Pyruvate (PYR) dehydrogenase complex (PDC) is an enzymatic system that plays a crucial role in cellular metabolism as it controls the entry of carbon into the Krebs cycle. From a structural point of view, PDC is formed by three different subunits  capable of catalyzing the three reaction steps necessary for the full conversion of pyruvate to acetyl-CoA. Recent investigations pointed out the crucial role of this enzyme in the replication and survival of specific cancer cell lines, renewing the interest of the scientific community. Here, we report the results of our molecular dynamics studies on the mechanism by which posttranslational modifications, in particular the phosphorylation of three serine residues , influence the enzymatic function of the protein. Our results support the hypothesis that the phosphorylation of Ser-264-α and Ser-271-α leads to (1) a perturbation of the catalytic site structure and dynamics and, especially in the case of Ser-264-α, to (2) a reduction in the affinity of E1 for the substrate. Additionally, an analysis of the channels connecting the external environment with the catalytic site indicates that the inhibitory effect should not be due to the occlusion of the access/egress pathways to/from the active site. In fact, recent experimental investigations pointed out that an increase in PDC activity can sustain the progression of some cancer types (for example prostate cancer and glioblastoma)8 as well as the survival of cancer stem cells9.Given its central role in cellular metabolism10. While the first two subunits are involved in the synthesis of acetyl-coenzyme A (Ac-CoA), the third subunit (E3) is a FAD/NAD+ dependent system performing redox recycling , E2 (dihydrolipoyl trans-acetylase) and E3 (dihydrolipoyl dehydrogenase)1 have shown that the E1 PDC subunit is formed by two different chains (α and β) and is characterized by the presence of a magnesium ion coordinated by three amino acid residues  and a thiamine diphosphate (ThDP) molecule acting as a cofactor. ThDP is directly involved in the enzymatic reaction where after activation by Glu59 and the consequent formation of a dipolar molecule bearing a formally negatively charged atom  the PDC global stoichiometry, (2) the influence of posttranslational modifications on the function of PDC and (3) a full characterization of the enzymatic reaction at an atomistic level are still unanswered.Given its importance in cellular metabolism, the PDC complex has been at the center of numerous structural and functional studies1 and that the level of phosphorylation is controlled by four pyruvate dehydrogenase kinases (PDKs1-4) and two pyruvate dehydrogenase phosphatases (PDP1-2).In regards to question (2), it has been extensively reported that the activity of PDC is regulated by the phosphorylation of three serine residues6.In particular, experimental data clearly indicate that the phosphorylation of specific serine residues  results in different degrees of enzyme inhibition, with the strongest effect observed with Ser-264-α-PIn vitro studies demonstrated12 that PDK1 is the only kinase that can phosphorylate all the three sites, even if with different preferences and kinetics, while PDK2, PDK3 and PDK4 only act on Ser-264-α and Ser-271-α. In contrast, a similar action on all the three sites was observed for the two phosphatases13.14 and that the phosphorylation of Ser-264-α drastically reduces the enzymatic activity, while the phosphorylation of Ser-271-α or Ser-203-α have less clear inhibitory effects16.Furthermore, other investigations have shown that these posttranslational modifications are independent and not sequential18 indicated that the levels of PDC phosphorylation are significant for several widely diffused diseases such as cancer18 diabetes and neuro-metabolic disorders20. Understanding, at an atomistic level of detail, how posttranslational modifications alter enzymatic activity could open the way to the development of innovative therapeutic tools based on PDC inhibition/activation.Finally, experimental investigations22. Kato et al.21 suggested that a perturbation of protein dynamics, in particular of two phosphorylation loops , as origin of the inhibition while Seifert et al.22 hypothesized that Ser-264-α-P could hinder the substrates access to the active site22.Two different hypotheses to explain the PDC inhibition induced by phosphorylation have been proposed in the scientific literature34 as they provide a detailed atomistic picture of the biomolecular dynamics. In particular, they have been recently used to obtain a low resolution model of the entire PCD structure35 and to investigate other aspects of this enzymatic complex37.Over the years, molecular simulations have become a valuable tool to answer these types of questionsThus, we carried out an MD simulation study in which the PDC-E1 system in its unphosphorylated and phosphorylated forms  was simulated for 1 μsec (MD). Finally, the trajectories were analyzed to investigate the effects of the posttranslational modifications on the protein structure, dynamics and on its ability to bind the substrate.The molecular dynamics trajectories for the different systems were compared by considering different quantities such as RMSD, RMSF, Lindemann coefficient and pyruvate binding energy, as well as, the number, position, length and diameter of the tunnels leading to the active site.A detailed analysis of our simulations Figs  clearly 38, which gives an estimation of the solid-liquid behavior of a protein. The values of the coefficients . In fact, while Ser-203-α-P stiffens both loops, Ser-264-α-P and Ser-271-α-P significantly increase the flexibility of loop B but not that of loop A. Concerning this point, it should be noted that Ser-264-α and Ser-271-α are inside loop A, while Ser-203-α is placed in loop B.To better understand the origins of these differences, we visually inspected the trajectories of all the simulations.This analysis showed that during the Ser-203-α-P simulation, this residue gets surrounded by three arginine residues  forming a cationic-hole Fig. . Interes40 have pointed out the role of specific amino acids residues in the catalytic activity of E1-PCD. We, therefore, analyzed our simulations focusing on the consequences of phosphorylation on the catalytic site structure and dynamics.Several experimental and theoretical investigationsThe RMSD values Fig. , computeet al.21 that also pointed out an increased disorder in the two phosphorylation loops (loop A and B).In summary, the analysis of our simulations shows that phosphorylation of the three investigated serine residues have marked structural and dynamics effects on the PDC-E1-α subunit. In particular, both Ser-264-α-P and Ser-271-α-P increase the flexibility of loop B, the loop that directly coordinates the magnesium ion interacting with the ThDP cofactor. These findings are in line with the conclusions of Kato At the same time, the stiffening of the protein structure observed in the presence of Ser-203-α-P, suggests that this posttranslational modification should not inhibit PDC or, alternatively, that the inhibition could have a different explanation.41 and conformational entropy42. We, therefore, used specific computational tools to analyze our simulations with respect to these properties.Phosphorylation of single residues can influence protein properties such as hydrophobicity, lipophilicityThe entropic contribution  to the total free energy (−ΤΔS) was estimated to be −3.4 ± 0.1, −5.5 ± 0.1 and 26.2 ± 0.1 kcal/mol for the systems bearing Ser-264-α-P, Ser-271-α-P and Ser-203-α-P, respectively. These values confirm a stabilizing effect of Ser-203-α-P and a slight destabilizing action of Ser-264-α-P and Ser-271-α-P.41, we also investigated the effects of phosphorylation on the protein hydrophobicity/lipophilicity by computing the molecular hydrophobicity potential (MHP) on multiple snapshots extracted from our MD simulations.Following the suggestion of Polyansky and ZagrovicThese calculations resulted in ΔMHP values of −994, −835 and 258 log P units for the systems with Ser-264-α-P, Ser-271-α-P and Ser-203-α-P, respectively. These results indicate a decrease in the hydrophobicity of the protein in the first two cases and an opposite effect in the last one.14, suggest that changes in the enzyme affinity for the substrate and/or the cofactors could be one of the consequences of the phosphorylation. Because of technical problems in the purification of phosphorylated proteins, experimental investigations have been carried out using glutamate and aspartate mutations of the serine amino acids to mimic the effects of phosphorylation. However, while these mutations effectively reproduce the negative net charge of the phosphate group, they less efficiently mimic other properties, in particular the amino acid size. Therefore, to better clarify this point, we estimated the affinity between PDC-E1 and PYR or ThDP in all the investigated systems by MM-GBSA  positive, i.e. repulsive, contribution from Ser-264-α-P and a reduction  in the contribution to the binding energy given by His63-α and His263-α. In fact, in the WT protein, these two residues are close to each other on the interior of the active site and are able to form stable H-bond interactions with PYR. In the system with Ser-264-α-P, the phosphate group interacts with His263-α, inducing its reorientation to the exterior of the active site and resulting in the breaking of the H-bonds with the substrate.In the case of Ser-203-α-P, the increase in the predicted affinity, with respect to the unphosphorylated protein, is due to a larger contribution of Phe61-α and an additional interaction with Gln128-β.The calculations carried out to estimate the protein affinity for ThDP did not show any negative effects of the phosphorylation of the three serine residues. In contrast, the computed affinities for the proteins bearing Ser-264-α-P and Ser-203-α-P were higher than for the unphosphorylated ones.In summary, MM-GBSA calculations indicated that Ser-264-α-P has a repulsive effect on PYR reducing its affinity for the catalytic site, while a similar effect was not observed for Ser-271-α-P and Ser-203-α-P. Moreover, the results indicated that serine phosphorylations do not affect the affinity for ThDP.et al.22, studying the structure of the a pseudophosphorylated variant in which Ser-264-α was mutated to glutamate, concluded that the Ser-264-α-P hinders the substrate access to the active site. However, their conclusions were drawn by structural investigation on a system bearing a phosphomimetic mutation and using an experimental technique, X-ray crystallography, that provides only limited information about the dynamics of the system. Furthermore, the study22 focused on only one of the three possible phosphorylations.Seifert 43, a software specifically designed to identify tunnels in protein and previously used in similar investigations45.Therefore, aiming to determine if this hypothesis could be confirmed by simulations of the different systems bearing an explicit phosphate group, we analyzed the 1 μs MD simulations outputs with Caver3.022 is topologically located near the protein region occupied by Ser-264-α. Moreover, a second channel  start from different positions but in their terminal portion, converge on the same pathway passing near the regions of the protein occupied by Ser-264-α and Ser-271-α. It is also worth noting that in these cases, the channel diameters are larger than those detected for the main channels of the WT system.Finally, in the case of Ser-203-α-P, the two best scoring channels detected by Caver were essentially identical to those observed in the WT, only with a reverse ranking.22, should represent the main pathway connecting the protein surface with the active site. In all the phosphorylated systems, this channel was demonstrated to be as viable as in the WT system. We can therefore conclude that the origin of the inhibitory effect of phosphorylations21 is not due to the closure of the access route to the active site.Taken together, the results of our analysis strongly suggest that the channel passing near Ser-264-α and Ser-271-α, corresponding to what is already described in the literatureIn this work, we focused on the effects of specific serine phosphorylations on the enzymatic activity of the PDC E1 subunit.+ ion.Our calculations highlighted an increase in the protein flexibility induced by the phosphorylation of Ser-264-α and Ser-271-α at both the global (entire protein) and local  levels, while the phosphorylation of Ser-203-α was associated with an increase in protein rigidity. In particular, in the Ser-264-α-P and Ser-271-α-P systems, we observed a significant increase in the flexibility of the phosphorylation loop B, which is directly involved in the binding of both the ThDP cofactor and of the ThDP coordinated Mg2Binding energy calculations helped to shed light on the consequences of the increased flexibility induced by the presence Ser-264-α-P and Ser-271-α-P. In fact, while the affinity for the ThDP cofactor is unaltered, Ser-264-α-P reduces the affinity of PYR for the catalytic site. A similar effect was not observed for Ser-271-α-P, while the affinity in the Ser-203-α-P system was significantly increased by the posttranslational modification.Finally, Caver calculations showed that the presence of the phosphate groups in the three considered serine residues does not induce a closure of the access/release pathway to/from the catalytic site.Taken together, our findings suggest that the strong inhibitory effect observed for Ser-264-α-P is due to both a perturbation of the active site structure and dynamics and to a repulsive interaction between the modified residue and PYR that leads to a reduced protein-ligand affinity.16.In the cases of Ser-271-α and Ser-203-α, the picture emerging from our studies is less clear. In fact, the phosphorylation of Ser-271-α, seems to show only a small increase in protein flexibility that could however be the cause of its experimentally observed milder inhibitory effectRegarding Ser-203-α-P, the effects of phosphorylation are completely different with respect to Ser-264-α-P and Ser-271-α-P. In this case, considering the debate about the strength of its influence on the PDC-E1 catalytic activity, we can hypothesize that this modification does not lead to enzyme inhibition or the inhibition is obtained by a different mechanism.In this work, because of the lack of an experimentally solved atomistic structure of E1 in complex with the other subunits, as well as, the too large computational resources required to perform long enough atomistic simulations of the entire PDC, we chose to focus our investigation on a ‘minimal model’ able to recapitulate the most important features of the complex. Consequently, once more complete structural information and more potent computational resources become available, additional investigations should be carried out to gain a deeper understanding the effects of phosphorylation in the context of the entire system.Finally, from a pharmacological point of view, the results of this work suggest that an allosteric modulation of the PDC-E1 is possible, and it could be a viable strategy to design new molecular tools aimed to modulate the enzymatic activity.21.In particular, small organic molecules or peptides could be designed to mimic the effects of Ser-264-α-P. Alternatively, in the case of PDHA1 deficiency, the inhibitory effects of the two posttranslational modifications could be selectively blocked, hindering the interaction of PDK(s) and the PDC-E1 subunit near Ser-264-α for which the most potent inhibition was reported46. The coordinates of PYR, not present in the 1NI4 crystal structure, were obtained by structural alignment with the structure of the E1-PDC protein from Geobacillus stearothermophilus (PDB ID: 3DV0)11. Furthermore, selenomethionine residues were mutated to methionine.Molecular dynamics simulations were carried out starting from the structure of the E1-PDC human protein (PDB ID: 1NI4)47 with a minimum distance of 10 Å from the protein surface. The charge neutrality of the systems was ensured by adding a proper number of counterions (Na+ ions).All molecular complexes were fully solvated in a TIP3P water box48 for the protein, phosaa1050 for the phosphorylated residues, GAFF51 for pyruvate and cofactors; the parameters for Na+ and Mg2+ were taken from references52 and53, respectively). Atomic charges for ThDP and PYR were computed with the AM1-bcc approach implemented in the ANTECHAMBER module of Amber16. Finally, MD simulations reported in this study were carried out with the GPU accelerated PMEMD code available in Amber1654.The systems were described by Amber Force Field  distance restraint.To avoid distortions of the complexes, the systems were first minimized for 10.000 steps or until an energy gradient of 0.2 kcal mol57, cpptraj58 and ALMOST59. The Lindemann coefficient38 was computed considering backbone atoms by the PCA suite program (http://mmb.pcb.ub.es/software/pcasuite/pcasuite.html). The Lindemann coefficient was first proposed to determine the solid or liquid character of an infinite system61 and subsequently applied to proteins by Karplus and coworkers38.MD trajectories were analyzed using specific tools implemented in the programs VMD43 on 1250 equally spaced snapshots (1 every 800 ps) extracted from the 1 μs MD simulations. All the water molecules and the pyruvate were removed from the structures. Furthermore, the origin of the tunnels was set to be the geometrical center of the following four atoms: (1) the nucleophile carbon of ThDP . Finally, considering the size of PYR, the search of tunnels was performed using a probe with a radius of 1.5 Å.Protein tunnel analysis was done with the Caver 3.0 software43.In Caver analysis, each tunnel is described as an ensemble of spheres whose radius can fit the tunnel diameter. All identified tunnels were clustered by hierarchical average link using the pairwise distances between the centers of the spheres. The predicted tunnels were ranked according to the Carver score, designed to give priority to wide paths with a reasonable length62.The affinity between the proteins and the substrate (PYR) was estimated using the MM-GBSA approach63. Per-residue decomposition (PRD) was used to estimate the contribution of single protein residues to the binding energy. All the calculations were run using the MMPBSA.py62 module available in AmberTools16.MM-GBSA calculations were run on 2500 frames extracted (one every 400 ps) from the entire trajectories according to the following procedure: (1) water molecules and counterions were removed, (2) the same parameters used during MD simulations were assigned to both protein and ligand, and (3) the polar contribution to solvation energy was computed by the Onufriev, Bashford and Case model setting the dielectric constant to 1 for the solute and 80 for the solvent65 is a well-established method to estimate entropy from MD simulations68. The influence of the protein phosphorylations on conformational entropy were assessed using the implementation of the method in the recently released PDB2ENTROPY program67. To address this aim, 2500 snapshots were extracted from the MD simulations and processed using the following command line options ‘-mi -nt 16 -ne 40’.The maximum information spanning tree (MIST) approach of Tidor and coworkersThe entropic contribution to the free energy caused by phosphorylation was defined as:69. The difference between the sum of the MHP values over the protein solvent-accessible surface calculated for the phosphorylated and unphosphorylated protein (ΔMHP) was considered as measure of the effect of phosphorylation.How phosphorylation influences the hydrophobicity/hydrophilicity of PDC-E1 was assessed by computing the molecular hydrophobicity potential (MHP) using the PLATINUM webserverSupplementary Information


The paper presents the processed results of Plate Load Tests and of Multichannel Analysis of Surface Waves for dispersive and semi-rocky soils at the sites with different soil conditions located in the Perm Region, Russian Federation. Unit weight and deformation modulus were calculated from the obtained data. The value of the data lies in their applicability for the prompt preliminary assessment of the site geotechnical situation. Soil under the foundation slab:2.−Medium strength loose fractured saturated argillite-like clay with pockets of low and medium strength sand rock;−Fine-grained loose fractured saturated sand rock of low and medium strength;Site No. 2. Highway. Site beside a pillar of bridge crossing:3.−Tough and medium-hard clay;Site No. 3. Site of the former factory that is free of constructions:4.−Gray-brown areneceous fluid clayey sand with veins and pockets of 3–5 cm fine gray saturated sand and very soft brown clayey sand;−Dark-gray heavy silty very soft sandy clay with up to 15% inclusions of well-decomposed black organic matter;Site No. 4. Base of the foundation plate for a residential building:5.−Brown fine-grained sand.Site No. 5. A test site of the chair “Construction operations and geotechnic” of PNRPU that is free of constructions:PLT tests and MASW surveys were performed at five sites with different soil conditions:Physical properties of the soils determined in the laboratory are presented in E5000 obtained for a plate of 5000 cm22 plate was transformed to the module E5000 using E600 is deformation modulus for the 600 cm2 plate; m is conversion factor depending on the void ratio e according to Plate Load Tests were performed in accordance with the standard procedure set out in the State Standard m in Eq. A5000 is the 5000 cm2 plate; Ai is the i cm2 plate area; n is reduction argument according to Annex B of n = 0.15–0.3, for sandy soil n = 0.25–0.5.According to Ref. Multichannel Analysis of Surface waves (MASW) is a non-expensive rapid non-invasive field method of wave analysis of the low velocity zone in the upper part of soil profile. The procedure of field survey and further data processing used by the authors is described in the papers The obtained values of the S-wave velocities in the tested soil layers were used to calculate initial shear moduli from the expression 3; z is layer base depth, m.It is worth noting that expression (6) proposed in paper


SE translocation (SET), an inhibitor of protein phosphatase 2A (PP2A), plays important roles in mitosis and possesses oncogenic activity in several types of cancer. However, little is known regarding its regulation. Here we reveal a novel phosphorylation site of SET isoform 1, and we have determined its biological significance in tumorigenesis. We found that the mitotic kinase cyclin-dependent kinase 1 (CDK1) phosphorylates SET isoform 1 in vitro and in vivo at serine 7 during antitubulin drug-induced mitotic arrest and normal mitosis. SET deletion resulted in massive multipolar spindles, chromosome misalignment and missegregation, and centrosome amplification during mitosis. Moreover, mitotic phosphorylation of SET isoform 1 is required for cell migration, invasion, and anchorage-independent growth in vitro and tumorigenesis in xenograft animal models. We further documented that SET phosphorylation affects Akt activity. Collectively, our findings suggest that SET isoform 1 promotes oncogenesis in a mitotic phosphorylation-dependent manner. PP2A plays a critical role in neoplastic transformation by negatively regulating many oncogenic signaling pathways, and it serves as a therapeutic target5. Accumulated evidence demonstrated that SET functions as an oncogene in several neoplasms and promotes tumorigenesis7. Overexpression of SET has been shown in breast cancer8, ovarian cancer9, leukemia11, lymphoma12, hepatocellular carcinoma13, prostate cancer14, colon cancer15, non-small-cell lung carcinoma16, and pancreatic cancer17. Furthermore, upregulation of SET correlates with poor clinical outcomes in hepatocellular carcinoma, ovarian cancer, and colorectal cancer18. In line with these clinical observations, SET silencing or pharmacological inhibition of SET significantly impaired tumor growth in various human malignancies6. Interestingly, ectopic expression of SET also promoted resistance to chemotherapeutics in colon cancer, nonsmall cell lung carcinoma, and hematologic malignancies19. Promisingly, several antagonists  that target the SET–PP2A interface have been shown to inhibit tumor growth and overcome therapeutic resistance in various preclinical models20. In addition to its oncogenic roles, SET is also involved in various other molecular processes, including histone modification, chromatin remodeling, DNA repair, gene transcription, and androgen synthesis9. Mechanistically, SET has been shown to interact with several PP2A-regulated oncogenic pathways, including the Akt, mitogen-activated protein kinase, and BCR-ABL pathways10. However, SET may regulate cellular physiology through PP2A-independent pathways.SET, also known as inhibitor 2 of protein phosphatase 2A (I2PP2A), directly binds to PP2A and inhibits its phosphatase activity22. Therefore, mitosis has been a long-standing anticancer drug target26. Interestingly, SET has been linked to the mitotic machinery. For example, SET associates with cyclin B and inhibits cyclin B-CDK1 activity28. Knockdown (KD) of SET delayed mitotic progression and inhibited G2/M transition29. Moreover, primarily located in the nucleus, SET has been demonstrated to protect histones from acetylation, modulation of chromosome condensation, and cohesion30. Overexpression of SET resulted in precocious separation of chromatids in mouse oocytes31. SET is centromere localized31 and forms a complex with linker histones and shugoshins during mitosis29. Several studies demonstrated that SET mediates timely resolution of sister chromatids during mitosis and functions as a mitotic chaperone32. These studies suggest that SET might exert its oncogenic function through dysregulation of mitosis.Although extensive studies have demonstrated the important roles for SET-PP2A signaling in tumorigenesis, the underlying mechanisms are less clear. Mitotic aberrations cause aneuploidy or genomic instability, which is a hallmark of human malignancy34. There are four protein isoforms of SET and the only difference is in the first exon among them35. Isoforms 1 and 2 are the most thoroughly characterized ones17. SET is a phosphoprotein, and two sites (S9 and S24 in isoform 2) have been identified as phosphorylated by protein kinase C in vivo33. Furthermore, casein kinase 2-mediated phosphorylation at S9 causes cytoplasmic retention of SET and induces hyperphosphorylation of tau in Alzheimer disease37. However, the regulation of SET in mitosis and its possible role in cancer have remained unknown. Given the critical roles of SET in mitosis and oncogenesis, we sought to elucidate the regulatory mechanisms of SET in mitosis. We found that the mitotic kinase cyclin-dependent kinase 1 (CDK1) phosphorylates SET at S7 (isoform 1) during mitosis. Moreover, we showed that mitotic phosphorylation of SET is required for precise mitosis and oncogenic activity.The human SET gene is located on chromosome 9q34 and contains 11 exons and 10 introns. It was originally identified as part of a fusion gene with nucleoporin Nup214 (CAN) in a patient with acute undifferentiated leukemia in 199238 with a neomycin-coding sequence.The full-length human SET cDNA clone  was purchased from GeneCopoeia . A point mutation (Serine 7 to Alanine) was generated by the QuikChange Site-Directed PCR Mutagenesis Kit  and verified by sequencing. To make the lentivirus-mediated SET expression construct, the above cDNA was cloned into the pSIN4-Flag-IRES-neo vector. The pSIN4-Flag-IRES-neo vector was made by replacing the puromycin-coding sequence of the pSIN4-Flag-IRES-puro vector39. Attractene  was used for transient overexpression transfections following the manufacturer’s instructions. Nocodazole (100 ng/ml for 16–20 h) and Taxol (100 nM for 16–20 h) were used to arrest cells at late G2 and prometaphase (G2/M), unless otherwise indicated. VX680 , BI2536 (Plk1 inhibitor), and MK2206 (Akt inhibitor) were purchased from Selleck Chemicals . U0126 (MEK1/2 inhibitor), SB203580 (p38 inhibitor), LY294002 (PI-3K inhibitor), rapamycin (mTOR inhibitor), and SP600125 (JNK1/2 inhibitor) were from LC Laboratories . RO3306 (CDK1 inhibitor) and Purvalanol A (CDK1/2/5 inhibitor) were from ENZO Life Sciences . MK5108 (Aurora-A inhibitor) and SB216763 (GSK3β inhibitor) were from Merck  and Sigma-Aldrich , respectively.HEK293T, HeLa, and RKO cell lines were purchased from American Type Culture Collection . The cell lines were authenticated at ATCC and were used at low (<20) passages. All other cell lines were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. HPNE  was kindly provided by Dr Michel Ouellette , who originally established the cell line and deposited it at ATCC40. Gene KD was achieved by an shRNA-mediated method. The MISSION shRNA plasmids targeting human SET were purchased from Sigma-Aldrich (TRCN0000063717). To make the shRNA-resistant (Res) SET cDNA, the target sequence (5′-CCACCGAAATCAAATGGAAATCT-3′) was changed into 5′-CaACtGAgATCAAATGGAAATCT-3′ by PCR mutagenesis. The mutated SET cDNA was then cloned into the pSIN4-Flag-IRES-Neo vector to generate a Flag-tagged shRNA-resistant SET construct.Stable overexpression and re-expression of SET (wild-type and S7A mutant) in SET-KD cells were achieved by lentivirus-mediated infection and selection32P-ATP . Purified CDK1/cyclin B1 complex from SignalChem  was also used for in vitro kinase assays using phospho-specific antibodies. The samples were resolved by SDS-PAGE, transferred onto PVDF , and visualized by autoradiography or detected by phospho-specific antibodies.The glutathione S-transferase (GST)-tagged proteins were bacterially expressed and purified on GSTrap FF affinity columns  following the manufacturer’s instructions. About 0.5 µg of GST-SET proteins were incubated with 10 U recombinant CDK1/cyclin B1 complex  in the presence of 5 µCi γ-The anti-SET monoclonal antibody from Santa Cruz Biotechnology  was used throughout the study. Rabbit polyclonal phospho-specific antibody against human SET S7 (isoform 1) was generated and purified by AbMart . The peptide for generating the phospho antibody is: APKRQ-pS-PLPPQ. The corresponding non-phospho-peptide was also synthesized for antibody purification. Anti-Flag antibodies were from Sigma-Aldrich. Anti-β-actin, anti-ERK1/2, anti-phospho-S380 RSK, anti-MAD1, anti-MAD2, anti-TTK, anti-CDC27, anti-CDC20, and anti-cyclin B1 antibodies were from Santa Cruz Biotechnology. Anti-Aurora-A, anti-Bub1, anti-BubR1, anti-Cyclin C, and anti-GST antibodies were from Bethyl Laboratories . The following antibodies were obtained from Cell Signaling Technology : Phospho-T288/T232/T198 Aurora-A/B/C, phospho-S10 H3, phospho-T202/Y204 ERK1/2, phospho-S127 YAP, phospho-S397 YAP, Yes-associated protein (YAP), ribosomal S6 kinase 1 (RSK1), phospho-S807/S811 Rb, Rb, phospho-S536 NF-kB p65, NF-kB p65, phospho-S176/S180 IKKα/β, IKKα/β, phospho-T308 Akt, phospho-S473 Akt, Akt, phospho-Y705 STAT3, signal transducers and activators of transcription 3 (STAT3), phospho-S727 STAT1, phospho-Y1034/Y1035 JAK1, Janus kinase 1 (JAK1), phospho-S33/S37/T41 β-catenin, β-catenin, CDK1, CDK3, CDK4, CDK5, Cyclin A2, Cyclin D1, Cyclin E1, p21, and CDC25C. Anti-β-tubulin (Sigma-Aldrich) and anti-γ-tubulin  antibodies were used for immunofluorescence staining.TM SDS-acrylamide gels were used as we described previously41. Western blotting, immunoprecipitation, and lambda phosphatase treatment assays were done as described40.Phos-tag42. IHC staining  in tumor tissues was performed according to protocols described44. Anticleaved caspase 3 (1:100) and p-T308 Akt (1:100) antibodies were from Cell Signaling Technology. Anti-Ki-67 antibody was from Thermo Scientific  and was used at 1:100 dilutions.Fluorescence staining and confocal microscopy were done, as previously described45. In vitro analysis of invasion and migration (1.0 × 105 cells) was assessed using the BioCoat invasion system  and Transwell system , respectively, according to the manufacturer’s instructions. The invasive and migratory cells were stained with 0.1% crystal violet and counted manually.Cell proliferation and colony formation assays in soft agar (anchorage-independent growth) were performed as described6 cells each line) were subcutaneously injected into both flanks of 6-week-old male athymic nude mice . Cells were suspended in phosphate-buffered saline (PBS) and mixed with Matrigel in 1:1 ratio (volume). Five animals were used per group. Tumor sizes were measured twice a week using an electronic caliper 10 days post injection. Tumor volume (V) was calculated by the formula: V = 0.5 × length × width2,45. Mice were euthanized by CO2 inhalation at the end of the experiment and the tumors were excised for subsequent analysis. The animals were housed in pathogen-free facilities. All animal experiments were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee.For in vivo xenograft studies, RKO cells  and examined the response of SET on a Phos-tag gel. SET proteins were shown as a doublet (isoform 1 and isoform 2) on an SDS-PAGE gel Fig. . InteresIn order to determine which upstream kinase(s) could be responsible for SET phosphorylation, we treated cells with various kinase inhibitors together with MG132 (stabilizes cyclin B1 and prevent cells from exiting mitosis). Interestingly, the most significant inhibition of phosphorylation of SET was observed in cells treated with RO3306 (a CDK1 inhibitor) and Purvalanol A (inhibits CDK1/2/5) Fig. , suggest32P incorporation into SET  identified serine 7 (followed by a proline) as a possible phosphorylation site in SET during mitosis47. Of interest, mutating S7 to alanine largely eliminated the phosphorylation (32P incorporation) of SET , microtubule/spindle formation (immunofluorescence staining with β-tubulin), and centrosome number (γ-tubulin staining) during mitosis in control cells Fig. . In contNext, we further explored the biological significance of mitotic phosphorylation of SET in cancer cell growth. First, wound scratch assays were performed in control, SET-KD, and SET-KD cells expressing SET or SET-S7A. As expected, KD of SET (both isoforms 1 and 2) significantly decreased the rate of wound closure for HeLa cells Fig. . SimilarSecond, we used Transwell and Matrigel systems to examine the migratory and invasive properties of these cells. Consistent with the data from Fig. Consistent with the results in migration and invasion, KD of SET significantly decreased cell proliferation and importantly, expression of wild-type SET isoform 1 completely rescued the cell proliferation defects Fig. . HoweverHaving demonstrated the role of mitotic phosphorylation of SET in cell culture models, we next evaluated the influence of SET and its mitotic phosphorylation on tumor growth in animals. Various RKO cell lines Fig.  were sub6. We found that SET (isoforms 1 and 2) expression is upregulated in colon cancer cells compared with immortalized colon epithelial cells  were significantly correlated with poor survival in various malignancies  is upregulated in cancer cell lines and clinical tumor sampleslls Fig. . We furties Fig. .50. We surveyed the expression of a panel of cell cycle regulators to determine which one(s) is affected by SET and its mitotic phosphorylation in our system. While most of them were not altered upon SET KD, we found p-S642 WEE1 levels were increased in SET-KD cells , extracellular-signal-regulated kinase/RSK (ERK/RSK), as well as β-catenin and YAP activities were not affected in SET-KD cells when compared to control cells  was greatly decreased upon SET inhibition in RKO cells  and phospho-proteomic studies revealed potential mitotic phosphorylation sites at the N-terminus of I1PP2A (T15/S17)54. The role and regulation of I1PP2A in mitosis have not been defined. We are also interested in investigating how I1PP2A is regulated and whether it also plays a role in mitosis similar to SET/I2PP2A. As an inhibitor of PP2A, SET promotes tumorigenesis mainly through forming an inhibitory protein complex with PP2A6. The activity of PP2A is tightly regulated by the C-terminal region of SET that is responsible for the binding to PP2A catalytic subunit PP2A-C2. Since mitotic phosphorylation occurs on the N-terminus of SET isoform 1, we do not expect that loss of oncogenic activity of SET-S7A is due to alterations in binding with PP2A. Indeed, our coimmunoprecipitation experiments revealed no significant difference between SET and SET-S7A in association with PP2A-C (data not shown). Therefore, it is currently unclear how phosphorylation of SET promotes its oncogenic activity and Akt signaling (Fig. 55 several of which are involved in mitotic cell cycle progression and tumorigenesis. Does SET phosphorylation affect these binding partners? The future elucidation of these mechanisms will provide novel insights underlying SET-driven tumorigenesis.SET is also called I2PP2A and interacts with PP2A and another closely related protein I1PP2A/PP32ing Fig. . In addi


Different epidemiological studies have found that backyard chickens are a reservoir for poultry diseases. Most backyard chicken flocks have a poor level of biosecurity, which increases the risk of spread of diseases. In recent years, the number of backyard chickens has been on the rise in Iran. However, the health status of backyard flocks is still poorly documented. Thus, this study aimed at examining the seroprevalence of antibodies against infectious bronchitis virus (IBV) and molecular surveillance and genotyping of IBV among backyard chickens (without vaccination history) in Mazandaran province, North of Iran, 2014.A total of 460 blood samples of unvaccinated backyard chickens in the mentioned area were tested for antibodies against IBV using commercial ELISA. Also, cecal tonsils were collected from 75 chickens in the same area. Real time RT-PCR (for detection) and RT-PCR and sequencing spike gene were performed.The seropositivity rate was 54.5%. In addition, we detected 793/B, Variant 2, and QX in the backyard flocks and performed phylogenetic studies on them. The phylogenetic study revealed that the detected genotypes had high homology with IBV strains that were infected broilers, pullets, and layers in Iran.There is a need for continuous monitoring of IBV among avian species to complete the epidemiological map and work on the pathogenesis of Iranian IBV strains in Iranian backyard chickens. Gammacoronavirus of the Coronaviridae family and is the etiologic agent of infectious bronchitis (IB), which is a major, highly complex infectious disease of poultry caused by multiple serotypes of IBV (Avian infectious bronchitis virus (IBV) belongs to the genus s of IBV . IBV poss of IBV . The S gs of IBV , 4. IB as of IBV , 6. IB hs of IBV . Therefos of IBV . The firs of IBV . IB is sMoreover, no preventive and controlling strategy has been undertaken against IB in backyard chickens in Iran –12.Mazandaran province is one of the 31 provinces of Iran and is located along the Caspian Sea in Iran’s Region 3, just east of Gilan province, west of Golestan province, and north of Tehran and Semnan provinces (36.5656°N 53.0588°E). Mazandaran is a major producer of poultry, and poultry farmers in this region provide an important economic addition to the traditional dominance of agriculture. For serology, we collected 460 sera from backyard chickens , and antibody titers obtained from samples were also evaluated. Laboratory results of IBV ELISA were entered and managed using Microsoft Excel (Windows 2010). Descriptive statistics for the ELISA antibody titers were performed using the same program.Viral genomes were extracted from cecal tonsil samples  according to manufacturer’s instructions. The extracted viral RNA was contained in a 1.5 mL sterile RNase and DNase- free microtube and stored at −70°C until further use.Viral RNA was reverse transcribed using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific); cDNAs were stored at −20°C until use.Real time RTPCR was conducted in a QIAGEN Rotor-Gene Q (Corbett Rotor-Gene 6000) . Forward primer (IBV5′GU391 5′-GCTTTTGAGCCTAGCGTT-3′), reverse primer (IBV5′GL533 5′GCCATGTTGTCACTGTCTATTG-3′), and Taqman® dual-labeled probe (IBV5′G probe 5′-FAMCACCACCAGAACCTGTCACCTC-BHQ1-3′) were used to amplify and detect a 143-bp fragment at the 5′UTR of the IBV genome .A pair of degenerated primers (SX1: CACCTAGAGGTTTGYTWGCATG and SX2: TCCACCTCTATAAACACCYTTAC) and 3 others (SX3: TAATACTGGYAATTTTTCAGATGG& SX4: AATACAGATTGCTTACAACCACC) were finally selected for use in the initial PCR and in the subsequent nested PCR . First rThe PCR products of all positive samples were purified using PCR Purification Kit  and were sent for sequencing . All sequences from a given sample were combined and used to construct alignments. ClustalX (Version 1.83) multiple sequence alignment analysis was performed to calculate the percentage of sequence similarity between our positive samples and sequences of referral strains and other IBV strains. Phylogenetic trees of sequences were constructed by the neighbor-joining method and the Kimura 2-parameter model by MEGA package, Version 5.1 . A bootsThe overall seroprevalence and ELISA titer of IBV antibodies revealed in this study were 54.5% and 4949, respectively. No clinical signs were observed in chickens at the time of blood sampling. The highest mean titer (7597) was observed in Sari, while the lowest 1373) was observed in Amol . Accordi373 was oIBV: 793/B (67.7%), (2) Variant 2 (25.8%), and (3) QX (6.5%). Variant 2, QX, and 793/B detected isolates in this study had 100%, 98%, and 99.35% to 100% homology, respectively. Homology percent with some selected references and Iranian IBV isolates based on the spike gene have been summarized in Backyard poultry makes approximately 80% of natural flocks in Africa and Asia. Keeping backyard poultry next to other birds in the north of Iran helps the economic level of the families significantly. Based on the Ministry of Agriculture statistics, the number of backyard poultry is 4.51 million. Mazandaran province is the center of Iranian poultry industry, especially in lines, preparents and breeders. There were many reports of high pathogenic avian influenza in backyard and rural bird populations, especially in the north of Iran, so veterinary council inspected the backyard flocks more. Several studies were performed on the serological prevalence of viral agents, such as Newcastle Disease and avian influenza, in backyard poultry . The fir


Biological effects of FPR2 stimulation include intracellular Ca2+ mobilization, cellular proliferation and migration, and wound healing. A systematic analysis of the phosphoproteome in FPR2-stimulated cells has not been yet reported. Herein, we describe a large-scale phosphoproteomic study in WKYMVm-stimulated CaLu-6 cells. By using high resolution MS/MS we identified 290 differentially phosphorylated proteins and 53 unique phosphopeptides mapping on 40 proteins. Phosphorylations on five selected phospho-proteins were further validated by western blotting, confirming their dependence on FPR2 stimulation. Interconnection between some of the signalling readout identified was also evaluated. Furthermore, we show that FPR2 stimulation with two anti-inflammatory agonists induces the phosphorylation of selected differentially phosphorylated proteins, suggesting their role in the resolution of inflammation. These data provide a promising resource for further studies on new signaling networks triggered by FPR2 and on novel molecular drug targets for human diseases.Formyl peptide receptors (FPRs) belong to the family of seven transmembrane Gi-protein coupled receptors (GPCR). FPR2 is considered the most promiscuous member of this family since it recognizes a wide variety of ligands. It plays a crucial role in several physio-pathological processes and different studies highlighted the correlation between its expression and the higher propensity to invasion and metastasis of some cancers. FPR2 stimulation by its synthetic agonist WKYMVm triggers multiple phosphorylations of intracellular signaling molecules, such as ERKs, PKC, PKB, p38MAPK, PI3K, PLC, and of non-signaling proteins, such as p47 FPR2 is highly expressed in cells of the immune system but it is also functionally expressed on cellular membranes of epithelial, neuronal and endothelial cells1. FPR2 was also detected onto nuclear membranes of anaplastic lung cancer CaLu-6 and human gastric adenocarcinoma AGS cells, where triggers intranuclear signaling cascades crucially involved in gene expression6. Furthermore, different studies highlighted the correlation between FPRs expression or function and inflammatory conditions, including inflammatory disorders, cancer, infections and other diseases9.Formyl peptides receptors  are members of the GPCR family coupled to inhibitory G (Gi) proteins sensitive to pertussis toxin (PTX)10. For instance, the interaction of W peptide with its receptor elicits the activation of phospholipase A2, phospholipase C, and phospholipase D 12, the stimulation of phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (PKB or Akt) pathway13, and the activation of protein kinase C (PKC), p38MAPK, extracellular response kinases 1 and 2 (ERK1/2) and c-Src15. Some of FPR2-dependent phosphorylated molecules are non-signaling proteins, such as the cytosolic subunits p47phox and p67phox of NADPH oxidase complex16 whose phosphorylation requires FPR2-dependent activation of ERKs, PKCα, and PKCδ17. Activated NADPH oxidase complex generates reactive oxygen species (ROS), which play a crucial role in tyrosine kinase receptors (TKRs) transactivation in several cell lines18. In CaLu-6 cell line, FPR2 stimulation by W peptide induces Epidermal Growth Factor Receptor (EGFR), p47phox and c-Src phosphorylation. Phosphotyrosine residues of EGFR provide docking sites for recruitment, phosphorylation, and triggering of Signal Transducer and Activator of Transcription 3 (STAT3) transcriptional factor16. In human prostate epithelial cell line PNT1A, FPR2 activation induces the phosphorylation of Y1313, Y1349 and Y1356 residues of Hepatocyte Growth Factor Receptor (c-Met) and triggers STAT3, PLC-γ1/PKCα and PI3K/Akt pathways19. Furthermore, N-fMLP stimulation of FPR1 induces the phosphorylation of cytosolic Y951, Y996, and Y1175 residues of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), which constitute the anchoring sites for signaling molecules. These, in turn, activate PI3K/Akt and PLC-γ1/PKC intracellular pathways20.FPR2 stimulation induces multiple phosphorylations of several signalling proteins, which modulate critical intracellular functions such as cell growth, proliferation, intracellular communication, migration, differentiation, apoptosis, and survival21 and about 30% of the total content of proteins in eukaryotic cells is phosphorylated at any given time22. However, in eukaryotic cells the overall level of tyrosine and threonine phosphorylated residues in proteins is very low compared to the level of phosphoserine23.Phosphorylation represents the most frequent reversible covalent modification of proteins observed in eukaryotic cells. About 2–5% of the human genome codifies for kinases and phosphatases24.Characterization of the phosphorylation status of cells plays a key role in the study of intracellular signaling cascades, in the understanding of the molecular mechanisms responsible of human disorders and, in turn, for the appropriate pharmacological approachAlthough intracellular signaling pathways triggered by W peptide in FPR2-expressing cells are widely investigated, a complete analysis of the phosphoproteome is not currently available. Herein, we apply a phosphoproteomics approach to investigate phosphorylated proteins in WKYMVm-stimulated CaLu-6 cells. We identified 290 phosphoproteins and 53 unique phosphopeptides mapping on 40 proteins and we prove that selective phosphorylation on Ser82 of Heat Shock Protein-27 (HSP-27), Ser139 of Mini-Chromosome Maintenance protein 2 (MCM2), Ser339 of Oxidative-Stress-Responsive kinase 1 (OSR1), Ser608 of Retinoblastoma (Rb), and Ser170 of Myristolated Alanine-Rich C-Kinase Substrate (MARCKS), specifically depends on FPR2 stimulation by WKYMVm. The interconnection between some of the signalling readouts identified was demonstrated by using specific signalling inhibitors. Furthermore, we show that FPR2 stimulation with Annexin A1 (ANXA1) or Lipoxin A4 (LXA4), two anti-inflammatory FPR2 agonists, induces the phosphorylation of the selected differentially phosphorylated proteins.Our overall data demonstrate that network-based global phospho-proteomic analysis can contribute to the identification of new intracellular pathways elicited by FPR2 which, in turn, could lead to the identification of new targeting strategies for FPR2-dependent human diseases.CaLu-6 cell line was obtained from American Type Culture Collection . Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% foetal bovine serum (FBS) (Invitrogen). Once reached 80% confluence, cells were serum starved for 24 hours, and stimulated for 5 minutes with WKYMVm  at the final concentration of 10 μM, or with Lipoxin A4  at the final concentration of 1 μM or with Annexin A1 (Abcam) at the final concentration of 10 nM. In other experiments, serum-deprived cells were preincubated for 16 hours with PTX (Sigma) at a final concentration of 100 ng/mL, or with 10 μM WRWWWW (WRW4)  for 15 minutes, or with 50 μM PD098059 (Sigma) for 90 minutes, or with 5 Rottlerin μM (Sigma) for 1 hour before the stimulation with W peptide. SDS-PAGE reagents were purchased from Bio-Rad . Anti-phosphoHSP-27(S82), anti-phosphoMCM2(S139) and anti-phosphoRb(S608) were from Cell Signalling Technology . Anti-phosphoOSR1(S339) was purchased from Signalway Antibody  and anti-phosphoMARCKS(S170) were from GeneTex . Anti-tubulin, anti-cyclin D, anti-cyclin E, anti-rabbit and anti-mouse antibodies were purchased from Santa Cruz Biotechnology . Protein A–horseradish peroxidase was from Thermo Scientific .The enrichment of phosphorylated proteins was performed by a phosphoprotein purification kit , accordingly to the manufacturer’s instructions. Briefly, whole lysates were obtained from 24-hours serum starved CaLu-6 cells stimulated or not with W peptide for 5 minutes. Cellular proteins were incubated with the appropriate amount of Phosphoprotein Lysis Buffer for 30 minutes at 4 °C and gently vortexed every 10 minutes. Lysates were centrifuged at 10000xg for 30 minutes at 4 °C and protein concentration was determined by using a Bio-Rad protein assay . Lysates (2.5 mg) were loaded onto Phosphoprotein Purification Column to allow the binding of phosphorylated proteins and the column was washed with the appropriate buffer as described in the manufacturer’s instructions. Finally, phosphoproteins were eluted by adding to the column 500 µL of Phosphoprotein Elution Buffer. This step was repeated four times and the concentration of the four enriched phosphoprotein fractions was determined. To concentrate samples, an ultrafiltration step was performed by centrifuging the eluted fractions, at 10000xg for 10 min.3CN), formic acid (FA) and water LC-MS grade were from Fisher Scientific Italia .Chemicals, tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin were from Sigma Chemical Co. . Acetonitrile (CH4HCO3 pH 8.2. Samples were then subjected to disulphide reduction with 10 mM DTT (1 h at 55 °C) and alkylation with 7.5 mM iodacetamide (20 min at room temperature in the dark). Enzymatic hydrolyses were performed on reduced and alkylated samples by adding TPCK-treated trypsin with an enzyme/substrate (E/S) ratio of 1:100 (w/w) for 3 h and 1:50 for 16 h at 37 °C.Aliquots of enriched phosphoprotein samples (~50 µg) from 24-hours serum starved CaLu-6 cells, treated or not with W peptide for 5 minutes, were precipitated by adding pre-chilled acetone (six volumes) for 16 h at −20 °C. Following centrifugation for 10 min at 8,000 × g at 4 °C, protein pellets were resuspended in 100 µL of 50 mM NH25 on a Q-Exactive Orbitrap mass spectrometer equipped with an EASY-Spray nanoelectrospray ion source  and coupled to a ThermoScientific Dionex UltiMate 3000RSLC nano system (Thermo Fisher Scientific).Mass spectrometry analyses of tryptic digests (2 µg) were performed as previously described25. The HCD MS/MS spectra were searched against the Homo sapiens database available in UniprotKB  assuming trypsin (Full) as digestion enzyme with two allowed number of missed cleavage sites and a minimum peptide length of six residues. The mass tolerances were set to 10 ppm and 0.02 Da for precursor and fragment ions, respectively. Carbamidomethylation (+57.021464 Da) of cysteine was set as static modification. Phosphorylation of serine, threonine, and tyrosine (+79.966 Da) and acetylation of lysine (+42.011 Da) were set as dynamic modifications. False discovery rates (FDRs) for peptide spectral matches (PSMs) were calculated and filtered using the Target Decoy PSM Validator Node in Proteome Discoverer with the following settings: maximum Delta Cn of 0.05, a strict target FDR of 0.01, a relaxed target FDR of 0.05 and validation based on q-value. Localization and best site probability for variable post-translational modifications within peptides were performed with the ptmRS tool, integrated in Proteome Discoverer26. The Protein FDR Validator Node in Proteome Discoverer was used to classify protein identifications based on q-value. Proteins with a q-value of 0.01 were classified as high confidence identifications and proteins with a q-value of 0.01–0.05 were classified as medium confidence identifications. The resulting protein table was then filtered based on the presence of phosphorylated peptides. Phosphopeptide changes following WKYMVm treatment were only considered when modifications were identified in at least two out of three replicate injections in treated sample and not identified in replicate injections of untreated sample.The acquired raw files were analysed with Proteome Discoverer 2.1 software (Thermo Fisher Scientific) using the SEQUEST HT search engine as describedhttp://funrich.org/index.html). Molecular functional enrichment of identified proteins based on gene ontology categories was performed by using the Network analyst software 27.Interaction network analysis of proteins identified by LC-MS/MS were performed by the FunRich open access software  and lysed by incubation with RIPA buffer  for 45 min at 4 °C28. Bio-Rad protein assay was used to determine proteins concentration . Fifty micrograms of whole lysates were resolved on 10% SDS-PAGE and proteins were transferred by blotting onto PVDF membranes. Membranes were incubated with phosphospecific primary antibodies and probed with appropriate horseradish peroxidase-conjugated secondary antibodies. Proteins were detected by autoradiography and bands densitometry was estimated using a Discover Pharmacia scanner equipped with a sun spark classic densitometric workstation29. The same filters were reprobed with an anti-tubulin antibody to normalize the amount of loaded proteins.Whole lysates were obtained as previously describedAll the data are representative of three or more independent experiments and are expressed as means ± Standard Error Mean (SEM). Statistical analyses were evaluated by one-way analysis of variance (ANOVA) performed with GraphPad Prism 7 . A p value of less than 0.05 was considered statistically significant.31.The global phospho-signaling response triggered by WKYMVm in CaLu-6 cells in comparison to untreated cells was investigated by nano-liquid chromatography coupled with high-resolution tandem mass spectrometry. The schematic representation of the experimental design is reported in Fig. In order to unravel the biological relevance of phosphorylation events triggered by W peptide stimulation, we focused our attention on a small number of peptides (53 mapping on 40 proteins) that were phosphorylated only in WKYMVm-stimulated cells Table .Table 1PThe molecular function enrichment analysis of these proteins, performed through the Network analysis software, revealed that kinase binding, protein kinase binding, enzyme binding, proteins involved in transcription processes were among the most significantly enriched categories Fig. . We used19. As shown, a large subset of proteins was mapped on a cluster converging on β-arrestin-1 (ARRB1), which plays a crucial role in GPCR signaling. This cluster also includes and displays connections with several phospho-proteins uniquely identified in WKYMVm-treated cells  for further validation by western blot analysis, on the basis of their involvement in the control of cell proliferation and in signal transduction cascades triggered by FPR2 activation that we identified by MS/MS analysis.FPR2 stimulation by WKYMVm exacerbates cell proliferation and the malignant phenotype of CaLu-6 cells, and its inhibition by PTX or WRWWWW significantly prevents these biological responses32 with anti-inflammatory and immunomodulatory actions, including the release of the anti-inflammatory cytokine IL-1033. The protein is phosphorylated on three residues  by MAPKAP kinase 2/3/5 via the activation of p38MAPK pathway34. Other kinases, such as PKB, PKC, PKD, and PKG also independently phosphorylate HSP-2735. In fact, in SH-SY5Y human neuroblastoma cells, carbachol-dependent HSP-27 phosphorylation on Ser82 and Ser78 residues are prevented by PKC and p38MAPK inhibition, and phorbol esters-dependent HSP-27(Ser82) phosphorylation is reversed by PKC or PKD inhibitors36. Rac is also involved in HSP-27 phosphorylation, as demonstrated by the observation that the inhibition of Rac-guanine nucleotide exchange factor interaction reduces the collagen-induced HSP-27 phosphorylation on Ser15, Ser78 and Ser82 residues37.HSP-27 (P04792) belongs to heat shock protein family and is expressed at basal level in many cell types and tissues. It is a chaperone38; (ii) prevent Fas-induced apoptosis39; (iii) promote actin filament polymerization40 and (iv) modulate estrogen signaling41. In addition, in a phospho-proteome analysis of tumors undergoing ischemia, HSP-27 results hyper-phosphorylated at the Ser82 residue suggesting the relevance of this site in ischemia42. Furthermore, in atherosclerotic lesions of hypercholesterolaemic rabbits a significant increase of HSP-27(Ser82) phosphorylation and pospho-p38MAPK was observed43.HSP-27 phosphorylation promotes the dissociation of HSP-27 multimers into monomers and dimers that can interact with other proteins. HSP-27 phosphorylation is highly dynamics and regulates several cellular processes in a context-specific manner. Indeed, phospho-HSP-27 is known to: (i) down-regulate the expression of intracellular glutathione and, in turn, the resistance against oxidative stress44, our MS/MS analysis revealed FPR2-dependent phosphorylation of HSP-27 on Ser82 residue  are unable to phosphorylate HSP-27, suggesting that the activation of p38MAPK/MAPKAPK/HSP-27 pathway is a response triggered by pro-resolving, and not by pro-inflammatory ligands, upon FPR2 activation47 both in vivo and in vitro, on Ser27, Ser41, and Ser139 residues48, whereas Cdks phosphorylate MCM2 but not on Ser139 residues47. In human cells, Cdc7 is activated by its regulatory subunits Dbf4 and Drf150 and Cdc7/Dbf4 complex is directly involved in the initiation of DNA replication by targeting MCM248. Casein kinase 2 (CK2) and salt-inducible kinase 1 (SIK1) also phosphorylate MCM2 on Ser139 in vitro, but there is not evidence that CK2 is responsible for this phosphorylation in vivo52. EGFR- and ERKs-dependent activation of CK2 phosphorylates phosphoglycerate kinase 1 (PGK1), resulting in PGK1/Cdc7 interaction. Cdc7-bound PGK1 converts the ADP in ATP thus removing ADP inhibition on Cdc7 and promoting MCM2 phosphorylation on Ser13953. Previously, we demonstrated that WKYMVm stimulation of CaLu-6 cells induces EGFR transactivation and ERKs phosphorylation16, and accordingly to EGFR-dependent activation of CK2/PGK1/Cdc/7 cascade, our results potentially explain the observed FPR2-dependent phosphorylation of MCM2 on the Ser139 residue (Table MCM2 (P49736) belongs to the protein family of “ATPases associated with diverse cellular activities” (AAA+). It is a component of the MCM2-7 complex, which is the putative replicative helicase necessary for DNA replication initiation and elongation in eukaryotic cells. MCM2 can be phosphorylated by different kinases and these phosphorylation events are involved in DNA replication, cell cycle progression and checkpoints regulation. Cdc7/Dbf4 phosphorylates MCM2 and other MCMsue Table .54. Such analysis demonstrated a phosphoMCM2-regulated functional network, suggesting that the deregulation of MCM2 phosphorylation is involved in lung cancer cell proliferation, cell cycle, and migration and that potential target cancer-specific phospho-proteins can be identified by the analysis of molecular interactions of phosphorylated MCM254. The role of phosphorylated MCM2 in cancer is also corroborated by a phospho-proteomic analysis of liver cell lines with different proliferation potential. The results show that MCM2 is hyper-phosphorylated in liver cancer in particular on a novel Thr27 phosphosite, but also on Ser139 residue55. In these cells, MCM2 promotes cell proliferation via the regulation of high mobility group protein HMG-I/HMG-Y (HMGA1) phosphorylation55.The regulatory role of MCM2 in lung cancer has been extensively investigated in a integrate analysis of phospho-proteome and proteome of overexpressed and silenced MCM2 lung cancer cells57. OSR1 is activated by “with no lysine” (WNK) protein kinase family, which phosphorylates a Thr185 residue in the T-loop kinase domain, and Ser325 and Ser339 residues in the S-domain of OSR1. The role of OSR1(Ser325) and OSR1(Ser339) phosphorylations is unclear58. Some evidence suggests that since the S-domain of OSR1 contains a WEW motif (aminoacids 336–338), essential for binding to the scaffolding protein MO25, the phosphorylation on serine residues adjacent to WEW motif (Ser339) could enhance binding to MO2558. PI3K-Akt signaling activates the WNK-OSR1 cascade59 by Akt-dependent phosphorylation of WNK on Thr60, which is prevented by PI3K inhibitors60. WNK3 is a direct target of Akt61 and is subjected to phosphorylation triggered by EGF-dependent PI3K-Akt pathway59. Akt activity is regulated not only by PI3K phosphorylation in the activation loop (Thr308) but also by mammalian target of rapamycin complex 2 (mTORC2) phosphorylation in C-terminal hydrophobic motif (Ser473)57. mTORC2 also phosphorylates OSR1 on Ser339 residue, increasing OSR1 activity62, and inhibition of mTORC2 does not prevent WNK activity, indicating that mTORC2 regulates OSR1 independently by WNK57. Accordingly, OSR1(Ser339) phosphorylation has been identified by MS in phospho-proteomic studies to define the signaling networks downstream of mTORC1 and mTORC264. A phospho-proteomics analysis of hydrogen peroxide-induced fibroblasts derived from normal individuals and A-T patients suggests that also ROS play a role in OSR1 phosphorylation65. OSR1 phosphorylation on Ser339 residue observed in WKYMVm-stimulated CaLu-6 cells  antibody shows an increase of OSR1(Ser339) phosphorylation in FPR2-stimulated cells, which is prevented by WRW4 or PTX  is a serine/threonine-protein kinase involved in the regulation of the solute carrier 12 family of cation-chloride cotransporters and thereby in the modulation of cellular ion homeostasis, blood pressure, hearing, and kidney functionsPTX Fig. .67. The Rb protein consists of several domains , as well as a large loop within the pocket domain (RbPL). Cdk-dependent Rb phosphorylation occurs at 13 consensus sites which include all these regions67. Unphosphorylated Rb on Ser608 residue has growth suppressive activity and specifically contact the COOH-terminus of MCM4. Phosphorylation of Ser608 in RbPL is sufficient for partial inhibition of E2F binding66, whereas Ser608/Thr373 phosphorylation produces unique conformational changes of the Rb structure that result in the allosteric and complete inhibition of the E2F binding67. Ser608/Ser612 phosphorylation induces an intramolecular association between RbPL and the pocket domain. This association obstructs the E2FTD binding site in the pocket, preventing the simultaneous binding of phosphorylated RbPL and E2FTD67.The retinoblastoma tumor suppressor protein, Rb (P06400), negatively regulates entry into S phase. The binding of Rb to E2F prevents the release of E2F, thereby inhibiting the entry of the cell cycle. Therefore, Rb acts as a cell cycle inhibitor and is activated by multisite Cdk-dependent phosphorylation in response to positive growth signals68. The role of Rb(Ser608) phosphorylation in the control of the progression of the cell cycle is further confirmed in a subset of tumor-derived cell lines, where conversion of unphosphorylated Rb in a phosphorylated form does not arise69. In these defective cells, a lack of dephosphorylation of Rb(Ser608) is observed. Several members of PP1 phosphatase family are implicated in the dephosphorylation, but there is no evidence for defective or deregulated expression of catalytic PP1 cores69. We decided to validate the identified Rb(Ser608) phosphorylation on the basis of its role in the control of cell cycle and, in western blot analysis, we observed that an anti-pRb(Ser608) antibody confirmed this phosphorylation in FPR2-stimulated cells, which was prevented by preincubation with WRW4 or PTX  is phosphorylated by CDK4/6 complexes in acute lymphoblastic leukemia cellsPTX Fig. .70. MARCKS consists of three conserved regions, which include the phosphorylation site domain (PSD), also called effector domain (ED), which is necessary for its association to PIP2. PKC-dependent phosphorylation on Ser159, Ser163, and Ser170 residues within PSD increases MARCKS detachment from the membrane and removes PIP2 association71.MARCKS (P29966) is a membrane-binding protein and is the main substrate of PKC in many cell types. It binds calmodulin (CaM), actin, synapsin and is involved in cell migration, regulation of cell cycle, actin-filament reorganization, adhesion, cytoskeletal control and neurosecretion70. MARCKS is preferentially phosphorylated by PKCα but also by PKCδ and PKCε, which phosphorylate Ser159, Ser163, and Ser170 residues in the PSD of human MARCKS73. These phosphorylations mask positive charges on the protein which, in turn, segregates from the plasma membrane to the cytoplasm. Rho kinase (ROCK) also phosphorylates PSD of MARCKS74 and, in mouse bone granulocytes, W peptide stimulation triggers ROCK activation75. We previously observed the ability of FPR2 to activate PKCα and PKCδ in several cell types19 and, accordingly, our phospho-proteomic analysis identified the FPR2-dependent MARCKS(Ser170) phosphorylation  phosphorylation  and enhances the levels of anti-inflammatory cytokines (IL-10 and TGFβ)79. FPR2 can to transmit both pro- and anti-inflammatory signals and this duality was originally established by the nature of the ligands. In general, short and flexible structures, like that of formylated peptides, activate a pro-inflammatory cell response, while ANXA1 and LXA4 are well-known anti-inflammatory FPR2 ligands. The shift between pro- and anti-inflammatory cell responses is caused by conformational modifications of FPR2 upon ligand binding, but are not merely due to ligand-receptor interaction. FPR2 can dimerize and this conformational change could account for its biological functions. Anti-inflammatory agonists enhance the formation of FPR2 homodimers and the release of inflammation-resolving cytokines, while inflammatory ligands, such as SAA, do not cause receptor homodimerization. Heterodimers of FPR2 with other members of the FPR family can transduce proapoptotic signals43. We stimulated CaLu-6 cells with two anti-inflammatory agonists and, in western blot experiments we observed that ANXA1 and LXA4 induce HSP-27(Ser82), OSR1(Ser339), Rb(Ser608), and MARCKS(Ser170) phosphorylation, which was prevented by PTX and WRW4 (Figs. Several data support the development of WKYMVm as a novel and effective anti-inflammatory therapeutic agent. It shows pleiotropic, immunomodulatory, anti-apoptotic, and anti-inflammatory effects in several pathological conditionsW4 Figs.  and 7A–DW4 Figs.  and 7E, 80, but in these cells, the phosphorylation of HSP-27 on a Ser82 residue has not been demonstrated. Anti-inflammatory ligands increase the formation of FPR2 homodimers and/or FPR1/FPR2 heterodimers and, in turn, the release of anti-inflammatory cytokines43. Since CaLu-6 cells don’t express FPR116, we can exclude FPR1/FPR2, but not FPR2/FPR3 heterodimerization.ANXA1 stimulation of FPR2-expressing cells induces the phosphorylation of p38MAPK/MAPKAP/HSP-27 cascade81, triggers c-Met and EGFR transactivation in PNT1A and CaLu-6 cells19, respectively, and elicits NOX2-dependent superoxide generation in IMR90 human fibroblasts17, suggesting a specific role of FPR2 in non-phagocytic cells. CaLu-6 is a human lung anaplastic cancer cell line which expresses high levels of EGFR as well as a biological functional FPR2. We already demonstrated that stimulation with W peptide exacerbates the malignant phenotype of these cells through a molecular mechanism which involves EGFR tyrosine trans-phosphorylation, p47phox phosphorylation, NADPH-oxidase-dependent superoxide generation, and c-Src kinase and STAT3 activity16. FPR2 inhibition significantly prevents cell proliferation and the malignant phenotype, suggesting the potential role of FPR2 signalling and, in turn, of phosphorylated proteins as drug targets in lung cancer epithelial cells16.FPR2 was initially detected in phagocytic leukocytes, but the receptor was subsequently identified in other cell types and tissues, where it is involved in many physio-pathological processes and where it shows further functions than those normally exerted in polymorphonuclear cells. For instance, FPR2 promotes the malignancy of colon cancerThe investigation of the complex and dynamic regulation systems of protein phosphorylation represents an attractive field of proteomic research. The outline of the cellular phosphorylation status is crucial for the identification of intracellular signalling pathways and, in turn, for the understanding of the molecular mechanisms responsible for cancer and other human disorders. Here we report the first phospho-proteomic analysis of FPR2-stimulated CaLu-6 cells that allowed the overall identification of 290 phosphorylated proteins; among them, we found 53 unique phosphopeptides that mapped on 40 proteins with crucial roles in metabolic processes and biological regulation. In particular, we identified FPR2-induced phosphorylated proteins involved in the control of cell cycle progression, cell division and apoptosis, and thereby implicated in molecular mechanisms of cellular invasion and metastasis. Interestingly, most of these phospho-proteins are transcriptional factors or are regulators of replication, recombination, transcription, splicing, translation processes and DNA repair. The heterogeneity and complexity of phospho-protein patterns are indicative of the activity of multiple signal transduction pathways triggered by FPR2 stimulation. Several novel phosphosites have been identified in this study, which represent an important basis for further functional studies that may contribute to the identification of new signaling networks. However we cannot exclude that the phosphorylation pattern that we observed is a general phenomenon that occurs during FPR2 activation in all FPR2-expressing cells, rather than specific of lung epithelial carcinoma CaLu-6 cells.43. Pro-resolving ligands, such as ANXA1 and Ac2-26, increase the formation of FPR2 homodimers and/or FPR1/FPR2 heterodimers, which led to the release of inflammation-resolving cytokines, while pro-inflammatory agonists do not cause homodimerization43. The receptor oligomerization is not limited to the FPR family members but also includes the scavenger receptor MARCO and is connected to agonist-evoked modifications in ERKs phosphorylation and cAMP levels82. We can exclude FPR1/FPR2 heterodimerization in CaLu-6 cells since these cells don’t express FPR1, but we cannot rule out the FPR2/FPR3 heterodimerization. Our results strongly suggest a potential FPR2 homodimerization upon WKYMVm stimulation, since FPR2 oligomer contributes to the activation of the p38/MAPKAPK/HSP-27 pathway80 and pro-resolving ligands, such as ANXA1, which increases the formation of FPR2 homodimers, induces the phosphorylation of HSP-27 and of other validated proteins (Fig. FPR2 can transmit both pro- and anti-inflammatory signals and this ability is determined by the nature of the ligands and by the formation of higher-order structures. For instance, FPR2 can to homodimerize or to heterodimerize with FPR1 or FPR3The identification of the intracellular signalling pathways triggered by FPR2, as well as of downstream phosphorylated proteins, will provide a better understanding of the role of FPR2 in non-phagocytic cells in physiological and pathological conditions. Furthermore, these results could provide new insights into FPR2 signalling as possible drug targets in human diseases, including inflammatory disorders, infections, and cancer, that involve this receptor.Supplementary Table 1Supplementary Table 2