The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance. Agrin, MuSK and lrp4 mutant mice fail to form NMJs and consequently die at birth due to respiratory failure89The vertebrate neuromuscular junction (NMJ) represents a special chemical synapse between a motor neuron and a skeletal muscle fibre. As such the NMJ converts nerve-elicit signals into muscle contraction. The formation of the NMJ is crucially linked to signalling events induced by the receptor tyrosine kinase MuSK24MuSK kinase activity is tightly regulated by the juxtamembrane Y553 and by the binding of Dok713To better understand the signalling network initiated by MuSK we have recently performed a quantitative phosphoproteomics screen to identify targets downstream of MuSKIn a previous study we have used a muscle C2C12 cell culture model system to identify and characterize the phosphoproteome during agrin-induced MuSK activation. We were able to show that MuSK induces the phosphorylation of a large set of proteins with a distinct temporal manner of regulationin vivo. We stained muscle sections with antibodies against pS751. Parallel sections were stained with antibodies against MuSK or antibodies against pY754/Y755 of the MuSK activation loop. All samples were co-stained with α-BGT to label AChRs. in vivo and that MuSK carrying pS751 is present at NMJs.Next we asked whether S751 is also phosphorylated in MuSK −/− muscle cells to study S751 during MuSK activation and downstream signalling2MuSK−/− muscle cell lines expressing these S751 mutants or MuSK wild-type were generated, differentiated into myotubes and stimulated with agrin  and MuSK kinase-dead (KD) lack kinase activity due to mutations that interfere with activation of the kinase1113131913Serine phosphorylation of RTKs not only regulates downstream signalling but also endocytosis and internalization of RTKs222324In a different set of experiments we asked whether mutation of S751 has an effect on MuSK protein stability. For this, we treated myotubes with cycloheximide to inhibit protein synthesis followed by an analysis of protein expression by immunoblotting. As shown in The hallmark of MuSK signalling is the clustering of AChRs to discrete patches on the muscle membrane. Changes in MuSK kinase activity or alterations in the downstream signalling pathway greatly influence AChR clustering11131413in vitro1314In vivo constitutive active MuSK induces ectopic postsynaptic structures reminiscent of NMJs independent of nerve and agrin262728MuSK kinase activity plays a crucial role during AChR clustering and NMJ formation28in vitro and in vivo: the juxtamembrane tyrosine Y553, tyrosines Y750, Y754, and Y755 within the activation loop, Y576 near the beginning of the kinase domain and Y812 within the C-terminal loop of the kinase domain1913in vitro phosphorylation, was unregulated and remained stable during all time points of agrin stimulationIt was previously reported that MuSK is phosphorylated on six of the nineteen intracellular tyrosine residues S751 lies within the activation loop between the critical tyrosines Y750, Y754 and Y755. The sequence motif surrounding S751 does not conform to motifs of well-known serine/threonine kinases such as PKA/G/C, proline directed MAPK (ERK1/2), JNK, CK1/2 or GSK332MuSK−/− muscle cells expressing wild-type, phosphomimetic S751D and phosphorylation-deficient S751A MuSK. Our data suggest that S751 has a modulatory role during MuSK activation and subsequent signalling. This is based on the observations that (1) Mutation of S751 to D751 increases basal MuSK phosphorylation. (2) Phosphorylation of MuSK S751D is significantly enhanced in response to sub-threshold levels of agrin. (3) Basal AChR phosphorylation is significantly increased as well as AChR phosphorylation after 15 minutes of agrin stimulation in MuSK S751D expressing muscle cells suggesting that the phosphomimetic mutant accelerates the downstream signalling. (4) Agrin-induced AChR clusters are bigger and disassemble more slowly in cells expressing MuSK S751D compared to cells expressing MuSK wild-type or MuSK S751A. (5) Myotubes expressing MuSK S751D form more microclusters than myotubes expressing MuSK wild-type or MuSK S751A.Serine phosphorylation sites with different functional roles modulating receptor kinase activation, receptor endocytosis or internalization, expression, cell survival, proliferation and differentiation have been discovered in different RTKs. It has been reported for several RTKs such as insulin receptor, platelet-derived growth factor receptor and estrogen receptor that serine phosphorylation regulates kinase activity3435222324Previous studies have shown that the activation loop carrying Y750, Y754, Y755 and the juxtamembrane region including Y553 are not only important for MuSK kinase activity but also responsible for autoinhibition. The crystal structure of unphosphorylated MuSK implied that the activation loop of MuSK provides a stringent autoinhibition to limit ligand-independent activation. In addition, a second autoinhibition mechanism involves the juxtamembrane region, which is largely disordered in MuSK and does not interact with the kinase domain1818381837Lowered autoinhibition or respectively a stabilized active conformation of the activation loop might play an important role during two developmental states of NMJ development, namely AChR prepatterning and NMJ maintenance. NMJs have to be stable and functional for the entire lifetime of an organism4547et al.49Previously, Cheusova Taken together, we identified a novel serine phosphorylation site in the activation loop of MuSK, which functions as regulatory site during kinase activation. We propose that phosphorylation of S751 lowers the autoinhibition of the activation loop. An increased basal kinase activity might play an important role during prepatterning or NMJ maintenance.Single amino acid substitutions were generated by site-directed mutagenesis using PCR. S751 residue in the rat MuSK cytoplasmic domain was either replaced with alanine (S751A) or aspartate (S751D). Constructs carrying a C-terminal HA-Tag were ligated into retroviral vector pBabe/puro2816171321The following antibodies were purchased from commercial sources: anti-phosphotyrosine PY99  and PY-100 , anti-AChR α (BD Biosciences), anti-AChR β (Sigma-Aldrich), anti-HA (Sigma-Aldrich), anti-Myc 9E10 (Sigma-Aldrich), anti-GFP (Santa Cruz), anti-Actin , anti-Tubulin (Sigma-Aldrich). Biotin- and Alexa 594-conjugated α-bungarotoxin (BGT), Alexa 488-conjugated secondary antibodies were obtained from Invitrogen . Horseradish-peroxidase-coupled goat anti-mouse/rat/rabbit secondary antibodies were purchased from Jackson ImmunoResearch . IRDye 680RD goat anti-mouse, IRDye 800CW goat anti-rabbit secondary antibodies were purchased form LI-COR . Streptavidin and Protein A agarose beads were obtained from Novagen  and Roche , respectively. The antibody MuSK-EC (#83033) was used for immunohistochemistry and was previously described1316132. Resulting peptide mixtures were fractionated using high resolution Strong Cation Exchange (SCX) Chromatography on a 25 cm × 1 cm column. Phosphorylated peptides were separated into 130 fractions whereas unphosphorylated peptides were split into 70 fractions. SCX fractions were separated on a 25 cm reverse-phase C18 column using a 60-minute gradient on-line coupled to an LTQ Velos Orbitrap ETD mass spectrometer via a nano-electrospray source. The mass spectrometer was operated in data-dependent mode acquiring a full scan in the Orbitrap at resolution 60.000 followed by MS/MS scans of the five most abundant ions in the LTQ and in the Higher-energy collisional dissociation (HCD) cell. Raw files were processed in Proteome Discoverer (version 1.4.0.282). Database searches were performed using Mascot (version 2.2) against a concatenated target-decoy database based on the mouse UniProt database (version 2012_11). Oxidation of methionine and phosphorylation of serine, threonine and tyrosine were set as dynamic modifications and methylthio-cysteine and iTRAQ at the N terminus and lysine were specified as fixed modifications. Trypsin was defined as the proteolytic enzyme allowing for up to two missed cleavages. A mass tolerance of 7 ppm was set as the precursor ion tolerance. The fragment ion tolerance values for HCD and Collision-induced dissociation (CID) spectra were set to 0.03 and 0.5 Da, respectively. Reporter ion intensities were extracted in Proteome Discoverer from the closest centroid mass within an integration tolerance of 5 mmu. PhosphoRS (version 3.0) was employed to determine the localization of phosphorylated residuesPhosphorylation at MuSK serine 751 was identified in a previously published quantitative phosphoproteomic study4)ADYYKADGC) with the pS751 residue at position 8 was synthesized by Fmoc-solid-phase synthesis, purified by high-pressure liquid chromatography and conjugated to a subunit of the carrier Keyhole Limpet Hemocyanin. 250 μg of antigen were used for the immunization of two rabbits. Reactivity of sera was tested by immunoblotting. The serum was purified by liquid chromatography. The column coated with the epitope peptide attached to maleimide activated POROS was washed with HEPES buffered solution (HBS), 12 mM HCl and afterwards re-equilibrated with HBS. Before injection to column, the serum was extracted with 1,1,2-Trichloro-1,2,2-trifluoroethane (Sigma-Aldrich) and filtrated with a 0.22 μm filter . After injection of serum, the column was washed with HBS until the baseline was stable. For the first elution step 1.5 M MgCl2, 50 mM sodium acetate pH 5.2 and for the second elution step 0.1 M glycine pH 2.45, 0.1 M NaCl was used. The eluates were buffered immediately with 2 M HEPES pH 7.9. Subsequently, the antibody solutions were dialyzed with phosphate buffered saline (PBS). The specificity of the antibodies was tested by immunoblotting MuSK−/− muscle cells was performed using pBabe/MuSK-WT, S751D and S751A constructs. Cells were maintained and differentiated as described1313HEK 293T cells were purchased from . Phoenix cells were kindly provided by Dr. Wilfried Ellmeier . Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with glutamine, 4.5 mg/ml glucose, 10% fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin at 37 °C and 5% CO2. HEK 293T cells were transfected using TurboFect (Thermo Fischer Scientific). Briefly, HEK 293T cells were plated on 6 well plates and transfected the next day with 2.2 μg DNA and 4 μl TurboFect in 200 μl DMEM. Frozen muscle sections were thawed at room temperature, briefly rehydrated with PBS and blocked with 10% FBS in PBS for 30 minutes. Sections were incubated overnight at 4 °C with primary antibodies, washed three times with PBS and incubated at room temperature for 2 h with Alexa 488-conjugated secondary antibodies (Jackson Immunoresearch) and Alexa 594-conjugated α-BGT to label synaptic AChRs. Finally, the muscle sections were washed three times with PBS, post-fixed in 1% PFA for 5 min and mounted with Mowiol 4–88 (Sigma-Aldrich). The stained sections were imaged with Leica TCS SP5 confocal laser scanning microscope using 63x1.40 glycerol immersion magnification objective.Differentiated myotubes were starved for 2 h in DMEM and then stimulated with neural A4B8 agrin for 15, 60, 240, 480, 960 minutes. Cells were lysed in RIPA buffer  supplemented with protease  and phosphatase inhibitors . Cleared lysates were incubated with anti-MuSK (C-terminus) antibodies for MuSK isolation or with biotin-conjugated α-BGT for AChR pull down as previously described2, 1 mM MgCl2, 5 mM KCL pH 7.4. Cells were washed with PBS two times for 5 min and lysed in 0.5% NP-40, 50 mM Tris/HCl pH 7.5, 1 mM EDTA, 100 mM NaCl supplemented with protease and phosphatase inhibitors. Protein A agarose was added for 1–3 hours. To analyse the stability of MuSK, myotubes were incubated with 7.5 ng/ml cycloheximide for 1, 2, 4, 6 h. Cells were lysed in RIPA buffer and proteins were analysed by immunoblotting. IRDye 680RD goat anti-mouse, IRDye 800CW goat anti-rabbit secondary antibodies were used and proteins were imaged with Odyssey Imaging System .After 2 h of starvation, myotubes were stimulated with neural A4B8 agrin for 30 min. Cells were washed two times with PBS and then incubated for 60 min in a cold chamber (10–12 °C) with anti-HA antibodies diluted in 1% BSA, 140 mM NaCl, 20 mM HEPES, 1 mM CaClTo induce AChR clustering, myotubes were stimulated overnight (16 h) with neural A4B8 and non-neural A4B0 agrin. Next day, samples were either processed for staining or washed with PBS for three times and incubated with differentiation medium lacking agrin for 6 h and 12 h. To label surface AChRs, cells were fixed with 4% PFA in PBS at room temperature, washed two times with PBS for 5 min and incubated with Alexa 594-conjugated α-BGT in 2% FBS/PBS for 30 min. Cells were washed two times with PBS for 5 min and mounted with Mowiol 4–88. AChR clusters were imaged with a LEICA DM-IRB fluorescence microscope using 63x oil immersion magnification objective. Metamorph  and ImageJ (NIH) software were used to acquire and quantify images.2 were measured. Finally, mean of feret, number of clusters/1000 μm2 and myotubes with microclusters  were statistically analysed using Graphpad Prism software . AChR clustering experiments were replicated in at least three independent experiments (n = 3) and presented as the mean ± SEM. Data were analysed using non-parametric Kruskal Wallis, parametric one-way ANOVA for two or more group comparisons and two-way ANOVA for two factor comparisons with Dunn’s, Tukey’s post-tests and with Sidak’s multiple comparison tests, respectively. GraphPad Prism version 6.0 or R version 3.3.1  were used for analysis. The values of p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) were considered statistically significant.To quantify MuSK and AChR β phosphorylation, the ratio of phosphorylated protein to total protein was quantified using Image Lab Software . For the temporal activation profile, the values for MuSK-WT, S751D, S751A after 1 h agrin stimulation were defined as 1.0 and used to calculate the relative increase for the other time points. AChR clusters were quantified using ImageJ. In each image the myotube area was selected, thresholded and all objects with a minimum size of 4 μmHow to cite this article: Camurdanoglu, B. Z. et al. Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop. Sci. Rep.6, 33583; doi: 10.1038/srep33583 (2016).


The present study investigated the interplay between selective inhibition (the ability to suppress specific competing responses) and nonselective inhibition (the ability to suppress any inappropriate response) during single word production. To this end, we combined two well-established research paradigms: the picture-word interference task and the stop-signal task. Selective inhibition was assessed by instructing participants to name target pictures  in the presence of semantically related  or unrelated  distractor words. Nonselective inhibition was tested by occasionally presenting a visual stop-signal, indicating that participants should withhold their verbal response. The stop-signal was presented early (250 ms) aimed at interrupting the lexical selection stage, and late (325 ms) to influence the word-encoding stage of the speech production process. We found longer naming latencies for pictures with semantically related distractors than with unrelated distractors (semantic interference effect). The results further showed that, at both delays, stopping latencies  were prolonged for naming pictures with semantically related distractors compared to pictures with unrelated distractors. Taken together, our findings suggest that selective and nonselective inhibition, at least partly, share a common inhibitory mechanism during different stages of the speech production process. For instance, many irrelevant thoughts may come to mind and need to be suppressed because they are contextually or socially inappropriate. In addition, we do not produce an infinite stream of speech, but instead are able to stop ourselves when we notice that someone else is about to speak. It thus seems that some processes in language production require different forms of inhibition that allow for the emergence of goal-directed speech behavior. The present study focuses on the interplay between selective and nonselective inhibition during single word production. We first discuss the main components of word production and which type of inhibition, selective and nonselective, may be necessary for these production processes. Next, we discuss what is known about the interplay between these two forms of inhibition in the nonverbal domain, which leads to our hypotheses for such an interplay in word production: the interest of our study.When we talk to another person, we not only have to think of what to say, but also of what The production of spoken words appears to be a complex cognitive task which entails a number of mental processes that take place between activating an idea and activating the sounds you need to express the idea ,2. In thinhibition. However, it has been proposed that inhibition is not a unitary construct but can be considered as a set of closely related abilities , given a particular SSD. For example, on the basis of the numbers in nth go RT–SSD’.Stop-signal RTs were estimated based on the independent horse-race model ,21. A grlme4 package )one-up one down procedure [One way to improve the current design is to use a dynamic tracking procedure . Typicarocedure . As suchrocedure ). Therefrocedure , for simA limitation of the present study was that the picture-word interference stop-signal task was restricted to the production of single words. This was mostly done in order to exclude the number of syllables as a possible confounding factor for stopping performance. However, there is reason to assume to stopping speech in the stop-signal paradigm is different from stopping speech in real-life . For insIn a similar vein, it would be interesting to see what happens if the stop-signal was more like a stop-signal that would occur in naturalistic communication, such as a spoken word. A speaker should stop talking when his or her interlocutor vocally indicates he/she wants the turn to speak. Previous research has shown that combining two tasks that are both linguistic in nature (picture naming and syllable categorization) is more difficult than combining a linguistic and nonlinguistic task (picture naming and tone categorization) . TherefoEven with these limitations, we believe the findings from the present study have several important theoretical implications. First of all, the present study adds to a growing body of research that shows stop-signal RTs are similar for inhibiting speech and inhibiting manual responses –28 and pSecondly, our results provide additional evidence for the involvement of inhibitory control in language production ,42. ThisThe present study provides behavioral evidence for the interplay between selective and nonselective inhibition during single word production. Combining a picture-word interference task (thought to tap selective inhibition) with a stop-signal task (nonselective inhibition), we found longer naming latencies for pictures with semantically related distractor than with unrelated distractors (semantic interference effect). In addition, we observed longer stopping latencies in the semantically related condition than in the unrelated condition, indicating that nonselective inhibition ability is influenced by selective inhibition. The results suggest that both types of inhibition share a common inhibitory mechanism, at least in part. Our findings have important theoretical implications for understanding the role of inhibition in language production.S1 Table(DOCX)Click here for additional data file.


Due to demographic changes, more and more fracture patterns involving anterior acetabular structures occur. The infra-acetabular screw is seen a useful tool to increase stability in fixation of the acetabular cup. However, the exact position of this screw in relation to anatomic landmarks which are intra-operatively palpable via an intra-pelvic approach has not yet been determined.This biomorphometric experimental study references the ideal screw position of an infra-acetabular screw to anatomic landmarks palpable via an intra-pelvic approach. Therefore, we created a computer tomography-based 3D-model of 40 patients  who received a computer tomography (CT) scan of the pelvis for any other reason than an acetabular fracture.The entry point of an ideal infra-acetabular was of high constancy. At mean, this point was 10.2 mm caudal and 10.4 mm medial of the ilio-pubic/ilio-pectineal eminence. This reference is independent of age, gender, or physical dimensions. However, we found gender-dependent differences for the angulation and the length of the screw.This study provides a comprehensive guideline to determine the ideal entry point for an infra-acetabular screw via an intra-pelvic approach. The entry point is located 10.2 mm caudal and 10.4 mm medial of the ilio-pubic/ilio-pectineal eminence.Z-2017-0930-1. Registered 04. Dec 2017.Clinical Trial Registry University of Regensburg  Treatment of fractures of the acetabulum is one of the most challenging procedures in trauma surgery. Achieving an anatomic reduction of the joint surface can be demanding. However, it is necessary to establish a rigid fixation to retain the position until the fracture has healed.Large register studies on the epidemiology of acetabular fractures have shown an increasing number of cases with involvement of the anterior acetabular column during the last 20 years . TherefoThe purpose of the study is to specify the ideal position of the infra-acetabular screw in relation to anatomic landmarks that are accessible via an intra-pelvic approach.The Ethics Committee at the University of Regensburg approved the study in January 2018 . The study is registered at the Clinical Trial Registry University of Regensburg Z-2017-0930-1. All procedures performed in studies were in accordance with the 1964 Helsinki declaration.This study is an experimental computer tomography-based 3D-model analysis to reference the ideal screw position of an infra-acetabular screw to anatomic landmarks. We investigated 3D-computer tomography (CT) scans of 40 patients  obtained by the Department of Radiology of our institution.Baseline characteristics of the study group are shown in Table x) and on a perpendicular (y) line  and axial (β) angles in relation to the pelvic inlet plane. The entry point of the straight line was referenced by measuring the relation to the apex of the IPE. Therefore, the distance was measured on a horizontal line .Since there is no previous data on the variance of the entry point, this preliminary study was designed as an exploratory pilot study without any a priori sample size calculation based on a primary endpoint. Based on the variance of the angle related to the anterior pelvic plane, a sample size of 40 patients was considered feasible and expected to have enough power.t test. The Spearmen’s rank correlation was used to investigate a relationship between age, height, and weight of the patient. Correlation coefficients ≥ .40 indicate a relevant relationship. Unless otherwise stated, descriptive data are given as mean ± standard deviation. The level of significance was at p < 0.05 for all tests.For comparison of mean values, we used the independent p = 0.000) respectively.Table p = 0.006 for the sagittal and p = 0.000 for the axial plane). We did not find any significant difference between the right and left side concerning the screw position, length of the corridor, or angulation. Figure The mean angle of both left and right screw related to the axial plane was 10.4° (± 1.6°) and to the sagittal plane 70.5° (± 3.2). The mean angle in female patients related to the axial plane was 11.2° (± 1.6) and to the sagittal plane 71.4° (± 3.6). Male patients had a mean angle of 9.6° (± 1.2°) related to the axial plane and 71.4° (± 3.6) to the sagittal plane. This differences between female and male patients were significant  , 20, 23.infra-acetabular screw was intended to minimize the risk of intra-articular placement of the screw. The entry point for this modified screw was reported to be 1 cm caudal to the IPE and in the middle of the pubis ramus. When this infra-acetabular screw position was introduced, the anterior ilio-inguinal approach which allows direct access to the pubic ramus circumference was the most frequent anterior approach [Historically, the posterior Kocher/Langenbeck approach was the standard approach to stabilize acetabular fractures , 16. Durapproach . Howeverapproach . Therefoapproach  are not The major advantage of the intra-pelvic approach is a direct inspection of the quadrilateral surface which is often involved in geriatric fractures , 19, 25.Letournel was the first to describe the construct of a peri-acetabular frame to improve stability in acetabular fracture fixation . They reRecently, a large biomorphometric CT-based study identified a viable infra-acetabular corridor with a diameter over 5 mm in 93% of specimens . It can This study has some limitations. The guideline for placement of the infa-acetabular screw is based on the identification of the IPE. This anatomic landmark is a large-area bony bump rather than a sharply marked spike. However, via an intra-pelvic approach, it is well-palpable and its tip can easily be identified . AnotherThis study provides a comprehensive guideline to determine the ideal entry point for an infra-acetabular screw via an intra-pelvic approach. The entry point is located 10.2 mm caudal and 10.4 mm medial of the IPE. This reference is independent of age, gender, or physical dimensions. However, we found gender-dependent differences for the angulation and the length of the screw. Further prospective studies are needed to the proof effectiveness of this guideline in a clinical setting.


Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a small transmembrane protein expressed exclusively in lymphocytes. LPAP is a component of a supramolecular complex composed of the phosphatase CD45, the co-receptor CD4, and the kinase Lck. In contrast to its immunologically important partners, the function of LPAP is unknown. We hypothesized that the biological role of LPAP may be determined by analyzing LPAP phosphorylation. In the present study, we identified LPAP phosphorylation sites by site-directed mutagenesis, phospho-specific antibodies, and protein immunoprecipitation coupled to mass spectrometry analysis. Our results confirmed previous reports that Ser-99, Ser-153, and Ser-163 are phosphorylated, as well as provided evidence for the phosphorylation of Ser-172. Using various SDS-PAGE techniques, we detected and quantified a set of LPAP phosphoforms that were assigned to a combination of particular phosphorylation events. The phosphorylation of LPAP appears to be a tightly regulated process. Our results support the model: following phorbol 12-myristate 13-acetate (PMA) or TCR/CD3 activation of T cells, LPAP is rapidly dephosphorylated at Ser-99 and Ser-172 and slowly phosphorylated at Ser-163. Ser-153 exhibited a high basal level of phosphorylation in both resting and activated cells. Therefore, we suggest that LPAP may function as a co-regulator of T-cell signaling. PTPRCAP), is a 206 amino acid (aa) small transmembrane protein with an apparent molecular weight of 32 kDa D[T/S][E/D]G, and though high levels of phosphorylation are present in the human protein, phosphorylation of murine PTPRCAP Thr-144 has not been identified. Presumably, this is due to the fact that after trypsinolysis, Thr-144 is placed in a 51-aa peptide with a molecular weight of 4750 Da, which is not easily analyzed by MS. Ser-153 is positioned within a typical CK2 recognition motif, SXXE/D; however, in LPAP, we observe the more specific motif [pS][D/E]X[D/E]X[D/E] .Unlike Ser-99 and Ser-172, which are phosphorylated dynamically, Ser-153 is believed to be stably phosphorylated. Ser-153 is positioned in the acidic Lck binding domain of LPAP ,37. The Ser-163 phosphorylation has been achieved by stimulating CD3/CD28 receptors , and is The cytoplasmic tail of LPAP contains three tyrosine residues, two of which, Tyr-64 and Tyr-115, are predicted to be phosphorylated with a high score by NetPhos. Tyr-115 is part of a consensus sequence, YxxV/L/I, for phosphorylation by Src family protein tyrosine kinase (PTK) , and a sIn contrast to protein kinases, Ser-Thr phosphatases from the most abundant group of PPP phosphatases do not have well-defined motifs and the prediction of their potential substrates is not straight-forward . There aConventionally, proteomic data concerns individual phosphorylation sites and does not reflect the presence of site combinations, but this information can be deduced from analysis of particular phosphoforms. Using 2D-PAGE and Phos-tag-PAGE, we resolved and quantitatively characterized at least six LPAP phosphoforms, providing the first quantitative measurement of LPAP phosphorylation at individual sites. Furthermore, we found that Ser-153 was phosphorylated in most LPAP molecules, whereas in resting CEM cells, Ser-99 and Ser-172 were phosphorylated in 35% and 15% of LPAP molecules, respectively.+ and CD8+ subsets. In line with this phospho-specific anti-p172 Abs gave poor reaction with LPAP from PBMC, possibly due to low stoichiometry of pSer-172 in these cells.It is important to determine whether LPAP phosphorylation characterized for CEM cells is valid for primary lymphocytes. We found that patterns of LPAP phosphorylation detected by SDS-PAGE in PBMCs resembled those in CEM cells. Therefore, we considered the phosphorylation sites identified in LPAP from CEM cells appropriate for LPAP from PBMCs. However, there is some uncertainty about phosphorylation of Ser-172 in lymphocytes. Thus, we could not detect a downward shifted band at 28 kDa in purified CD4In order to understand how proximal or distal LPAP phosphorylation takes place in a signaling pathway, we compared the kinetics of LPAP phosphorylation changes after PMA stimulation with the time courses of other PMA-induced processes. PMA is a direct PKC activator that promotes rapid (<5 min) redistribution of PKC from the cytosolic to the membrane compartment . PMA stiWe suggest that the phosphorylation of LPAP is involved in signal transduction in lymphocytes for the following reasons. First, LPAP is localized to the cell membrane. Second, LPAP is tightly associated with the following molecules involved in the regulation of lymphocyte signaling: protein tyrosine phosphatase CD45 , co-receIn summary, our study demonstrated that LPAP in lymphocytes exists in several phosphoforms, which we detected through site-directed mutagenesis, phospho-specific antibodies, and MS analysis. We have shown that phospho-modification of LPAP is altered dynamically and site-specifically upon cell stimulation with the PKC activator, PMA. We believe that the changes in the phosphorylation status of LPAP are important for TCR activation and signal transduction in primary human lymphocytes, and its comprehensive analysis may ultimately uncover a true function of this small enigmatic molecule.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.


Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities. Chromosome translocations are one of the most common types of genetic rearrangements induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies. The presence of disease-specific chromosome translocations, especially in hematological malignancies such as the t or Philadelphia chromosome in chronic myelocytic leukemia, has been reported. Molecular studies of the breakpoints of such disease-specific chromosome translocations have revealed the clustering of the breakpoints in specific regions, designated as the breakpoint cluster region (BCR). In lymphoid malignancies, the involvement of the physiological recombination of immunoglobulin and T-cell receptor genes in chromosome translocations has been suggested, due to the presence of signal sequences for the recombination at the breakpoints. However, the molecular mechanisms of chromosome translocations in other cell types remain largely unknown.Chromosome translocations arise as a consequence of errors in the repair of DNA double strand breaks (DSBs). Eukaryotic cells utilize a variety of repair pathways for DSBs, including two major ones, non-homologous end joining (NHEJ) and homologous recombination repair (HR). In the absence of these canonical pathways, the activation of the alternative NHEJ (Alt-EJ) pathway and the inactivation of DNA polymerase theta are implicated in chromosomal translocations  Mizuno . In contDNA damage leads to the activation of the DNA damage response and repair pathways. The ataxia-telangiectasia mutated (ATM) protein regulates the DNA damage response in reaction to DSBs, through its kinase activity . AlteratINO80 forms a chromatin remodeling complex with more than 15 subunits . Among tIn our previous study, we showed that ATM deficiency resulted in the overloading of INO80 and RAD51 onto the BCR of the MLL gene after etoposide treatment . The finThe substrates of ATM contain the core sequence with an SQ or TQ motif . By searTo confirm that Ser412 is the site in ARP8 that is phosphorylated after etoposide treatment, we produced an ARP8 mutant by replacing Ser412 with alanine. An immunoprecipitation analysis revealed that the etoposide-induced phosphorylation was completely abolished by the S412A substitution, indicating that Ser412 is the sole site within ARP8 that is phosphorylated in response to etoposide treatment .Next to investigate whether the etoposide-induced phosphorylation of ARP8 is regulated by ATM, we compared the phosphorylation status of ARP8 in ATM-deficient BIVA and ATM-proficient 11–4 cells after etoposide treatment. An immunoblotting analysis revealed that the etoposide-induced ARP8 phosphorylation in BIVA cells was lower than that in 11–4 cells . MoreoveSince ATMi did not completely abolish the etoposide-induced phosphorylation of ARP8, we next examined the relevance of ATR, another PI3-family kinase member sharing the same phosphorylation motif with ATM. ATR is responsible for the phosphorylation in the DNA replication stress response, and is activated by ATM after ionizing radiation . The etoHaving established that ARP8 is phosphorylated after etoposide treatment, we investigated the role of ARP8 in INO80 loading onto the MLL BCR . The enrTo determine whether the phosphorylation of ARP8 affects the loading of INO80 onto the MLL BCR through its interaction with INO80, we performed an immunoprecipitation analysis using U2OS cells. We found that etoposide treatment increased the interaction between INO80 and ARP8 in the ATM-proficient U2OS cells . The etoIn our previous study, we detected the excess loading of RAD51 onto the BCR of the MLL gene in etoposide-treated ATM-deficient BIVA cells . In yeasWe then studied the effect of ARP8 phosphorylation on the regulation of RAD51 binding to the BCR after etoposide treatment. The expression of the ARP8 phosphorylation-deficient mutant S412A increased the RAD51 binding to the BCR of the MLL gene after etoposide treatment in ATM-proficient cells . In ATM-Having established that ARP8 regulates the loading of RAD51 and INO80 onto the BCR after etoposide treatment, we decided to examine the involvement of ARP8 in 11q23 chromosome translocations. A two-color fluorescence in situ hybridization (FISH) analysis, covering the upstream and downstream regions of the MLL BCR, revealed that the number of BIVA cells carrying split FISH signals after etoposide treatment was significantly reduced by the siRNA-mediated depletion of ARP8, suggesting the involvement of ARP8 in 11q23 chromosomal abnormalities in ATM-deficient cells . This fiNext, to investigate the role of the ATM-dependent phosphorylation of ARP8 in preventing the 11q23 chromosome abnormalities, we performed the FISH analysis of ATM-deficient BIVA cells expressing the phospho-mimicking ARP8 S412D mutant. The FISH analysis revealed that the expression of ARP8 S412D reduced the number of cells exhibiting split FISH signals after etoposide treatment in ATM-deficient cells . In contSince the etoposide-induced phosphorylation of ARP8 was strongly repressed by an ATR inhibitor in 11–4 cells , the ATRNext, we examined the effect of an ATRi on the etoposide-induced 11q23 chromosome translocations in ATM-proficient 11–4 cells, by the dual color FISH analysis using the MLL gene probes . The incOur present results are the first to identify ARP8 as the phosphorylation target regulated by ATM and ATR after the induction of DNA damage by etoposide treatment. ARP8 facilitates the binding of INO80 and RAD51 to the BCR of the MLL gene, while the phosphorylation of ARP8 suppresses it through a reduction of its interaction with INO80. The incidence of etoposide-induced 11q23 translocations is reduced by the expression of the phospho-mimicking ARP8 mutant in ATM-deficient cells. Moreover, the expression of the phospho-deficient ARP8 in ATM-proficient cells increases chromosome translocations. ATR was not involved in the regulation of RAD51 binding to the BCR after etoposide treatment. These findings strongly suggest that ATM represses the 11q23 chromosome translocations by regulating the binding of INO80 and RAD51 to the BCR of the MLL gene at functionally appropriate levels, via the phosphorylation of ARP8.In response to DNA damage, the DNA repair process is facilitated by the phosphorylation of various proteins, including DNA repair factors, cell cycle regulators, and chromatin remodeling factors, through the positive regulation by ATM and ATR  Cimpric Clouair. In contThe phosphorylation of ARP8 is relatively slow, and occurs more than 2 hr after etoposide treatment, as compared to the early activation of ATM within 1 hr . Since tThis study revealed that ATR is likely to be the major kinase responsible for the etoposide-induced ARP8 phosphorylation. However, unlike the inhibition of ATM, ATR inhibition failed to increase the RAD51 binding to the BCR of MLL after etoposide treatment , suggestSeveral lines of evidence have suggested the association of HR with genomic instability . RAD51-dThe wild-type and the phospho-deficient mutant of ARP8 show increased interactions with INO80 after etoposide treatment, but the phospho-mimetic mutant does not  and 2. HChromosome abnormalities involving the MLL gene are one of the most frequent chromosomal aberrations observed in secondary leukemia associated with cancer therapy. We have shown that the etoposide-induced DNA damage in ATM-deficient cells facilitates the illegitimate recombination at the MLL gene, through the excessive binding of RAD51 and INO80 to the BCR. Our findings highlight the importance of the ATM-dependent modulation of recombination repair to avert 11q23 chromosome translocations. Further studies to investigate the mechanism that maintains the fidelity of DNA repair activity, using the chromosome translocations observed in secondary malignancy will provide new insights into the general mechanisms of carcinogenesis.The SV40-transformed AT fibroblast cell line AT5BIVA and its ATM-proficient derivative, AT5BIVA cells reconstituted with chromosome 11 (11-4), were kindly provided by Dr. S. Matsuura . The AT5The antibodies used for chromatin immunoprecipitation, immunoblotting, and immunofluorescence staining were rabbit anti-phospho ATM/ATR substrate motif , mouse anti-phospho ATM , mouse anti-HA, mouse anti-γH2AX (Merck Millipore), rabbit anti-RAD51 , rabbit anti -RPA2 S4/8 and rabbit anti-INO80 , mouse anti-RPA34 , rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) , rabbit anti-H2AX , and mouse anti-β-actin (Sigma-Aldrich) and rabbit anti-ARP8 .NotI/HindIII sites, followed by inserting the PCR-amplified HA between the ApaI and XhoI sites of pcDNA3.1/Myc-His (-) C. Plasmid transfections were performed using the GeneJuice transfection reagent , according to the manufacturer’s instructions. For rescue experiments, the siRNA-resistant ARP8 expression vector was co-transfected with the siRNA, using the Lipofectamine 2000 transfection reagent (Invitrogen) for 2 or 3 days. The mutant vectors were constructed by site-directed mutagenesis, using the indicated oligonucleotides: For the siRNA-resistant HA-ARP8 mutant, 5'-ctcaacaaaatgccaccatcgttcagacgtataattgaaaatgtggatg-3', for the HA-ARP8 S418A mutant, 5'-TTGCAGCACAGAGCTCAGGGCGATCCTG-3', and for the S418D mutant, 5'-TTGCAGCACAGAGATCAGGGCGATCCTG-3'. The sequences were confirmed using a BigDye Terminator v3.1 Cycle Sequencing Kit and an Applied Biosystems Genetic Analyzer, model 3130.All siRNAs were Ambion Silencer Select siRNAs . The siRNAs were s41201 for ARP8, s57219 for ATM, s3638 for CK2, s29257 and s224310 for INO80. Select Negative Control siRNA was used as the control. The siRNA interference experiments were performed 2 days after transfection with 0.2–0.3 nM siRNA, using Lipofectamine RNAiMAX (Invitrogen). The pcDNA 3.1 vector bearing the HA-tagged ARP8 was constructed by inserting the PCR-amplified ARP8 cDNA into the HindIII and KpnI sites of the pcDNA5/FRT/TO vector. For the induction of the HA-ARP8 expression, a final concentration of 2 μg/ml tetracycline was added for 24 hr.To generate cells stably expressing U2OS, the pcDNA 3.1 plasmid encoding the HA-tagged wild-type or S412A mutant of ARP8 was transfected using the GeneJuice transfection reagent, with G418 selection . After the confirmation of stable expression by immunofluorescence staining and immunoblotting, pools of single clones were used for experiments. For the generation of the inducible expression of wild-type or phosphorylation mutant ARP8 in 11–4 cells, the Flp-In System (Thermo Fisher Scientific) was used, according to the manufacturer’s instructions. The siRNA-resistant HA-tagged wild-type,or S412A ARP8 fragment from pcDNA 3.1 HA-ARP8 was inserted between the FISH analyses were performed using the 11q23 chromosome translocation probe  and the LSI MLL Dual Color, BreakApart Rearrangement Probe , according to the manufacturers’ protocols. For the DNA FISH analysis using the probe from Metaystems, the images were acquired on an Axio Imager Z2 microscope  equipped with the MetaSystems software. Subsequently, at least 2000 etoposide-exposed or DMSO-exposed cells were counted by the Metafer platform, MetaCyte, and the cells containing split signals (separated by >1 μm) were monitored. For the DNA FISH analysis using the probe from Vysis, the images were acquired on a Zeiss AxioplanII microscope using an AxioCamMRm controlled by Axiovision. At least 200 etoposide-exposed or DMSO-exposed cells were counted. All FISH analyses were repeated three times.In brief, the cells were fixed by adding formaldehyde to a 1% final concentration for 10 min at 25°C. The cells were then sonicated to prepare chromatin suspensions of DNA fragments that were roughly 300–500 bps in length. Immunoprecipitations were performed using antibodies against INO80 and RAD51. Normal rabbit IgG was used as the negative control. Real-time PCR reactions were performed using SYBR premix Ex Taq . The dissociation curve analysis of the melting temperature of the amplified DNA showed that each primer set gave a single, specific product. The immunoprecipitation data were normalized to those of a control region in the GAPDH or β-globin gene, to correct for experimental variation. The relative immunoprecipitation value represents the ratio of the immunoprecipitated DNA after chemical treatment to the immunoprecipitated DNA after vehicle treatment. All ChIP analyses were repeated at least three times, and in each experiment, quantitative PCR reactions were performed in duplicate. Values represent the means ± SE. The primers for real-time PCR were: bt56 forward: 5 '-TACTCTGAATCTCCCGCA-3' bt56 reverse: 5'-CGCTCGTTCTCCTCTAA-3' t56 forward: 5'-TTGCCAAGTCTGTTGTGAGC-3' t56 reverse: 5'-CAGAGGCCCAGCTGTAGTTC-3'GAPDH forward: 5'-TCTCCCCACACACATGCACTT-3'GAPDH reverse: 5'-CCTAGTCCCAGGGCTTTGATT-3'.β-globin forward: 5'-TTGGACCCAGAGGTTCTTTG3'β-globin reverse: 5'-GAGCCAGGCCATCACTAAAG3'After fixation in 4% paraformaldehyde in 1X phosphate-buffered saline (PBS) for 10 min at room temperature, the cells were permeabilized with 0.1% sodium dodecyl sulfate (SDS)−0.5% Triton X-100 in 1x PBS for 5 min. For the detection of HA-tagged ARP8, fixed cells were incubated for 30 min at 37°C with a mouse anti-HA antibody (1:600) in 1% bovine serum albumin (BSA)/1 XPBS. Cy3-conjugated goat anti-mouse  antibodies were used as the secondary antibodies. Cells were mounted using Vectashield containing DAPI and observed with a BZ-X700 microscope (Keyence).The nuclear fraction was prepared as described previously . The whoGM0637 cells were transfected with the HA-tagged wild type or S412A or S412D mutant of ARP8 or empty vector for 24 hr, fixed with 2% paraformaldehyde in PBS for 10 min at room temperature and permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. The cells were incubated with the rabbit anti-INO80 and mouse anti-HA antibodies diluted in 0.1% BSA for 30 min at 37°C in a moist chamber. Proximity ligation was then conducted in situ, according to the manufacturer’s instructions . We used the PLA probe anti-rabbit PLUS and the PLA probe anti-mouse MINUS. To visualize the interaction between two proteins, the samples were incubated for ligation and amplification. After serial SSC (sodium/sodium citrate) washes, nuclei were stained with DAPI. The slides were mounted with Vectashield (Vector Labs). PLA signals were detected with an LSM780 confocal microscope (Carl Zeiss), with a 63 × 1.40 NA plan-apochromat objective, and counted in at least 400 cells with the Image J software. All PLA analyses were repeated four times.Data in all experiments are represented as mean ± SE. Statistical analysis was performed using the two-tailed unpaired t- test. For the FISH analysis, the percentages of cells with split signals were determined by the Z test of homogeneity for independent samples. Results reaching p<0.05 were considered to be statistically significant . In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Jerry Workman as the Reviewing Editor and Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "ATM phosphorylates ARP8 to prevent chromosome translocations by counteracting repair protein loading to damaged sites" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this manuscript, the authors addressed the phosphorylation of ARP8, a subunit of INO80-C, upon etoposide treatment, and how this phosphorylation affects the MLL translocation, which occurs frequently in leukemia. The authors established that ATM regulates phosphorylation of ARP8 S412 after etoposide treatment by an anti-ATM/ATR substrate antibody. Arp8 phosphorylation detected by this antibody was sensitive to the phosphatase treatment and it was abolished upon mutation of ARP8 S412 to alanine, suggesting that S412 is the only phosphorylation site detectable in ARP8. They further showed that S412 phosphorylation is sensitive to treatment with the ATM inhibitor KU55933 and to knockdown of ATM siRNA, suggesting that ATM regulates S412 phosphorylation.To explore the molecular role of ARP8 S412 phosphorylation, the authors first tested its influence on INO80-ARP8 protein interaction upon etoposide treatment. They found that ATM inhibition or ARP8 alanine mutation at S412 of ARP8 enhanced INO80-ARP8 interaction. These findings indicate that ATM dependent phosphorylation on ARP8-S412 reduces ARP8 INO80 interaction.To elucidate consequences of changed ARP8 INO80 interaction, the authors investigated the binding of INO80 and a HR protein RAD51 to a chromosome breakpoint clustered in BCR MLL locus that is associated with chromosome translocation. They demonstrated by ChIP analyses that increased INO80 and RAD51 association to the break point upon etoposide treatment is sensitive to ARP8 silencing and phospho-mimetic S412D ARP8 mutation in ATM deficient BIVA cells. Conversely, they showed that an ARP8 S412A non-phospho mutation increases INO80 and RAD51 binding to the break point in ATM-proficient cells. These results suggest that the phosphorylation of ARP8 S412, which minimizes the INO80 interaction, reduces INO80 and RAD51 association to the break point.The authors further tested the ARP8 mutants on translocation. They showed by FISH analysis that 11q23 chromosome translocations after etoposide treatment in ATM-deficient cells (BIVA) are significantly reduced upon siRNA-mediated knockdown of ARP8, whereas translocations are increased in ATM-proficient cells (11-4). Expression of the ARP8 S412A or D mutant affects the number of chromosome translocations in an opposite manner, consistent with the notion that the ARP8 S412 phosphorylation reduces translocation events.Overall, the data presented in the manuscript support a model that ARP8 phosphorylation mediated by ATM suppresses 11q23 translocation by reducing the binding of the INO80 and RAD51 HR protein to the break site. However, authors did not provide any evidence that ATM directly targets ARP8, nor ATM and ARP8 function in the same pathway.Essential revisions:1) In the current manuscript, we did not find data that shows direct phosphorylation of ARP8 S412 by ATM, as the title of this manuscript indicates. The antibody probes for ATM or ATR targets and ATM inhibition only partially inhibits ARP8 phosphorylation detected by the antibody. Furthermore, ARP8 S412 phosphorylation status in ATM deficient BIVA cells compared to the ATM proficient cells was not shown. The onset of S412 phosphorylation after etoposide treatment is rather late. To correlate ATM action in this event, authors should compare the kinetics of ATM activation and another ATM target in parallel to ARP8 S412. As in the Discussion, it is plausible that ATR is also responsible for the ARP8 S412 phosphorylation. Phosphorylation analysis in combination with an ATR inhibitor would help to answer this question. To evaluate the phenotype that is influenced by ARP8 S412 phosphorylation and ATM, you should analyze the epistatic relation. Is S412A mutation additive or not to the ATM deficiency or ATM inhibition? For example, does ARP8 S412A mutation increase INO80 and RAD51 binding to the break point and 11q23 translocation increased in BIVA cells or by ATM inhibition in ATM proficient cells? Basically, we feel that there are many over-interpretations of these data throughout the manuscript .Under the current data set, the authors should not state "ATM phosphorylates ARP8", rather than "ATM regulates ARP8 phosphorylation", otherwise authors should provide an in vitro ATM kinase assay on ARP8.2) Data showing that ARP8 phosphorylation is attenuated by ATMi could be bolstered by examining phosphorylation status of ARP8 in ATM-deficient cells (ATVBIVA). A possible role of ATR in this phosphorylation should be investigated at least by using specific ATRi.3) Since gammaH2AX recruits INO80 to DSBs, the ChIP analyses in Figure 2 could be extended to gammaH2AX to confirm that etoposide induces DSBs specifically at the BCR.4) Does the S412D mutant fail to interact with INO80 in co-IPs? That could help explain the lack of INO80 enrichment in Figure 2D.5) Is the increased INO80 enrichment in Figure 2C altered by ATMi?6) In Figure 3B, it is difficult to compare interaction of WT and S412A ARP8 as the two data sets are on separate blots. Why does the interaction of WT become apparent only at 4 h, unlike at 2h in earlier blots?7) Figure 1—figure supplement 1A – it is unclear what is being shown in Figure 1—figure supplement 1A. On the anti-Ino80 IP, the upper band appears to be phosphorylation in untreated conditions and the lower band appears to be a background band that copurifies with IgG. Which is the band we should be noticing? A molecular weight marker is needed on all gels using the ATM/ATR substrate antibody.8) Figure 1C – why is the reduction of Arp8 phosphorylation not to the same degree as the phospho-Rpa2? Is the ATM/ATR substrate antibody detecting some other phospho substrate?Similar comment for Figure 1—figure supplement 1E – even after treatment with siATM, there seems to be increased signal compared to the ctrl (-etoposide) sample. The reason for this needs to be resolved.9) Figure 2 – while ChIP results are noted as statistically significant, the change in enrichment appears modest. What are the absolute values  of enrichment? How does this (modest) fold change alter downstream genome stability? Similar questions apply to many of the ChIPs in this manuscript.10) Figure 3B and Figure 3—figure supplement 1B – interaction of Ino80 appears modest. In Figure 3B it looks like it peaks at 2h then is reduced. Please explain why would interaction be reduced after 2 hours?11) Figure 5 – the translocation results look clear, yet modest. Can the authors provide a point of reference to better judge these results? For example, what translocation rate would be expected upon overexpression or deletion of a repair protein?12) The model of Arp8 phosphorylation weakening the interaction with Ino80 was a bit speculative, and the IP westerns not terribly convincing as is. We suggest probing for additional subunits in the IP westerns. I believe there are a couple of commercially available antibodies that can be used.13) The authors should clearly discuss how ATM-mediated inhibition of INO80 and RAD51 loading at BCR serves to suppress translocations.14) The authors should discuss if such a suppression of RAD51 loading is specific to BCR regions or is applicable to DSBs in general.eLife. Your revised article has been favorably evaluated by Jessica Tyler (Senior Editor), Jerry Workman (Reviewing Editor), and three reviewers.Thank you for resubmitting your work entitled "ATM regulates ARP8 phosphorylation to prevent chromosome translocations by counteracting DNA repair protein loading at damaged sites" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:eLife.Generally the authors have gone to great lengths to perform the requested experiments. Most are carried out adequately, although the results were not fully incorporated into the text and conclusions. In general, the findings are novel and important, as they provide relevant insight into the crosstalk of checkpoint kinases and chromatin remodelers in DNA repair and the phenotypes they monitor are indeed, cancer associated (chromosome translocation). Thus the significance of the paper is appropriate for Major Comments:1) New data provided in the revised manuscript suggests that a major kinase responsible for ARP8 phosphorylation after etoposide treatment is ATR . ATM has a slight effect, but ATR (assuming the inhibitor used is specific) is more important. It is unclear still whether either kinase modifies directly or perhaps is upstream of another kinase. But given the fact that their own inhibitor study implicates ATR more strongly than ATM, the authors should test the effect of ATR inhibition on chromosome translocation. The effect might be more pronounced than ATM ablation. This is a minimal requirement for publication.2) If ATR inhibition does not affect Rad51 loading then ATM is likely functioning locally at the translocation site, although ATR phosphorylates ARP8 globally. If acute ATR inhibition also influences chromosome translocation, authors should extensively re-write the manuscript so that both checkpoint kinases, ATM and ATR, are implicated in the phosphorylation of ARP8 and the prevention of chromosome translocation. The authors need to provide more detailed information on the conditions used for ATM and ATR inhibitor treatment. KU55933 and ATR inhibitor IV  were shown to be highly selective but ATP competitive inhibitors are non-specific at the high dose.3) Finally, based on the outcome of testing ATR knockdown or inhibition in some of the assays already included, the authors should change the text, main title and subtitles to recognize their own ATR results  In the current manuscript, we did not find data that shows direct phosphorylation of ARP8 S412 by ATM, as the title of this manuscript indicates. The antibody probes for ATM or ATR targets and ATM inhibition only partially inhibits ARP8 phosphorylation detected by the antibody. Furthermore, ARP8 S412 phosphorylation status in ATM deficient BIVA cells compared to the ATM proficient cells was not shown. The onset of S412 phosphorylation after etoposide treatment is rather late. To correlate ATM action in this event, authors should compare the kinetics of ATM activation and another ATM target in parallel to ARP8 S412. As in the Discussion, it is plausible that ATR is also responsible for the ARP8 S412 phosphorylation. Phosphorylation analysis in combination with an ATR inhibitor would help to answer this question. To evaluate the phenotype that is influenced by ARP8 S412 phosphorylation and ATM, you should analyze the epistatic relation. Is S412A mutation additive or not to the ATM deficiency or ATM inhibition? For example, does ARP8 S412A mutation increase INO80 and RAD51 binding to the break point and 11q23 translocation increased in BIVA cells or by ATM inhibition in ATM proficient cells? Basically, we feel that there are many over-interpretations of these data throughout the manuscript .Under the current data set, the authors should not state "ATM phosphorylates ARP8", rather than "ATM regulates ARP8 phosphorylation", otherwise authors should provide an in vitro ATM kinase assay on ARP8.Thank you very much for the constructive comments to improve our manuscript. We agree with the reviewers that our findings are not sufficient to indicate the direct phosphorylation of ARP8 S 412 by ATM.Following the reviewers’ advice, we compared the kinetics of ATM activation and other ATM targets, H2AX and RPA2, in parallel with ARP8 S412. As a result, the phosphorylations of ATM and H2AX were detected at 30 min after the induction of DNA damage. In contrast, the phosphorylations of RPA2 and ARP8 were relatively slow, and increased at 2 hrs and peaked at 4 hrs after etoposide treatment . These findings suggest that ATM may activate other kinases to phosphorylate ARP8 after etoposide treatment. This notion was also supported by the results from an experiment to compare the etoposide-induced phosphorylation status of ARP8 in ATM-deficient BIVA cells and ATM inhibitor-treated ATM-proficient 11-4 cells . The phosphorylation of ARP8 was not completely abolished in ATM-deficient and ATM inhibitor-treated cells.Next, we examined the involvement of ATR in the phosphorylation of ARP8 after etoposide treatment, according to the reviewers’ suggestion. An ATR inhibitor also repressed the phosphorylation of ARP8 after etoposide treatment. This finding suggests the involvement of ATR in the regulation of ARP8 phosphorylation after etoposide treatment . Taken together, these results suggest that ATM and ATR regulate the phosphorylation of ARP8 after etoposide treatment.Following the reviewers’ advice, we also examined the effect of ATM inhibition on the ARP8 S412A mutant by the ChIP analysis of 11-4 cells after etoposide treatment, using antibodies against INO80 and RAD51. We could not detect the additive effect of the ATM inhibitor on the ARP8 S412A mutation . ATMi treatment failed to enhance the etoposide-induced 11q23 translocations in ARP8 S412A mutant-expressing cells . This finding suggests that ATM regulates ARP8 phosphorylation at S412, to regulate the loading of INO80 and RAD51 on BCR after etoposide treatment. In the revised manuscript, the original Figures 4A and 4B are presented as Figure 4—figure supplement 1C and Figure 4A, respectively. We have also replaced original Figure 4—figure supplement 1B with data from six independent experiments.Taken together, these findings suggest that the etoposide-induced phosphorylation of ARP8 at S412 is regulated by ATM and ATR. Accordingly, we have changed the title to “ATM regulates ARP8 phosphorylation to prevent chromosome translocations by counteracting repair proteins loading at damaged sites”, according to the reviewers’ suggestions.2) Data showing that ARP8 phosphorylation is attenuated by ATMi could be bolstered by examining phosphorylation status of ARP8 in ATM-deficient cells (ATVBIVA). A possible role of ATR in this phosphorylation should be investigated at least by using specific ATRi.Following the reviewer’s comment, we examined the phosphorylation status of ARP8 in ATM-deficient BIVA cells after etoposide treatment . As a result, the etoposide-induced phosphorylation of ARP8 was repressed in BIVA cells. Although the basal level of ARP8 phosphorylation in BIVA cells was higher than that in ATM-proficient 11-4 cells , this finding confirms the involvement of ATM in the regulation of the etoposide-induced phosphorylation of ARP8.We also examined the effect of ATRi on the phosphorylation status of ARP8 after etoposide treatment in ATM-proficient 11-4 cells . We found that ATRi repressed the phosphorylation of ARP8 in ATM-proficient cells. These findings suggest that ATM and ATR regulate the etoposide-induced ARP8 phosphorylation. Accordingly, we have revised manuscript by including these findings.3) Since gammaH2AX recruits INO80 to DSBs, the ChIP analyses in Figure 2 could be extended to gammaH2AX to confirm that etoposide induces DSBs specifically at the BCR.Following the reviewer’s comment, we performed a chromatin immunoprecipitation assay using anti-gammaH2AX antibodies before and after etoposide treatment. The enrichment of gammaH2AX on BCR was observed after etoposide treatment, confirming that etoposide induces DNA damage specifically at the BCR. Accordingly, we have presented this result in the revised Figure 2—figure supplement 1A.4) Does the S412D mutant fail to interact with INO80 in co-IPs? That could help explain the lack of INO80 enrichment in Figure 2D.Following the reviewer’s suggestion, we performed the immunoprecipitation analysis to examine the interaction between S412D and INO80 in ATM-deficient cells . The interaction of the ARP8 S412D mutant with INO80 was weaker than that of ARP8 WT. The weaker interaction of the ARP8 S412D mutant with INO80 was also confirmed by the Proximity ligation assay (PLA) assay, as shown in Figure 3—figure supplement 2 in the revised manuscript. Accordingly, we have revised the manuscript by including these figures.5) Is the increased INO80 enrichment in Figure 2C altered by ATMi?We performed a chromatin immunoprecipitation analysis of INO80 using ATMi treated 11-4 Flp-in cells expressing either ARP8 wild-type or S412A mutant. The INO80 enrichment onto MLL BCR was increased by ATMi in ARP8 wild-type expressing cells, while no significant changes were observed in ARP8 S412A mutant expressing cells. This finding suggests the epistatic relation between ATM and ARP8 S412 phosphorylation. Accordingly, the data are presented in Figure 2C and D in the revised version.6) In Figure 3B, it is difficult to compare interaction of WT and S412A ARP8 as the two data sets are on separate blots. Why does the interaction of WT become apparent only at 4 h, unlike at 2h in earlier blots?Following the reviewer’s advice, we applied two sets of samples on one blot. The amounts of INO80 and HA-ARP8 from three independent experiments were quantified, using the Image J software. The S412A mutation increased the interaction of ARP8 with INO80. The gel images and quantitative results are presented in Figure 3B in the revised version.7) Figure 1—figure supplement 1A – it is unclear what is being shown in Figure 1—figure supplement 1A. On the anti-Ino80 IP, the upper band appears to be phosphorylation in untreated conditions and the lower band appears to be a background band that copurifies with IgG. Which is the band we should be noticing? A molecular weight marker is needed on all gels using the ATM/ATR substrate antibody.We apologize for the confusing data. We have added a molecular weight marker, and denoted the position of INO80 using an arrow in the revised Figure 1—figure supplement 1A. The upper bands detected by the anti-ATM/ATR substrate antibodies showed no significant difference after etoposide treatment. Since the size of the band is consistent with that of INO80, this upper band could be INO80 with background phosphorylation. The other possibility is that the upper band is non-specific. We agree with the reviewers that the lower band is a non-specific background band.Accordingly, we have revised Figure 1—figure supplement 1A.8) Figure 1C – why is the reduction of Arp8 phosphorylation not to the same degree as the phospho-Rpa2? Is the ATM/ATR substrate antibody detecting some other phospho substrate?Similar comment for Figure 1—figure supplement 1E – even after treatment with siATM, there seems to be increased signal compared to the ctrl (-etoposide) sample. The reason for this needs to be resolved.Thank you very much for pointing out this important issue. There are two possible explanations for this issue. There could be a difference in the detection efficiency of the phosphorylated forms of ARP8 and RPA2. We used antibodies against ATM/ATR substrates for the detection of phosphorylated ARP8. In contrast, we applied antibodies against RPA2 to detect the phosphorylated form of endogenous RPA2, as slowly migrating bands. The other possibility is that ATR is more deeply involved in the regulation of ARP8 phosphorylation than that of RPA2 after etoposide treatment.The phosphorylation of ARP8, as detected by the ATM/ATR substrate antibodies, was completely abolished by the S412A mutation. Therefore, the ATM/ATR substrate antibody should detect the phosphorylation of ARP8 at S412.As the reviewer pointed out, the phosphorylated form of ARP8 was still detected in ATM-depleted cells after etoposide treatment in Figure 1—figure supplement 1E. Since we found that ATR is also responsible for the phosphorylation of ARP8 after etoposide treatment, from the experiments suggested by the reviewer , ATR could facilitate phosphorylation of ARP8 even in ATM-depleted cells after etoposide treatment.9) Figure 2 – while ChIP results are noted as statistically significant, the change in enrichment appears modest. What are the absolute values  of enrichment? How does this (modest) fold change alter downstream genome stability? Similar questions apply to many of the ChIPs in this manuscript.In the revised manuscript, we have added the absolute values of enrichment in the ChIP analysis, in Figure 2—source data 1.As the reviewer pointed out, the change in enrichment of INO80 and RAD51 at BCR is modest, but increases chromosome translocations involving BCR. The possible explanation for this is that the binding of chromatin remodelers and DNA repair factors on damaged chromatin is dynamic, not static. Such a dynamic binding could reduce the ChIP value.10) Figure 3B and Figure 3—figure supplement 1B – interaction of Ino80 appears modest. In Figure 3B it looks like it peaks at 2h then is reduced. Please explain why would interaction be reduced after 2 hours?As in the original Figure 3B, the interaction of INO80 with the ARP8 S412A mutant also apparently peaks at 2h and then is reduced, in the revised Figure 3B. Factors associated with the homologous recombination repair pathway (HR) may be involved in the regulation of INO80 binding to ARP8. The other possibility is that the phosphorylation of ARP8 may facilitate the dissociation of the INO80 complex from damaged chromatin after the appropriate remodeling of damaged chromatin for DNA repair to avoid illegitimate recombination, leading to chromosome abnormalities. These possibilities are discussed in the second paragraph of the Discussion.11) Figure 5 – the translocation results look clear, yet modest. Can the authors provide a point of reference to better judge these results? For example, what translocation rate would be expected upon overexpression or deletion of a repair protein?Following the reviewer’s suggestion, we cited two papers here. One study showed the increased chromosome translocations in RAD51-overexpressing cells . The other paper from Mizutani’s lab showed the increased incidence of 11q23 translocations in ATM-deficient cells . We have reported that the percentage of the ATM-deficient BIVA cells with the 11q23 chromosome splitting signal is increased from 0.17% to 2.5% by etoposide treatment, and in etoposide-treated ATM-proficient cells from 1.33% to 5.83% after ATMi treatment .12) The model of Arp8 phosphorylation weakening the interaction with Ino80 was a bit speculative, and the IP westerns not terribly convincing as is. We suggest probing for additional subunits in the IP westerns. I believe there are a couple of commercially available antibodies that can be used.I agree that the IP westerns are not convincing. Following the reviewer’s comment, we reprobed the blots with antibodies against ARP5. As a result, similar to INO80, we could observe a slightly increased amount of ARP5 in the S412A precipitated blot, as compared with that of wild-type ARP8 in ATM proficient cells. Similar results were obtained in wild-type ARP8, but not in S412D-expressing, ATM-deficient BIVA cells. Moreover, we also confirmed the role of the phosphorylation of ARP8 in the interaction with INO80 using a Proximity ligation assay, as described above. We would like to present the following data to the reviewers.Examination of the interaction between INO80 and ARP8 in U2OS cells expressing HA-tagged wild-type or S412A ARP8. The endogenous ARP8-depleted cells were treated with etoposide for 15 min. After the cells were washed, they were placed in fresh medium and harvested at the indicated time points. The nuclear extracts were incubated with anti-HA-conjugated anti-mouse IgG Dynabeads. The precipitates were electrophoresed through a gel and probed by western blotting with an anti-INO80 or an anti-HA or an anti-ATM/ATR substrate antibody. The amounts of INO80 and HA-ARP8 were quantified, using the Image J software. The results of quantitative analysis are shown as the relative values as compared to the DMSO control from three independent experiments.Co-immunoprecipitation analysis of ARP8 and INO80. ATM-deficient BIVA cells were co-transfected with the siARP8 and siARP8-resistant HA-tagged wild-type ARP8 or phospho-mimetic S412D ARP8 mutant. After etoposide removal, the cells were recovered at the indicated time points. The nuclear extracts were incubated with anti-HA-conjugated anti-mouse IgG Dynabeads. The precipitates were electrophoresed through a gel and probed by western blotting with either an anti-INO80 antibody or an anti-HA antibody. The amounts of INO80 and HA-ARP8 were quantified, using the Image J software. The results of quantitative analysis are shown as the relative values as compared to the DMSO control, from three independent experiments.13) The authors should clearly discuss how ATM-mediated inhibition of INO80 and RAD51 loading at BCR serves to suppress translocations.According to the reviewer’s comment, we have rewritten the second and third paragraphs of the Discussion, as follows:“In response to DNA damage, the DNA repair process is facilitated by the phosphorylation of various proteins, including DNA repair factors, cell cycle regulators, and chromatin relaxing factors, through the positive regulation by ATM and ATR   Clouaire et al., 2017; Shiloh, 2003; Shiloh and Ziv, 2013). […] The phosphorylation of ARP8 after etoposide treatment could be regulated by multiple steps and factors, for the precise control of DNA repair activity to maintain chromosome stability.”14) The authors should discuss if such a suppression of RAD51 loading is specific to BCR regions or is applicable to DSBs in general.According to the reviewer’s comment, we have added the following sentences at the end of the fourth paragraph of the Discussion:“INO80 and ARP8 have been shown to regulate the RAD51 loading to damaged chromatin in yeast    . […] Therefore, this regulation of HR by ARP8 in human cells may also be applicable to DSBs in general.”Major Comments:1) New data provided in the revised manuscript suggests that a major kinase responsible for ARP8 phosphorylation after etoposide treatment is ATR . ATM has a slight effect, but ATR (assuming the inhibitor used is specific) is more important. It is unclear still whether either kinase modifies directly or perhaps is upstream of another kinase. But given the fact that their own inhibitor study implicates ATR more strongly than ATM, the authors should test the effect of ATR inhibition on chromosome translocation. The effect might be more pronounced than ATM ablation. This is a minimal requirement for publication.Thanks for the important comment. Following the editor’s advice, we examined the effect of the ATR inhibitor VE821 on the etoposide-induced 11q23 chromosome translocations in ATM-proficient 11-4 cells, by the dual color FISH analysis using the MLL gene probes . The FISH analysis revealed that the increase of the split signal positive cells by ATRi was less than that by ATMi. Although ATR is suggested to be the major kinase responsible for ARP8 phosphorylation after etoposide treatment, this finding suggests that the effect of ATRi on the etoposide-induced chromosome translocations is limited. Moreover, no additional effects of ATRi on the increase of the chromosome translocations by ATMi were observed. Taken together, these findings strongly suggest that ATM is likely functioning locally at the translocation site, while ATR phosphorylates ARP8 globally. Accordingly, we have added a section “ATM, but not ATR, negatively regulates RAD51 loading onto the BCR after etoposide treatment to repress 11q23 chromosome translocations” in the “Results”.2) If ATR inhibition does not affect Rad51 loading then ATM is likely functioning locally at the translocation site, although ATR phosphorylates ARP8 globally. If acute ATR inhibition also influences chromosome translocation, authors should extensively re-write the manuscript so that both checkpoint kinases, ATM and ATR, are implicated in the phosphorylation of ARP8 and the prevention of chromosome translocation. The authors need to provide more detailed information on the conditions used for ATM and ATR inhibitor treatment. KU55933 and ATR inhibitor IV  were shown to be highly selective but ATP competitive inhibitors are non-specific at the high dose.We examined the effect of ATR inhibition on the RAD51 loading on the BCR of MLL in 11-4 cells after etoposide treatment, by a ChIP analysis. In contrast to the increased loading of RAD51 onto the BCR of MLL after etoposide treatment by ATMi, ATRi failed to do so . This finding is consistent with the results obtained by the FISH experiments described above. As the editors suggested, ATM is likely functioning locally at the translocation sites, while ATR is functioning globally. Accordingly, we have discussed this point in the fourth paragraph in the “Discussion”.We apologize for providing insufficient information about the conditions used for the ATM and ATR inhibitor treatments. We now provide detailed information in the “Materials and methods” and the figure legends in the revised manuscript. As the reviewer speculated, the ATR inhibitor we used was VE821.3) Finally, based on the outcome of testing ATR knockdown or inhibition in some of the assays already included, the authors should change the text, main title and subtitles to recognize their own ATR results . These results suggest that the ARP8 phosphorylation regulated by ATR is functionally distinct from that by ATM. Accordingly, we have changed the title to “Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations”.


Syk activity relies on its phosphorylation status and its multiple phosphorylation sites predict several Syk conformations. In this report, we characterized Syk structural changes according to its phosphorylation/activation status by Surface Enhanced Raman Spectroscopy (SERS). Unphosphorylated/inactive and phosphorylated/active Syk forms were produced into two expression systems with different phosphorylation capability. Syk forms were then analysed by SERS that was carried out in liquid condition on a lithographically designed gold nanocylinders array. Our study demonstrated that SERS signatures of the two Syk forms were drastically distinct, indicating structural modifications related to their phosphorylation status. By comparison with the atomic structure of the unphosphorylated Syk, the SERS peak assignments of the phosphorylated Syk nearest gold nanostructures revealed a differential interaction with the gold surface. We finally described a model for Syk conformational variations according to its phosphorylation status. In conclusion, SERS is an efficient technical approach for studying  In SERS, Raman scattering efficiency is drastically enhanced at the surface of noble metal nanostructures by the generation of a locally enhanced electromagnetic (EM) field following excitation of Localized Surface Plasmon Resonance (LSPR)19202123242526In this work, we applied the SERS based technique to analyse Syk conformational changes related to its phosphorylation status. After evaluating both phosphorylation and kinase activity levels of Syk produced in two distinct expression systems, the SERS spectra were acquired in liquid on a gold nanocylinders array. SERS signatures of phosphorylated and unphosphorylated Syk forms, which reflected interaction between gold surface and Syk amino acid residues exposed at the surface of the nanostructure, were drastically different. With the help of atomic structural data of unphosphorylated Syk, SERS results allowed us to propose a model of Syk conformational changes according to its phosphorylation/activation.To study Syk structural variations as a function of its phosphorylation status, we first confirmed the relationship between the phosphorylation status and the kinase activity with a wild type (WT) human Syk protein produced either in prokaryotic BL21 bacteria or in eukaryotic Sf9 insect cells systems. Syk expression and its global tyrosine phosphorylation levels were detected by western blot using anti-Syk (α-Syk) and anti-phosphotyrosine antibodies (α-pY), respectively.Due to the lack of post-translational modifications machinery in bacteriaAltogether, these data demonstrated that Syk protein produced in bacteria was un-phosphorylated and inactive  whereas that produced in insect cells was phosphorylated and kinase active .After having checked the quality of the fabricated nanostructures , SERS ex−1 for unP-, deP- and KD-Syk whereas P-Syk had a radically different SERS spectrum. With similar biochemical properties in terms of phosphorylation and kinase activity statuses, unP-Syk and deP-Syk samples showed rather equivalent spectral features. Importantly, this common spectral signature strongly excluded a contribution of the expression system used (bacteria vs insect cells) as well as the tags (His vs GST-His). Indeed, SERS spectrum of the GST protein  residues nearby. Peaks around 620 and 715 cm−1 correspond to C-S stretching vibration. All these peaks were detected in the spectra of KD-, unP- and deP-Syk but not in the spectrum of P-Syk. According to the recent atomic structural analysis of unP-Syk−1 on the Rayleigh scattering background in the unphosphorylated Syk spectra could be assigned to S-Au vibration by a contact of the thiol group of the Cys101 to the gold surface  that renders the kinase inactive−1 that included peaks assigned to aromatic amino acid residues that were not substituted by the mutation. Therefore, the observed spectral distinctions, which probably result from structural modifications caused by the mutation, implied proximity of the Syk kinase domain to gold nanostructures. This model was reinforced by the presence of peaks corresponding to a disulfide bond between two Cys residues in KD-Syk, unP-Syk and deP-Syk spectra as described above. While peaks around 965 cm−1 for Trp and 1170 cm−1 for Tyr or Phe were detected in both unP- and deP-Syk, small contributions from Phe or Trp were detected at 1014 cm−1 in the P-Syk spectrum.The bands associated with amino acids carrying aromatic rings in their side chains, such as tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe) and histidine (His), are usually the main components of protein SERS spectra. Peaks around 750, 1530, 1580 cm−1 and were assigned to aliphatic amino acids with bulky hydrocarbons, such as valine , leucine (Leu) and isoleucine (Ile). These spectral features were strongly detected in all Syk spectra and dominant for the P-Syk spectrum. In contrast, the amide I bands around 1630–1680 cm−1, which are typical of protein secondary structure originated from polypeptide backbones, were poorly or not detected in all Syk spectra. The signal from amide I bands are normally the strongest among other amide bands, therefore bands around 1550 cm−1 for amide II and from 1230–1300 cm−1 for amide III should have little contribution to all the spectra. Thus, these silent amide bands were likely to come from the large distance of the Syk peptide bonds from the gold surfaceTo further analyse our Syk SERS spectra, we next focused our attention on the peaks from aliphatic carbons that were also observed between 1290 and 1490 cmCollectively, global and detailed comparisons of the four Syk SERS spectra showed differential patterns corresponding to the structural variations of their biophysical properties especially between unphosphorylated/inactive Syk and phosphorylated/active Syk.SERS spectrum mainly reflects adsorbed part of proteins onto gold nanostructures with an EM field generated by LSPR excitation exponentially decaying from a gold surfaceThe phosphorylation impact on Syk structure was then examined based on the distinct SERS spectra between unP-Syk and P-Syk. Interestingly, these spectral variations were larger than the ones observed between unP-Syk and KD-Syk, indicating that Syk phosphorylation allowed a larger structural change as compared to those created by a point mutation in the Syk kinase domain. The structural change resulted in another interaction between P-Syk and the gold surface. Indeed, peaks corresponding to thiol and disulfide bonds were no longer detected in P-Syk, reflecting a large modulation of the link between Syk and the gold surface. Moreover, peaks from the Tyr and Trp residues were weakly detected in P-Syk spectrum, revealing that these amino acids were not anymore in close proximity to the gold surface. Furthermore, aliphatic carbon signatures dominated the P-Syk SERS spectrum and their hydrophobic properties might explain how Syk interacts with the gold surface. Thus, the hydrophobic part that is folded inside and masked in the compact structure of Syk might be uncovered following phosphorylation. Flexible interdomains are thought to induce a local conformational change through phosphorylation of tyrosine residues in these regionsOur results are summarized and illustrated in 13In this report, we studied the conformational differences between phosphorylated and unphosphorylated forms of the protein. It is worth noting that spectral similarities between unP-Syk and deP-Syk suggested that phosphorylated Syk was able to reverse its conformation once it was unphosphorylated following phosphatase treatment. Additionally, the presence of multiple phosphorylation sites in Syk suggested intermediate phosphorylation statuses that might translate into diverse SERS spectra. In a preliminary study, SERS spectra of other Syk forms, which showed intermediate phosphorylation levels between unP-Syk and P-Syk species by western blot (data not shown), shared spectral features of both unP-Syk and P-Syk. Indeed, a gradual transition of the SERS peaks from unP-Syk to P-Syk was observed when the phosphorylation levels were increased . WithoutHis-tag human unphosphorylated wild type (WT) (unP-Syk) and kinase-dead (KD-Syk) Syk recombinant proteins were produced in BL21 bacteria (Invitrogen) and purified over magnetic Nickel beads (Millipore). GST-His WT phosphorylated Syk form (P-Syk) was produced in baculovirus infected Sf9 cells and purified (Active Motif). Purified proteins were then concentrated using columns with a 30 kDa-cut-off (Millipore) before loading by SDS-PAGE for western blot analysis and onto gold nanostructures for SERS analysis.2, 10 mM NaVO3, 1 mM DTT, supplemented or not with 100 mM ATP (Sigma). After 1 hour incubation at room temperature, reactions were stopped by cooling down on ice. Unphosphorylated and phosphorylated Syk forms were separated by SDS-PAGE, transferred into nitrocellulose membrane and blots incubated with monoclonal mouse anti-phosphotyrosine antibody , mouse anti-Syk antibody  or rabbit anti-phospho-specific tyrosine Syk antibodies  followed by a goat anti-mouse or -rabbit secondary antibodies coupled to Horse Radish Peroxidase enzyme (BioRad) before revelation with the Enhanced Chemiluminescence kit (Pierce).UnP-Syk and P-Syk were incubated in a buffer containing 50 mM Tris-HCl, 25 mM MgClcf. above).P-Syk was incubated in the presence or not of 10 units of Calf Intestinal Phosphatase (CIP) in a buffer containing 50 mM Tris-HCl and processed as described in the kinase assay  was built as SERS substrates by electron-beam lithography following a classical lift-off process on a glass substrate with 3 nm of chromium adhesion layer. Ten microliters of protein solution were deposited on the gold nanocylinder substrate and incubated for 18 hours at 4 °C in a humidified chamber. The substrate was set nanostructured side down on a glass substrate with a cavity filled with PBS 1X. SERS spectra were recorded with a commercial Raman microspectrometer . The excitation laser (λ = 660 nm) was focused through a microscope 60× objective  and Raman scattering was collected through the same objective. The laser power was 1 mW at the sample position. For one SERS spectrum, signal was accumulated twice for 60 sec.How to cite this article: Cottat, M. et al. Phosphorylation impact on Spleen Tyrosine Kinase conformation by Surface Enhanced Raman Spectroscopy. Sci. Rep.7, 39766; doi: 10.1038/srep39766 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.