of whether the phosphorylation state of threonine 124 might also affect the cooperativity of double-hexamer assembly on the viral origin.One class of T antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has been reported to display properties similar to those of the T124A mutant and the hyperphosphorylated form of T antigen (54). Class 4 mutant proteins are defective in viral DNA replication in vivo and in vitro, bind to the viral origin as double hexamers and alter the local DNA conformation, and have helicase activity but do not unwind closed circular duplex viral DNA. The replication and unwinding defects could be due to faulty phosphorylation patterns or to other malfunctions not dependent on phosphorylation status.The work presented here was undertaken to reevaluate the assembly of wild-type and T124A T antigen on SV40 origin DNA by using more-sensitive quantitative assays and to compare them with the class 4 mutants. We report that cooperativity of T124A T antigen in double-hexamer assembly on the viral origin is impaired. The class 4 mutant T antigens were also found to have defects in cooperativity of double-hexamer assembly. T124A T antigen inhibited the ability of the wild-type protein to unwind closed circular duplex origin DNA. Both T124A and the class 4 mutants displayed dominant-negative phenotypes in viral DNA replication in vitro. Based on these observations, we propose that the N-terminal cluster of phosphorylation sites and the DNA binding domain mediate cooperative hexamer-hexamer interactions during assembly on the viral origin and speculate that these regions of T antigen may interact during origin DNA unwinding.     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.     

Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3-related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis).In eukaryotes, the reversible phosphorylation of serine, threonine, and tyrosine residues within proteins is a wide-spread post-translational modification, essential for controlling a multitude of cellular processes. During the last decade, sequencing projects unexpectedly unraveled that plant genomes encode for a considerable larger number of protein kinases than the other kingdoms of life. Arabidopsis thaliana contains 1112 PKs (4% of all genes), twice the number encoded by the human genome (518 or 2% of all genes) and other plants have an even higher number of kinases (1).Phosphatidyl inositol 3′ kinase related kinases are important players in DNA damage response (DDR)¹ and crucial for genome integrity (2). Key to DNA double strand break (DSB) repair is a chain of events starting with detection of the lesion, activation of a signaling cascade, cell cycle arrest, and recruitment of the repair machinery. The cascade is triggered by the Phosphatidyl inositol 3′ kinase related kinases family kinases ataxia telangiectasia-mutated (ATM) (3) and Ataxia telangiectasia-mutated and Rad3-related (ATR) (4). Both kinases are conserved across eukaryotes. Their downstream targets have been systematically identified in yeast (5) and human cells (6, 7). Their essential role in mediating DNA repair in higher plants has been established (8–10). In Arabidopsis, loss of function mutants are viable (11); however, atm mutants are highly sensitive to genotoxic stress and have a reduced fertility. atr mutant plants have a cell-cycle checkpoint defect upon exposure to genotoxic chemicals (12). Somatic growth under nonchallenging conditions is not affected in the double mutant but plants are sterile, highlighting the role of both kinases coordinating meiotic DNA repair. In plants, systematic phosphoproteomic studies of the involved pathways have not been reported but would contribute to further elucidating the molecular mechanism of the observed phenotypes. Interestingly, plants lack clear homologs for many downstream regulatory components in the signaling cascade (e.g. CHK1, CHK2, p53, and MDC1) (13). In this context, it should be noted that DNA-PKcs (DNA-dependent protein kinase), another Phosphatidyl inositol 3′ kinase related kinases family member involved in DNA repair, has not been identified in plant genomes (14, 15), underscoring the significance of ATM and ATR as master regulators.ATM is recruited to DSBs via its interaction with NBS1/XRS2, a member of the MRN/X complex (MRE11/RAD50/NBS1-XRS2). In plants, the detailed molecular base for ATM recruitment has remained unknown. The complex acts as damage sensor in yeast, first to be detected at DNA double strand break (DSB) sites and essential for resection of DNA (16). In all organisms analyzed, the MRN/X complex is required for genotoxic stress resistance (17). The Mre11 endonuclease activity is critical for ATM activation, likely triggered by the generation of short oligo-nucleotides (18). In higher eukaryotes, ATM activation relies on MRN binding to DSBs via MRE11, subsequent tethering of DSB ends via RAD50 and recruitment of ATM. This interaction leads to monomerisation of inactive ATM dimers, followed by autophosphorylation. The MRN subcomplex member NBS1 interacts with monomeric ATM leading to its localization in close proximity of the DSB site (19). NBS1, H2AX, the checkpoint kinase CHK2, and the trimeric replication protein A (RPA) are important downstream targets of ATM (6).In yeast, ATR is activated by RPA coated single-stranded DNA (ssDNA) that is generated by 5′ resection mediated by MRX/N, Exo1, Sgs1, and Dna2 during DSB processing (20). Furthermore, ssDNA may become exposed because of replication fork break down during DNA replication or nucleotide excision repair (21). Exposed ssDNA is rapidly bound by RPA, attracting ATRIP, and the Rad17-RFC complex. ATRIP interacts with ATR and is essential for its activation and function (22). The Rad17-RFC complex is functional in loading the 9–1-1 protein complex (Rad9, Rad1, and Hus1) to 5′ dsDNA-ssDNA junctions, in turn stimulating ATR activity at the site of the exposed ssDNA (23). In human cells, TopBP1 is required for activation of ATR and localizing to DNA lesion sites. Rad17, TopBP1, RPA, and the checkpoint kinase CHK1 are known downstream targets of activated ATR.The core effectors of DNA repair (e.g. RAD51), the proteins detecting DNA damage and mediating initiation of repair (e.g. MRX) and the two master regulators ATM and ATR are conserved in plants but many downstream components have diverged considerably. Yet, a comprehensive model for DDR in plants requires identification of all components to delineate the involved signaling pathways and their cross-talk with other regulatory processes.Mass spectrometry-based methods are powerful and hypothesis-free approaches for protein characterization, enabling high-throughput studies of protein complexes (24), protein expression profiling (25), or large-scale identification of protein kinase targets (26). Also, the identification and quantification of thousands of phosphopeptides has become feasible by technological and methodological advances. As a consequence, system-wide analyses of signaling networks has become possible (27). Despite above mentioned advances, comprehensive phosphoproteomic studies remain challenging in regards to sample preparation and phosphopeptide enrichment. An additional complication in large-scale studies is imposed by the requirement for correct automatic localization of phosphorylation sites (28). Abundant metabolites make sample preparation in plants especially difficult; however, a number of large-scale phosphoproteomic studies have been reported (29–34).Phosphorylation sites in proteins are in most cases substoichiometric. As a consequence, the comprehensive analysis requires enrichment of phosphopeptides prior to LC-MS/MS. From the large number of developed methods (35), metal-based affinity chromatography such as immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) have become most widely used. Both materials have different specificities, resulting in a substantial increase of identified phosphopeptides when employed consecutively (36).Here we delineate a methodology to identify and relatively quantify phosphorylation events proteome-wide in higher plants (Fig. 1). We demonstrate its applicability in the context of ATM and ATR dependent DNA damage repair in Arabidopsis thaliana. The approach combines filter assisted sample preparation (FASP) (37), isobaric labeling via iTRAQ, phosphopeptide enrichment using IMAC and TiO₂, strong cation exchange (SCX) chromatography, followed by LC-MS/MS. We compared the relative differences between the phosphoproteomes of wild type plants with the double mutant (atm atr), studying both irradiated and nonirradiated plants. In addition, we performed an independent analysis based on peptide generation after protein precipitation.Open in a separate windowFig. 1.Workflow for identification of ATM/ATR dependent and independent phosphorylations. Wild type and atm atr double mutant plants were either exposed to irradiation or grown under regular conditions. Extracted proteins were purified via FASP and labeled with iTRAQ. Phosphopeptides were enriched by consecutive application of IMAC and TiO₂. Both, the phosphopeptide-enriched fraction and the flow-through of the TiO₂ chromatography, were separated by SCX chromatography and fractions were analyzed by reversed phase LC-MS/MS.All together, we identified 10,831 proteins. Four-hundred and 13 phosphoproteins are phosphorylated upon ionizing radiation, among them 108 in an ATM/ATR dependent manner. The acquired data-set represents a unique resource for plant researchers and extends the current knowledge on ATM/ATR dependent DNA repair pathways.     

The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

Genomic stability requires a functional Fanconi anemia (FA) pathway composed of an upstream “core complex” (FA proteins A/B/C/E/F/G/L/M) that mediates monoubiquitination of the downstream targets FANCD2 and FANCI. Unique among FA core complex members, FANCM has processing activities toward replication-associated DNA structures, suggesting a vital role for FANCM during replication. Using Xenopus egg extracts, we analyzed the functions of FANCM in replication and the DNA damage response. xFANCM binds chromatin in a replication-dependent manner and is phosphorylated in response to DNA damage structures. Chromatin binding and DNA damage-induced phosphorylation of xFANCM are mediated in part by the downstream FA pathway protein FANCD2. Moreover, phosphorylation and chromatin recruitment of FANCM is regulated by two mayor players in the DNA damage response: the cell cycle checkpoint kinases ATR and ATM. Our results indicate that functions of FANCM are controlled by FA- and non-FA pathways in the DNA damage response.Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Cells from FA³ patients show hypersensitivity to DNA interstrand cross-links and have highly elevated chromosomal breakage rates, indicating a role for FA proteins in the cellular DNA damage response. The FA pathway consists of an upstream FA core complex containing at least eight proteins (FANCA, -B, -C, -E, -F, -G, -L, and -M) that is required for the DNA damage-induced monoubiquitination of two downstream proteins, FANCD2 and FANCI. Although the molecular function of the FA pathway is unknown, the identification of additional FA genes FANCD1 (BRCA2), FANCN (PALB2), and the DNA helicase FANCJ (BRIP1) as breast cancer (BRCA) susceptibility genes suggests convergence of the FA/BRCA pathway with a larger network of proteins involved in DNA repair (reviewed in Ref. 1).In addition to monoubiquitination by the FA core complex, FANCD2 and FANCI are phosphorylated by the two major cell cycle checkpoint kinases, ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related),y in response to DNA damage (2–6). ATM-dependent phosphorylation of FANCD2 occurs following ionizing irradiation and is required for activation of the ionizing irradiation-induced intra-S phase checkpoint (4). ATR-dependent phosphorylation of FANCD2 is triggered by various types of DNA damage, including replication stress, and is required for the interstrand cross-link-induced intra-S phase checkpoint response (2, 3). Moreover, phosphorylation by ATR is required for efficient FANCD2 monoubiquitination in response to DNA damage, suggesting that the FA pathway might participate in ATR-dependent coordination of the S phase of the cell cycle (3, 7).The recent identification of a highly conserved FA core complex member, FANCM (8, 9), indicates a direct role of FA pathway proteins in repair steps at sites of DNA damage. FANCM is a homolog of the archaebacterial Hef protein (helicase-associated endonuclease for fork-structured DNA) and contains two DNA processing domains: a DEAH box helicase domain and an XPF/ERCC4-like endonuclease domain. FANCM has ATP-dependent DNA translocase activity and can dissociate DNA triple helices in vitro (8). Moreover, FANCM binds Holliday junctions and DNA replication fork structures in vitro and promotes ATP-dependent branch point migration, suggesting that FANCM might be involved in DNA processing at stalled replication forks (10, 11). In human cells, FANCM localizes to chromatin and is required for chromatin recruitment of other FA core complex proteins (8, 12). FANCM is phosphorylated during both the M and S phases and in response to DNA-damaging agents (8, 12, 13). Interestingly, DNA damage-induced phosphorylation of FANCM is independent of the FA core complex (8), suggesting that FANCM is controlled by other, as yet unknown upstream components of the DNA damage response. Here, we used cell-free Xenopus egg extracts to investigate the role of FANCM during replication and in the DNA damage response. We show that Xenopus FANCM (xFANCM) binds chromatin in a replication-dependent manner and is phosphorylated during unperturbed replication as well as in response to various DNA damage structures. Both chromatin recruitment and phosphorylation of xFANCM are partially controlled by xFANCD2, suggesting feedback signaling from xFANCD2 to the upstream xFA core complex via regulation of xFANCM. In addition, chromatin recruitment during unperturbed replication and activation of xFANCM in response to DNA damage are controlled by the xATR and xATM cell cycle kinases.     

Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD₅₀ increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD₅₀ increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044.Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications in both eukaryotic and prokaryotic organisms. It is best known that protein phosphorylation is a reversible enzyme-catalyzed process that is controlled by various kinases and phosphatases. The aberrant functions often result in irregular protein phosphorylation and ultimately lead to serious disease states such as malignant transformation, immune disorders, and pathogenic infections in mammals (1, 2). Recently, accumulating evidences suggest that Ser/Thr/Tyr phosphorylations also contribute to regulate a diverse range of cellular responses and physiological processes in prokaryotes (1). Among them, tyrosine phosphorylation in encapsulated bacteria has been discovered to play key roles in capsular polysaccharide (CPS1; K antigen) biosynthesis, which leads to virulence (3, 4). This thick layer of exopolysaccharide on many pathogenic bacteria can act as a physical boundary to evade phagocytosis and complement-mediated killing and further inhibit complement activation of the host (1, 5, 6).In 1996, Acinetobacter johnsonii protein-tyrosine kinase (Ptk) was first discovered and categorized under the bacterial protein-tyrosine kinase (BY-kinase) family (1, 7, 8). Shortly after, its function in bacterial exopolysaccharide production and transport was characterized (1, 7, 8). From then on, many more bacterial tyrosine kinases such as Wzc of Escherichia coli (1, 9) and EpsB of Pseudomonas solanacearum (10, 11) were found to possess this conserved property; deletion of such tyrosine kinases will result in the loss of exopolysaccharide production (12). Therefore, several experiments were conducted to investigate the role of the downstream substrates of the tyrosine kinases in different strains of bacteria, and some targeted proteins were found to participate in the exopolysaccharide anabolism (13, 14). These findings demonstrated a direct relationship between bacterial tyrosine phosphorylation and exopolysaccharide biosynthesis that was directly reflected in the strain virulence.In the past, the functional roles of the critical components involved in protein phosphorylation were defined by basic biochemical and genetic approaches (1). However, there exists a salient gap between the growing number of identified protein-tyrosine kinases/phosphatases and the relative paucity of protein substrates characterized to date. Genomic sequence analyses and advanced high resolution/high accuracy MS systems with vastly improved phosphopeptide enrichment strategies are among the two key enabling technologies that allow a high efficiency identification of the scarcely detectable site-specific phosphorylations in bacterial systems (15). Mann et al. (16) were the first to initiate a systematic study of the phosphoproteome of B. subtilis in 2007 followed by similar site-specific phosphoproteomics analyses of E. coli (17), Lactococcus lactis (18), and Halobacterium salinarum (19). These pioneering works have since set the foundation in bacterial phosphoproteomics but have not been specifically carried out to address a particular biological issue of causal relevance to virulence or pathogenesis.Klebsiella pneumoniae is a Gram-negative, non-motile, facultative anaerobic, and rod-shaped bacterium. It is commonly found in water and soil (20) as well as on plants (21) and mucosal surfaces of mammals, such as human, horse, and swine (22, 23). It was demonstrated that CPS on the surface of K. pneumoniae is the prime factor of virulence and toxicity in causing pyogenic liver abscess (PLA), a common intra-abdominal infection with a high 10–30% mortality rate worldwide (24–29). There are also variations in virulence in regard to different capsular serotypes; K1 and K2 were found to be especially pathogenic in causing PLA in a mouse model (30) compared with other serotypes, which show little or no effect (31–34). The K. pneumoniae NTUH-K2044 (K2044) strain, encapsulated with K1 antigen (35), was isolated from clinical K. pneumoniae liver abscess patients. It has become an important emerging pathogen (36) because it usually complicates metastatic septic endophthalmitis and irreversible central nervous system infections independent of host underlying diseases (30, 34). The transmission rate is high (37), and it often rapidly leads to outbreaks of community-acquired infections, such as bacteremia, nosocomial pneumonia, and sepsis, common in immunocompromised individuals (38).In this study, we wanted to prove that the biosynthesis of CPS is mediated through tyrosine phosphorylation of a subset of proteins. An MS-based systematic phosphoproteomics analysis was conducted on K2044 to identify tyrosine phosphorylated proteins that are also associated with CPS biosynthesis. We further validated the relationship between tyrosine phosphorylation on those proteins and virulence of K2044 by site-directed mutagenesis, CPS quantification, serum killing, and mouse lethality assay.