Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser1412, and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic stroke or neurodegeneration.Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser Nitric oxide synthases (NOSs) are the enzymes responsible for ·NO production, a biological signaling molecule involved in the control of cardiovascular, immune and nervous system physiology 916 located at -2 position within its PDZ-binding motif , which in turn controls Kidins220/ARMS transport and localization at the neuronal plasma membrane 1412, leading to increased ·NO production, hence establishing a novel role of PKD in the regulation of ·NO synthesis.Protein kinase D1 (PKD1) belongs to a family of phorbol ester/diacylglycerol-stimulated Ser/Thr kinases constituted by two additional members, PKD2 and PKD3 Animal procedures were approved by “Consejo Superior de Investigaciones Científicas” - CSIC Ethics Committee and performed in compliance with European Directive 2010/63/EU. Animals used were kept to a minimum, they were sacrificed by deep anesthesia, and all efforts were made to minimize suffering.G-nitroarginine methyl ester (L-NAME), N-Methyl-D-aspartate (NMDA), glycine, cytosine β-D-arabino furanoside (AraC), poly-L-lysine, L-laminin, Protein A/G-Sepharose, 2′,5′-ADP–Sepharose, adenosine 2′(3′)-monophosphate mixed isomers, DNA single stranded from salmon testes for hybridization, and 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside  and 4,5-Diaminofluorescein diacetate (DAF2-DA) were from Sigma Co. . Nerve growth factor was from Alexis Corp. . Ni-NTA resin was from Qiagen . L-Arginine and Gö6976 were purchased from Calbiochem . [γ32P]-ATP (370 MBq/ml) was from PerkinElmer, Inc. . Mouse monoclonal anti-Myc, anti-GST and rabbit polyclonal antibodies recognizing total PKD1/2 and phospho-Ser916 were from Cell Signaling Technology . Anti-β-tubulin I monoclonal antibody was purchased from Sigma and rabbit polyclonal anti-neuronal specific enolase (NSE) from ICN Biomedicals . We produced an antibody against nNOS immunizing rabbits with purified rat nNOS following standard procedures. Rabbit polyclonal anti-nNOS-phospho-Ser1412 was purchased from Upstate-Merck Millipore . Mouse monoclonal antibody recognizing total VASP and rabbit polyclonal antibody anti-VASP-phospho-Ser239 were from Santa Cruz Biotechnology . Rabbit polyclonal anti-GFP was obtained from Invitrogen-Life Technologies . Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were from General Electric . Oligonucleotide primers were from Invitrogen-Life Technologies . All other reagents were from standard suppliers or as indicated in the text.HEK293T, COS-7, and PC12 cells were obtained from American Type Culture Collection ATCC . Phorbol-12, 13-dibutyrate (PDBu), 8-Br-cGMP, L-NIn vitro kinase reactions after phosphorylating nNOS by a recombinant protein containing the active catalytic domain of PKD1 fused to GST (GST-PKD1-cat) were digested with trypsin and analyzed by HPLC followed by MALDI TOF/TOF and peptide fragmentation and de novo sequencing in the Proteomic Studies Unit  following standard procedures. MALDI-TOF MS analysis was performed in a 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer . The MALDI-TOF/TOF operated in positive reflector mode with an accelerating voltage of 20000 V. Selected peptides, were subjected to MS/MS sequencing analyzes using the 4800 Proteomics Analyzer . Suitable precursors from the MS spectra were selected for MS/MS analysis with CID on (atmospheric gas was used) 1 Kv ion reflector mode and precursor mass Windows ± 4 Da. The plate model and default calibration were optimized for the MS/MS spectra processing. De novo sequencing from fragmentation spectra of peptides was performed using De novo tool software (Applied Biosystems), tentative sequences were manually checked and validated.−/Trp−/His−SD plates in the presence of 12 mM 3-amino triazole (TDO plates) as well as in Leu−/Trp−/His+. Interacting proteins expressed within the same yeast resulted in colonies that could rescue growth in the absence of His. These colonies were subsequently screened in the X-Gal assay. Blue colonies corresponded to a positive interaction whereas white colonies corresponded to absence of interaction. The complete PKD1 active catalytic domain (Gly-557 to Leu-918) or shorter C-terminal sequences (Pro-591 to Leu-918) containing the PDZ-binding motif of wild-type PKD1 or the phospho-mimetic mutant PKD1-Ser916Glu , according to the manufacturer's specifications, and collected for processing 48 h later. Cell were transfected with empty vector pEFBOS-GFP or containing GFP fused to PKD1 wild-type (PKD1), kinase-inactive , constitutively active , PDZ-ligand mutants  and deletion mutants lacking the PH-domain (PKD1ΔPH) or the CRD domain (PKD1ΔCRD) that have been used previously 1412Ala (nNOSS1412A) were kindly provided by Dr. G. A. Rameau and Dr. E. B. Ziff 2. Cells were treated with nerve growth factor (75 ng/ml) for 2 days post-transfection. For transfection and immunofluorescence, HEK293T and PC12 cells were seeded at 50–60% confluence on poly-L-lysine (10 µg/ml)-coated glass coverslips. Cells were transfected as above and 48 h later cells were treated with PDBu (200 nM) for 15 min, fixed and processed for immunofluorescence.HEK293T or COS-7 cells were cultured in Dulbecco's modified Eagle's medium , supplemented with 10% (v/v) foetal calf serum, and 2 mM glutamine at 37°C in a humidified atmosphere containing 5% CO5 and incubated at 37°C in an atmosphere of 5% CO2. Neurons grown in vitro for 14 days (DIV14) were pretreated with Gö6976 (5 µM) for 1 h and left unstimulated or stimulated with the NMDAR agonist NMDA (50 µM) and its coagonist glycine (10 µM) for 5 min.Cultures of dissociated E19 rat cortical neurons were prepared from the cerebral cortex of 19-day-old Wistar rat embryos as described For immunofluorescence cells grown on coverslips were fixed for 10 min in 4% paraformaldehyde in phosphate-buffered saline at room temperature. After blocking  cells were incubated with the corresponding primary antibodies for 1 h at room temperature, and immunoreactivity was detected with the suitable fluorophore-conjugated secondary antibody before mounting in slides with ProLong . Images are single sections of z-series acquiring each channel in a sequential mode using an inverted Zeiss LSM710 confocal microscope with a 63X/1.40 Plan-Apochromatic objective. Pictures were processed with ZEN 2009 light Edition (Carl Zeiss MicroImaging) and Adobe CS3 Extended  software.S1412A were immunoprecipitated with anti-Myc antibody during 4 h at 4°C. Equal amounts of total lysates or equivalent volumes of immunocomplexes were analyzed by SDS-PAGE followed by transfer to nitrocellulose filters and immunoblot. Membranes were blocked in TBST  plus 5% low-fat milk powder and incubated with the different primary and secondary antibodies in blocking solution and immunoreactive bands were visualized by enhanced chemiluminescence .Preparation of lysates and immunoprecipitation assays were performed as described previously Using rat nNOS as a template, the N-terminal half of nNOS comprising the heme-oxygenase domain (residues 1 to 759) or the C-terminal half of nNOS comprising the reductase domain (residues 715 to 1429) were amplified and NdeI and XbaI sites were introduced at the 5′and 3′end respectively. The PCR bands were digested with NdeI/XbaI and ligated in the corresponding sites of 6His-pCWori 5′-AAA AAG CAG GCT CCG GAT CCA ACT CAC ACA AAG ATA-3′) and 3′ (5′-AGA AAG CTG GGT TTT TGA CAG ATT AGA GGG GAT GGA-3′). The PCR product was cloned in pDONR201 by a recombination reaction with BP clonase , to generate the construct pENTR-PKD1cat. After automated sequencing, PKD1cat was subcloned in pDEST15 using LR clonase. This vector for procaryotic expression generates PKD1cat fused to glutathione S-transferase  of approximate molecular weight of 65 kDa that was purified following standard methods and stored at −20°C. This protein is constitutively active since it lacks the regulatory autoinhibitory domain.The C-terminal region of PKD1  containing the full-length catalytic domain was amplified using as template pBS-PKD1 using oligonucleotides 5′ , and subjected to an in vitro kinase assay for 30 min at 30°C in the presence of 100 µM final concentration of [γ32P] ATP or non-radioactive ATP. Samples were analyzed by SDS-PAGE and Ponceau staining, autoradiography or immunoblot as indicated in the text.PKD or nNOS were immunoprecipitated from cultures of primary rat cortical neurons DIV14, and PKD phosphorylation activity was determined performing similar 916) becomes autophosphorylated in the active enzyme 916 in active PKD1 could convert this domain in a bona-fide PDZ-binding motif for the PDZ domain of nNOS. To check this idea, and given that nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, we first examined the possible association of both enzymes in mature neurons performing co-immunoprecipitation assays. Cultured primary rat cortical neurons grown in vitro for 14 days (DIV14) were lysated and PKD and nNOS were immunoprecipitated using specific antibodies (in vitro kinase assay (IVK) in the presence of [γ-32P]-ATP before being resolved in SDS-PAGE gels and transferred to a nitrocellulose membrane. This filter was first subjected to Western blot analysis to examine the presence of PKD or nNOS in the immunoprecipitates. As shown in Proteins known to bind to nNOS PDZ domain must present a hydrophobic amino acid such as Val, Leu or Ile at the final position together with an acidic residue at position -2 or - 3 tibodies . These iTo further support PKD-nNOS association, we analyzed if we could detect the association of PKD and nNOS by co-immumoprecipitation and Western blot using epitope-tagged versions of both proteins transfected into mammalian cells. Here, we also examined how PKD1 activation could affect their association. HEK293T cells were transfected with Myc-nNOS together with GFP-PKD1 wild-type (PKD1wt) and 48 h later were left untreated or treated with the phorbol ester PDBu in order to activate PKD . After i916 autophosphorylation within its PDZ-binding motif, we further examined the contribution of this phosphorylation to the association of PKD1 with nNOS. To this end, HEK293T cells were cotransfected with Myc-nNOS together with a phospho-mimetic mutant GFP-PKD1-Ser916Glu (PKD1S916E) or a non-phosphorylatable mutant GFP-PKD1-Ser916Ala (PKD1S916A). Co-immunoprecipitation analysis performed as above showed that mutation of this residue did not alter the association of PKD1 with nNOS under basal conditions and in the absence of PDBu  rendered a similar result in which PKD1 was able to co-immunoprecipitate with nNOS preferentially after kinase activation by PDBu treatment (Because PKD activation leads to Ser of PDBu . Importareatment . These r916IL) at its very C-terminal end, and used it as bait to screen its binding to the PDZ domain of nNOS , presenting the autophosphorylated PDZ-stretch . HEK293T cells transfected with these mutants together with nNOS for 48 h were untreated or treated with PDBu for 15 min to activate PKD1. Cellular lysates were immunoprecipitated with an anti-Myc antibody to detect PKD1 and nNOS co-immunoprecipitation. As shown in ΔPH mutant is constitutively active and autophosphorylates at the PDZ-ligand Ser916We continued examining the participation of other PKD1 domains that could be mediating PKD and nNOS association transfecting into mammalian cells GFP-PKD1 mutants where the PH or CRD domains had been deleted  in vitro kinase assays using radioactive [γ32P]-ATP and purified enzymes. As shown in Our initial d by PKD , IVK. Sein vitro kinase reaction, performed with non-radioactive ATP, was digested with trypsin and subsequently subjected to HPLC and peptide fragmentation by MALDI TOF/TOF IAFIEESKK and the phosphorylated residue was unambiguously assigned to the Ser residue present at the fifth position  as the serine phosphorylated by PKD1 . Furthermore, various amino acids present at nNOS PKD1 phosphorylation sequence are identical to those present in other known PKD1 substrates. In addition to the conserved Arg residue at position -3, nNOS phosphorylation sequence displays a Ser at -2 position . We performed in vitro kinase assays as above followed by immunoblot analysis. The nNOS-pSer1412 antibody only detected purified nNOS when it had been pre-incubated with active PKD1 catalytic domain in the presence of ATP  or the non-phosphorylatable mutant nNOS-Ser1412Ala (nNOSSA) together with a constitutively active mutant of PKD1 fused to GFP (PKD1ca). Levels of ectopically expressed GFP-PKD1ca or mutated or wild-type Myc-nNOS were similar in all cellular total lysates. In agreement with our mass spectrometry results, after immunoprecipitating nNOS with anti-Myc antibodies we detected that Ser1412 was phosphorylated in vivo only in cells that had been cotransfected with constitutively active PKD1 , which has been suggested to represent a biochemical marker of ·NO levels in intact cells 239, but also in other two residues 239 migrate in SDS-PAGE gels with an apparent molecular weight of 45 kDa and additional phosphorylations in either one or both of the other residues produces a shift up to 50 kDa. Therefore, depending on the activation of this signaling cascade, the antibody recognizing VASP-pSer239 will detect a double band in immunoblot analysis. Importantly, if the phosphorylation of the protein decreases significantly, total VASP will mainly be detected as a single band of 45 kDa. Changes in the intensity of VASP-pSer239 band or in the mobility of the protein correlate with the degree of phosphorylation and consequently of the activation/inactivation of this pathway by ·NO. To determine the influence of PKD1 activity on this parameter, we transfected HEK293T cells with Myc-nNOS and kinase inactive GFP-PKD1ki (PKD1ki)  for the last 24 h. As a positive control, we triggered VASP-pSer239 phosphorylation by incubating the cultures with the cGMP homologue 8-Br-cGMP (100 µM) for 30 min. The immunoblot image and its quantification analysis showed that VASP-pSer239 levels were almost undetectable in untreated and that the doublet signal only appeared after 8-Br-cGMP treatment  phosphorylated at Ser(PKD1ki) . Before reatment . Regardiol cells . Noteworh Gö6976 . In summh Gö6976 .916 at -2 position in active PKD1, therefore presenting a negatively charged phosphorylated residue at this site 916 within this motif. Instead, the PH domain of PKD1 is absolutely required for its association with nNOS, mediating a direct interaction of both enzymes. Several years ago we discovered that the PH domain of PKD1 is autoinhibitory since point mutations or complete deletion of this domain render a constitutively active kinase 916 within the PDZ-ligand, PKD1 mutant lacking the PH domain (PKD1ΔPH) is unable to associate with nNOS. Importantly, α-syntrophin, which interaction with nNOS through its PDZ domain was first identified in vivo, in addition to its PDZ domain Among the three enzymes involved in ·NO synthesis , the neuronal isoform nNOS is the only one bearing a PDZ domain. It is widely accepted that PDZ domains present selective interaction with specific PDZ-ligands. Particularly, the PDZ domain of nNOS was proposed long time ago to display a preference for PDZ-binding motifs bearing acidic residues at -2 or -3 position 744 and Ser748463 phosphorylation within PKD1 autoinhibitory PH domain and provokes a molecular switch that allows Src-mediated Tyr95 phosphorylation, the formation of a complex with PKCδ facilitating activation loop phosphorylation and correlated PKD1 activation The PH domain of PKD1 was first identified to mediate direct interactions preferentially with protein kinase C (PKC) novel isoforms, PKCη and PKCε Despite the list of PKD substrates is increasing, very little is known about the biological significance of their association to the kinase. For most PKD substrates, like Hsp27 1412in vitro and in vivo in living cells, stimulating its enzymatic activity and increasing ·NO production. In this context, it must be mentioned that this is an atypical site for PKD phosphorylation. This kinase usually recognizes a consensus motif presenting a hydrophobic residue such as Leu/Val/Ile at -5 position and Arg/Lys at -3 position referred to the phosphorylatable Ser residue 1412). Regardless of this fact, our data show that PKD1 is an activatory partner of this isoform by phosphorylating Ser1412. This residue is part of a motif positioned at the very C-terminal end of the reductase module and recent crystallographic data indicate that it adopts a helical conformation 1412 is directed toward the negatively charged flavin mononucleotide-binding domain residues Glu916 and Asp9181412 activates electron transfer from the reductase domain towards the oxygenase domain of nNOS thus augmenting ·NO synthesis and cGMP formation In addition, we demonstrate here that active PKD1 phosphorylates nNOS in the activatory Ser1412 by Akt/PKB 1179 and Ser1177 in bovine and human eNOS, respectively), located at the C-terminus (presumably an α-helix as well), was early recognized as a phosphorylation site. This serine residue is immersed within a consensus Akt/PKB-dependent phosphorylation consensus sequence (RXRXX(S/T)X, herein RLRSESI in nNOS and RIRTQSF in eNOS). We and other authors reported the phosphorylation of eNOS by Akt/PKB in vitro and in a cellular environment. Furthermore, the activity of at least six protein kinases .Similarly, it has been reported that nNOS is also phosphorylated on Ser1412 which is blocked after pharmacological inhibition of PKD. Other authors have reported previously that protein kinase Akt/PKB activation in response to NMDARs stimulation also results in nNOS phosphorylation at Ser1412 and increases its enzymatic activity in vivo, PKD inhibitors might be useful drugs in cases of nitric oxide associated neurotoxicity.Our data also show that activated PKD1 and substrate nNOS colocalize and are able to co-immunoprecipitate. This is not unprecedented, since the homologous eNOS can be co-immunoprecipitated with several protein kinases, such as Akt/PKB 1412 enhancing this way nNOS activity and ·NO production. Considering that the corresponding serine in eNOS is subjected to similar mechanisms of phosphorylation-dependent activation, PKD emerges as a common regulator of the enzymatic activity of constitutive NOS isoforms and ·NO synthesis. These novel findings add to the list of biological relevant roles of PKD a crucial one in the regulation of ·NO synthesis and the plethora of physiological and pathological processes where this mediator is involved.In conclusion, herein we reveal that PKD1 interacts with nNOS and phosphorylates SerFigure S1Association of PKD2 with nNOS.(TIF)Click here for additional data file.


Cch alone increased ASMC proliferation at 24 and 48 hrs. However, combination of Cch with other mitogens exhibited a dual effect, synergistic proliferation effect in the presence of EGF (5 ng/mL) and 5% FBS and inhibiting the proliferation induced by 10% FBS, EGF (10 ng/mL) and TNF-α (10 ng/mL). To determine the MAChR subtype involved in these biological responses, a titration curve of selective muscarinic antagonists were performed. The Cch stimulatory and inhibitory effects on ASCM proliferation was blocked by AF-DX-116 (M2AChR selective antagonist), in greater proportion than 4-DAMP (M3AChR selective antagonist), suggesting that the modulation of muscarinic agonist-induced proliferation is M2AChR mediated responses. Thus, M2AChR can activate multiple signal transduction systems and mediate both effects on ASMC proliferation depending on the plethora and variable airway microenvironments existing in asthma and COPD.Airways chronic inflammatory conditions in asthma and COPD are characterized by tissue remodeling, being smooth muscle hyperplasia, the most important feature. Non-neuronal and neuronal Acetylcholine acting on muscarinic receptors (MAChRs) has been postulated as determinant of tissue remodeling in asthma and COPD by promoting proliferation and phenotypic changes of airway smooth muscle cells (ASMC). The objective was to evaluate proliferative responses to muscarinic agonist as carbamylcholine (Cch) and to identify the MAchR subtype involved. ASMC were isolated from tracheal fragments of Sprague–Dawley rats by enzymatic digestion. Proliferation assays were performed by MTS-PMS method. Viability was confirmed by trypan blue exclusion method. Mitogens as, epidermal growth factor (EGF), Tumor necrosis factor-alpha (TNF-α) and fetal bovine serum (FBS) increased ASMC proliferation ( Chronic inflammatory conditions of the airways are usually associated with the development of structural changes of the airways; a phenomenon commonly described as airway remodeling. This process is seen in both asthma and Chronic Obstructive Pulmonary Disease (COPD), albeit the nature, localization and extent of the remodeling are variable -4. AirwaASMC are multifunctional cells that have high phenotypic plasticity. These cells can shift between different phenotypes depending on the stimulation conditions. Accordingly, mitogenic factors can induce the reversible transition to “synthetic-proliferative” phenotype, characterized by high capacity for cell proliferation and secretion . SeveralEGF binds to receptors with intrinsic tyrosine kinase activity . All EGFThe biological effects of TNF-α are mediated through two receptors of similar affinity  . In expe2AChR) and muscarinic receptor type 3 (M3AChR), whose activation promote synthetic functions, proliferation and phenotypic changes depending on stimulation conditions . After, the exposure time finished, 100 μL of medium plus 10 μL MTS [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-PMS [Phenazine Methosulfate]/well, and incubated for additional 90 min. The cell proliferation were determinated measuring optical density (OD) at λ = 492 nm, which is proportional measure to the number of viable cells in wells.ASMC proliferation was estimated by the nonradioactive method (MTS-PMS assay) based on the formation of tetrazolium salts  using, C0/G1 phase. After 12–24 hrs, cells were incubated at 37°C and 5% CO2 with solution containing each compound as experimental condition described. After 72 hrs, the cells were detached using trypsin/EDTA and centrifuged at 500xg for 15 min. The cells were vigorously resuspended in 1 mL of medium. The number of cells was estimated in a mixture of 10 μL of cell suspension plus 90 μL of 0.4% trypan blue [dilution factor (DF) 1:10] by 5 min, then 20 μL of this mixture was placed in the hemocytometer and observed under light microscopy. All cells in central quadrant and four quadrants of corners were counted. Cells number was estimated and the viability was determined (considering that only dead cells capture the dye) using the equations previously described [Proliferation and viability was confirmed by trypan blue 0.1% exclusion method . A fixedescribed .t-test. Differences were considered to be significant as p < 0.05. The parameters log IC50 ± SE were estimated using the GRAPH PAD® program.Values reported for all data are means ± SE. The statistical significance of differences between means was determined by an unpaired two-tailed Student’s 3cells/well in 96 well plates to evaluate its basal proliferation and response to mitogens using MTS-PMS method as shown in Figure p < 0.05). In addition, EGF and TNF-α, showed a dual effect increasing ASMC proliferation reaching maximum effect at 10 ng/mL and higher concentration the proliferation effect decreased (data not shown).Rat ASMC were incubated by 24, 48 and 72 hrs at 1 × 105cells/well and mitogens as FBS, EGF, TNF-α, which stimulated ASMC proliferation. However, there was not a clear cut correlation between cell viability with cell proliferation. In this sense, TNF-α (10 ng/mL) increased cell proliferation but decreased cell viability , indicating that TNF-α induces cell division and also promotes cell death (apoptosis). The mechanism of cell death induced by TNF-α could not be assessed with the methodology here applied.Viability and cell proliferation were also evaluated by trypan blue dye exclusion method as shown in Figure 2O2 treatment of airways myocytes successively stimulates the MAP kinase superfamily members, which are important in transduction of mitogenic signals to the nucleus [ASMC proliferation has been studied for in several conditions using various experimental animal and human models. Fetal Bovine Serum (FBS) is a potent mitogenic, that effect has been explained by the Reactive oxygen species (ROS) generation . Thus, e nucleus . This ha nucleus . In our Another mitogen studied was Tumor necrosis factor (TNF-α), which is a potent proinflammatory cytokine and its role as a potential mediator in asthma has been well described ,37. Morein vitro[EGF is a mitogen that been reported to stimulate ASMC growth in vitro and thisin vitro. In our 3cells/well in 96 well plates with increasing doses of Cch in the presence and absence of 10% FBS     and AF-DX-116-DS (for M2AChR) as shown in Figure p < 0.05) as shown in Figure 50 for 4-DAMP and AFDX-116, which were similar as 4-DAMP + FBS = −7.34 ± 0.21 AFDX-116 + FBS = −7.26 ± 0.51. These results may be explained by some inverse agonist actions described by these muscarinic drugs [To determine the MAchR subtype involved in these opposite effects displayed by Cch. ASMC at 1x10ic drugs . This ex50 for the 4-DAMP + FBS + Cch = −7.38 ± 0.42 and AFDX-116 + FBS + Cch = −8.99 ± 0.45. From these data, there is two order of magnitude of difference between these values supporting a pharmacological profile that AFDX 116 > 4-DAMP, that belongs to an M2AchR.Interestingly, in the case of the synergistic Cch and FBS proliferation effect, both muscarinic antagonists were able to inhibit such proliferation activity displaying different values of the log IC2AChR antagonist), which was more efficient than 4-DAMP (M3AChR antagonist) to reverse this novel Cch inhibition activity as shown in Figure -5 M) action (p < 0.05). Thus, the Log IC50 ± SE were 4-DAMP = −7.11 ± 0.71 and AFDX- = −9.40 ± 0.37 were estimated. The difference between these log IC50 values is more than 2 orders of magnitude, that support a pharmacological profile is AFDX 116 > > 4-DAMP clearly belongs to an M2AchR.Trying to understand this novel finding on the ability of Cch to exhibit anti-proliferative properties especially at high mitogen concentration (10% FBS). It was found, this anti-mitogenic Cch effect was reversed, in a dose-dependent manner, by preferential muscarinic antagonists as AF-DX-116  stimulatory effect by itself, 2) synergistic effect, at low concentrations of mitogens , and 3) inhibitory effect, at maximum concentration of mitogens  as above described.2+) that trigger several intracellular signal cascades that cross-talk involving protein-protein interactions and reversible posttranslational modifications (phospho/dephosphorylation processes) to activate or regulate the nuclear factors involve in the DNA duplication and cell division, which is a fast growing research field in the last 20 years.It is complex matter to explain these diverse biological effects that are initiate by the binding of a neurotransmitter (ACh) to muscarinic receptors (GPCR) at sarcolema of ASMC, with the involvement of some intracellular seconds messengers  [Muscarinic agonist synergistic effect on rat ASMC proliferation in the presence of low doses of EGF and 5% FBS was observed. The co-administration of muscarinic agonists with EGF in human ASMC induces a synergistic proliferative stimulus. This effect was associated with sustained activation of p70 S6 kinase ,44, an e (GSK-3) .3AChR mediated [2AChR) [3AChR) suggest that synergistic effect is mediated through the activation of M2AChR (log IC50 AF-DX-116 < IC50 4-DAMP). Our results suggest that constitutive activity of M2AChR could be necessary to maintain ASMC proliferation in 5% FBS response. Additionally, M2AChR is coupled to a pertussis toxin (PTX) sensitive Gi/o protein. Consequently, in several studies have been reported that mitogenic effect of Cch on human ASMC is antagonized by PTX [i/o, Gs, PDGF → Gi/o) or indirectly interaction (PDGF/EDG1 → Gi/o) [2AChR/Gi/o protein/p21Ras and MAPK signal cascade in response to Cch might be more efficient to potentiate the signal transduction of growth factors present in FBS. Nonetheless, synergistic effect of muscarinic agonists may involve a sustained activation of p70S6K in promotion of protein synthesis related with cell cycle transition [Similar synergistic effects of muscarinic agonists in human and bovine ASMC are antagonized by 4-DAMP and DAU 5884, in consequence appears to be Mmediated . However [2AChR)  and 4-DAd by PTX ,48. To ed by PTX  as repor → Gi/o) . Src and → Gi/o) . Therefoansition  rather tansition . Moreoveansition .S6K and ERKs. During the preparation of this manuscript, it was reported that muscarinic agonists (metacholine) exhibits synergistic effects on TGF-β1-induced proliferation, which were reduced by tiotropium and the M2AChR subtype antagonist gallamine, but not the M3AChR antagonist DAU5884. Moreover, pertussis toxin treatment also prevented the potentiation of TGF-β1-induced proliferation by methacholine, via an M2AChR coupled to Gi/o protein. These authors concluded that exposure to TGF-β1 induces ASMC proliferation, which is enhanced by M2AChR stimulation [2AChR are similar to the ones here described for rat ASMC.Briefly, muscarinic agonist synergism with other mitogens on rat ASMC proliferation could be mediated by interactions between several pathways and signal transduction effectors as GSK-3β, p70mulation . These e2AChR from pharmacological profile responses (log IC50 AF-DX-116 < log IC50 4-DAMP).Muscarinic agonist inhibition of rat ASMC proliferation is an original experimental finding of this work. This effect was observed when ASMC were incubated with Cch plus mitogens at its maximum doses for proliferation responses such as 10% FBS. This inhibitory novel response seems to be also mediated by M2AChR coupled to a Gi/o protein-dependent process, that augmented NO-sGC activity in bovine smooth muscle, independently of nitric oxide (NO) [3AChR/Gq protein complexes have been associated with the NPR-GC-B activation [Cip1/Waf1[Muscarinic agonist inhibition mechanism may be the result of the activation of two signaling pathways: 1) The cGMP/PKG activation cascade -58, and ide (NO) ,56. Liketivation ,56 produ2AChR-dependent inhibitory effect on ASMC proliferation is under intense investigation in our laboratory.The involvement of these two GCs (sGC and/or NPR-GC-B) in rat ASMC in response to Cch is supported by some additional experimental evidences (data not shown): 1) Cch induced proliferation is blocked by sodium nitroprusside . 2), Cch blocked synergistic effect on ASMC proliferation induced by ODQ, a selective inhibitor of NO-sGC, and 3) Cch potentiates the inhibitory effect of natriuretic peptide type-C  on ASMC proliferation. All these additional data suggest that inhibitory effect of Cch on mitogen-induced ASMC proliferation could include the activation of ones of these cGMP-dependent signaling pathways above mentioned. The identification of the members of the cGMP-PKG cascades related to this Mint) or mitogenic pathways over-activation (e.g. p38MAPK or JNK) trigger cell death [3AChR and p21Cip1 Waf1 expression was involved in cell cycle arrest. The dual effects of Cch on proliferation have also been reported for others cell systems [The fact that ASMC proliferation inhibition by Cch was observed at high doses of mitogens leads to ask whether inhibition is due to certain level of mitogenic pathways activation, which are associated in parallel with anti-proliferative pathway facilitation  [3AChR transcription and expression in ASMC that express contractile proteins and generate a basal lamina rich in laminina. Airway remodeling models suggest that M3AChR mediated ACh effects “in vivo” [3AChR may constitute an “in vivo” mechanism for ASMC phenotype modulation, which is crucial event in tissue remodeling rather proliferation stimulation [2AChR [2/M3AChR ratio is 4:1, which has been described in intact airway smooth muscle cells [ASMC phenotype present in these assays may be another factor that can influence these results. ASMC express both Mn (~80%) . Prolongin vivo” -20. Chromulation ,28. By on [2AChR , which sn [2AChR . Thus, tle cells ,65.3AChR subtype, could be beneficial in attenuating airway remodeling in chronic asthma [3AChR was inhibited and the M2AChR was elevated by the administration of tiotropium bromide. Our results on the role of M2AChR inhibiting the mitogen-induced proliferation may be relevant, which may be similar environment to the ones present in chronic asthma, which can explain these interesting experimental results of tiotropium bromide on M3/M2AChR expression [The exact cellular mechanisms underlying MAChR-modulated DNA synthesis and ASMC proliferation are not fully understood. ASMC proliferation involves several intracellular pathways leading to DNA replication and cell division as above discussed. In relation to this novel inhibitory effect of muscarinic agonists here described may improve our understanding of the intracellular mechanisms underlying the activation of mitogenic and anti-mitogenic pathways and provide insights for therapeutic drug development. Recent evidence suggests that ACh acting through muscarinic receptors may play an inhibitory role in the airway remodeling. The anticholinergic drug tiotropium bromide, which selectively antagonizes the Mc asthma . These apression .2AChRs through activation of distinctive and multiple signal transduction pathways, which may depend on the cell phenotype and the type and mitogen concentration used. These findings are important in ASMC proliferation induced by ACh “in vivo” especially on cells with synthetic-proliferative phenotype. If, mitogenic and anti-mitogenic effects are both mediated by the same receptor, leads us to propose an ASMC proliferation modulation by ACh, via M2AChRs. Our data here reported support the rationale about the need for the developing of new muscarinic antagonists-derivated from tiotropium bromide or similar compound that preferentially antagonize the putative M3AChR involves in the mitogen-induced ASMC proliferation associated to the airway remodeling presents in chronic asthma and COPD and increase the level of expression of this novel action of M2AChR acting as anti-proliferation receptor.Muscarinic agonist has three effects on “in vitro” rat ASMC proliferation: 1) stimulation, 2) synergism, and 3) inhibition. Interestingly, both biological actions seem to be mediated by MACh: Acetylcholine; ASMC: Airway smooth muscle cells; Cch: Carbamylcholine; COPD: Chronic obstructive pulmonary disease; cGMP: Cyclic guanosine monophosphate; EGF: Epidermal growth factor; EGFR: EGF Receptor extracellular signal-regulated kinases; ERKs: Fetal bovine serum; FITC: Fluorescein-1-isothiocyanate-conjugated; GPCR: G protein-coupled receptors; GSK-3 β: Glycogen synthase kinase-3β; IC50: half maximal inhibitory concentration; MAPK: Mitogen-activated protein kinases; mAchR: Muscarinic receptors; OD: Optical density; NO: Nitric oxide; NPR-GC: Natriuretic peptide receptor sensitive guanylyl cyclase; PDGF: Platelet-derived growth factor; PTX: Pertussis toxin; PDK-1: 3′-phosphoinositide-dependent kinase-1; PI3-K: Phosphoinositide 3-kinase; PKC: Protein kinase type C; NO-sGC: NO sensitive soluble guanylyl cyclase; TNF-α: tumor necrosis factor-alpha.The authors declare that they have no competing interests.FPU, CAFA, RFR, RGA, ILB, MJA. FPU, CAFA and RFR participated in the design and performance of tissue cell cultures and proliferation assays. FPU and MJA participated in the design of the study and performed the statistical analysis. FPU and MJA conceived the study. RGA participated in its design and coordination and drafted the manuscript. ILB helped to draft and the final version of the manuscript. All authors read and discussed and approved the final version of this manuscript.FPU: PhD in Cell Biology. Specialist in ASCM and MAPK expert. Actually is a Assistant professor at Biomembranes Section form Instituto de Medicina Experimental (IME). Faculty of Medicine. Central University of Venezuela (UCV).CAFA: MD and Postgraduate at Biomembranes Section form Instituto de Medicina Experimental (IME). Faculty of Medicine. Central University of Venezuela (UCV).RFR: MD and Postgraduate at Biomembranes Section form Instituto de Medicina Experimental (IME). Faculty of Medicine. Central University of Venezuela (UCV).RGA: Ph D. in Biochemistry Molecular and Cell Biology from Cornell University. Actually she is Full Professor in Biochemistry Molecular and Cell Biology at Biomembranes Section form Instituto de Medicina Experimental (IME). Faculty of Medicine. Central University of Venezuela (UCV).ILB: MD. Specialist in Biochemistry and Cell Biology from UCV. Actually she is Full Professor in Biochemistry and Cell Biology at Biomembranes Section form Instituto de Medicina Experimental (IME). Faculty of Medicine. Central University of Venezuela (UCV).MJA: MD. & Ph D. in Biochemistry Molecular and Cell Biology from Cornell University. Actually, he is Full Professor in Biochemistry Molecular and Cell Biology at Biomembranes Section and Director of Instituto de Medicina Experimental (IME). Faculty of Medicine. Central University of Venezuela (UCV).


In addition, although the expected enhanced phosphorylation of Src at Tyr-416 (activation site) is present in the most aggressive prostate cancer cell lines, unexpectedly high phosphorylation levels at the Tyr-527 inhibitory site are observed as well. The latter, rather than representative of inhibited enzyme, is more indicative of primed Src responsive to local phosphorylated binding partners.Metastatic progression is a multistep process that involves tumor growth and survival, motility and invasion, and subsequent proliferation in an inappropriate environment. The Src protein tyrosine kinase has been implicated in many of the biochemical pathways that drive these behaviors. Although Src itself is only rarely mutated in human tumors, its aberrant activity has been noted in various cancers and suggested to serve as a barometer of metastatic potential. With these features in mind, we examined Src kinase regulation at the structural, enzymatic, and expression levels as a function of progressively invasive prostate cancer cell lines. Surprisingly, both total Src content and kinase activity decrease with increasing cell line aggressiveness, an observation that appears to be inconsistent with the well-documented role of Src in the signaling pathways that drive growth and invasion. However, we do observe a direct correlation between Src kinase  Upon interaction of the SH2 or SH3 domains of the Src enzyme with other proteins, the inhibitory pTyr-527 residue is released, which generates fully active pTyr-527 Src per se, but rather of an enzyme that is potentially primed and ready to be “switched on” by interaction with an appropriate accessory protein. In short, high pTyr-527 Src levels could represent inhibited enzyme, activated enzyme, or some variant in-between. This uncertainty highlights the need to sample Src kinase directly via its ability to phosphorylate a target substrate. In addition to phosphorylation, the Src kinase is regulated at the protein level via ubiquitination and thus degradation by the proteasome-mediated pathway The Src protein kinase is the founding member of the Src kinase subfamily of nonreceptor protein tyrosine kinases. The catalytic activity of these enzymes is regulated, in part, via phosphorylation Src is strongly implicated in the pathways that control many of the characteristic cell behaviors responsible for tumor invasion and progression. For example, Src mediates adhesion and cytoskeleton dynamics and thereby controls cell migration Tyr-Gly-Glu-Phe-Orn(Ac)-amide (1), where Tyr is the site of phosphorylation. In addition, ornithine (Orn) residues were placed on each terminus of the peptide sequence, in the event that the peptide proved to display poor selectivity for Src versus other protein kinases. We’ve previously shown that side chain modification of active site-directed peptides with unnatural substituents can dramatically enhance the selectivity for the target protein kinase (vide infra) 2)] of the peptide were prepared via solid-phase peptide synthesis. 5-FAM (5-carboxyfluorescein) was chosen as the fluorophore for substrate and product detection by LIF.Based, in part, on a Src substrate sequence identified from an oriented peptide library 1 emerges at 240 sec, whereas the phosphorylated product 2 appears at 290 sec. Growth of the latter was monitored, as a function of time, by incubating the substrate (1) with active Src kinase. The 290 sec peak was confirmed to be the phosphorylated product by spiking with synthetic phospho-peptide 2. The kinetics of phosphorylation was obtained by evaluating collected time point samples via CE-LIF and quantifying the extent of phosphorylation via integration of the individual peaks. Peptide 1 phosphorylation is essentially complete within 3 min under the assay conditions that employed pure Src enzyme (vide infra) proceed at a more leisurely pace. In the latter case, initial rate kinetics (<10% peptide phosphorylation) were acquired within 10 min of cell lysate incubation.The separation of the non-phosphorylated substrate and the phosphorylated product by CE-LIF is shown in c enzyme . By cont1 with lysates, we monitored the stability of the peptide as a function of time. Cells were lysed using Pierce’s M-PER mammalian lysis buffer and, in the absence of ATP and Mg2+ (i.e. no observable kinase activity), the lysate was added to peptide 1. Only approximately 10% of the peptide is degraded after 1 h  than with whole cell lysates containing Src (1 phosphorylation in the absence of Src could be one or more of the eight other members of the Src family kinases (SFK). Indeed, several SFK family members, such as Fyn, Brk, Lyn Lck and Yes are known to present in prostate cell lines 1 phosphorylation by Src-free lysates, we decided that peptide 1 is, for our present needs, sufficiently Src selective.We examined the Src-selectivity of d of Src . Althoug1/CE-LIF method to detect Src activity by using a combination of two known Src inhibitors. Imatinib (marketed as Gleevec), which is used to treat chronic myelogenous leukemia and other cancers, is an Abl-selective inhibitor with demonstrated weak anti-Src activity. Imatinib blocks kinase activity by serving as a competitive inhibitor of ATP. Like imatinib, saracatinib (AZD0530) is a competitive inhibitor of ATP, but displays a high selectivity and robust potency for Src. For example, the IC50 values of imatinib and saracatinib with isolated Src enzyme are 24.4 µM 1 phosphorylation with a submicromolar IC50 (0.35±0.08 µM) whereas the correspondingly poor Src inhibitor imatinib displays an IC50 that is in excess of 100 µM  than the former (IC50 = 2.7 nM) against pure enzyme IC50 = 0.35±0.08 µM) and imatinib (>100 µM) for Src in DU145 cell lysates analogous to the reported difference under simple buffer conditions. Finally, we investigated whether the CE strategy could be used to observe Src activity and inhibition in single cells  or in cells preincubated with 1 µM of the weak inhibitor imatinib . By contrast, no peptide phosphorylation was observed in cells that had been preincubated with 1 µM of the potent inhibitor saracatinib (IC50 = 0.35±0.08 µM).Generic ELISA- c kinase  and prosc kinase . In the c kinase . The CE le cells . DU145 cWith the ability to sample Src activity using purified enzyme, in cell lysates, and in single cells, we examined Src activity from noninvasive, invasive, and androgen-independent prostate cell lines . Unexpecet al have shown that the more invasive the prostate cell line the higher the miR-205 level Why is the total Src content decreased in the most aggressive cell lines? One possible explanation is the known sensitivity of activated Src, relative to its non-activated counterpart, to ubiquitin-mediated degradation Src phosphorylation status has been assumed to correlate with activity where pTyr-416 is taken as active and pTyr-527 as inactive forms of the enzyme. With this in mind, we measured the levels of pTyr-416 via Western blot analysis and plotted pTyr-416 Src/total Src  versus cell line aggressiveness. As is evident from A high pTyr-527/total Src ratio should be, according to the conventional model, indicative of an overall lower fraction of active enzyme. However, we’ve found that this ratio is higher in aggressive lines as well . One posinactive Src in CaP cell lines. Rather, the latter ratio appears to be a much better predictor of active Src. From a mechanistic point of view, this might reflect the presence of phosphorylated proteins that can associate with (via Src’s SH2 domain) and thereby rapidly activate the inhibited enzyme. Consequently, the activity status of pTyr-527 Src should be viewed as “primed for activity” rather than statically inactive.There has been and continues to be a herculean effort to identify CaP markers that correlate with heritability as well as acquired somatic mutations. For example, more than 40 susceptibility loci have been assigned to approximately 25% of the heritable risk. In summary, we have developed a sensitive and selective Src kinase activity sensing system. Using a series of nonaggressive, aggressive, and androgen-independent prostate cell lines, we’ve found that total Src activity decreases as a function of increasing prostate cell line aggressiveness. However, this unexpected (and ultimately misleading) observation is a consequence of a corresponding decrease in total Src content in the more aggressive cell lines. When the latter is taken into account, a direct correlation exists between cellular aggressiveness and the fraction of cellular Src present in the active state.3)4 (3 equiv) in CHCl3/AcOH/N-methylmorpholine (37∶2:1). The Orn residues were then acetylated using acetic anhydride/DIPEA (4∶1) and the N-terminal Fmoc protecting group was subsequently removed. The free N-terminus was then exposed to 5-FAM (5 equiv), HOBT (10 equiv) and N,N’-Diisopropylcarbodiimide  in DMF overnight, and incubated with 30% piperidine for 15 min. The peptides were cleaved with TFA/H2O/TIS (triisopropylsilane) (95∶2.5∶2.5) and purified via HPLC (Waters), and their structures confirmed by ESI-Mass Spectrometry (Agilent Technologies). C81H97N13O27 (peptide 1) Exact Mass calculated for 1683.7, found (ESI+) 843.0 (M+2H)2+, 562.5 (M+3H)3+ and C81H98N13O30P (peptide 2) Exact Mass calculated 1763.6, found (ESI+) 883.0 (M+2H)2+.Common analytical grade reagents were purchased from Fisher or Sigma-Aldrich. Common amino acids, O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), N-Hydroxybenzotriazole (HOBT) and TGR resins were obtained from NovaBiochem. Fmoc-Orn(Aloc)-OH was obtained from Bachem. 5-carboxyfluorescein (5-FAM) was purchased from Anaspec. The non-phosphorylated peptide–resin Fmoc-Orn(Aloc)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ile-Tyr(tBu)-Gly-Glu(OtBu)-Phe-Orn(Aloc)-amide-Resin and the phosphorylated peptide–resin Fmoc-Orn(Aloc)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ile-Tyr(PO(OBzl)OH)-Gly-Glu(OtBu)-Phe-Orn(Aloc)-amide-Resin were synthesized using a standard Fmoc peptide synthesis protocol on the Prelude automatic peptide synthesizer (Protein Technologies). Amino acid (5 equiv), HCTU (5 equiv) and DIPEA (10 equiv) were mixed with resin in DMF and reacted for 10 min at room temperature. Fmoc deprotection was achieved using 20% piperidine in DMF for 20 min. The Aloc protecting group on the Orn residues was selectively removed with Pd(PPHPrimary antibodies Src (36D10): #2109, Yes: #2734, Phospho-Src Family (Tyr416): #2101, Phospho-Src Tyr527): #2105, and α-tubulin (DM1A): #3873 were purchased from Cell Signaling Technology; anti-Fyn: 610163 and anti-Lck: 551047 were purchased from BD Pharmingen Biosciences; anti-Lyn (H-6): sc-7274 was obtained from Santa Cruz Biotechnology. Secondary antibodies were obtained from GE Healthcare. SDS-PAGE gel electrophoresis was performed using NuPage 4–12% Bis-Tris precast gel (Invitrogen), running at 200 V for 50–60 min. Gels were transferred using iBlot dry blotting system (Invitrogen) for 7 min. Blocking, antibody incubation, and washes were performed on SNAPid protein detection system (Millipore). Blots were detected by Amersham ECL Plus  and chemiluminescent images were obtained by the FluoChemFC2 system (Alpha Innotech). Band densitometry was performed using AlphaView software. The number of western blots reported for each data point in 27: #2105Src was captured by incubating cell lysate (500 µg)-antibody  mixtures with Pierce protein A/G agarose (Pierce Classic IP kit); and a BSA (5 µg) control was mixed with Pierce control agarose resin for 1 h at 4°C. Both treatments (with antibodies and with BSA) were separately repeated (3 h exposure time to antibodies/BSA and 1 h agarose-capture). Finally, the pre-cleared lysate was collected and concentrated using a Vivaspin 500 centrifugal concentrator, 10 kD  for western blot analysis and kinase assays.Capillary electrophoresis was performed on a ProteomeLab PA800 system equipped with a laser-induced fluorescence (LIF) detector (Beckman Coulter). The LIF was excited using the 488 nm argon laser. A fused-silica capillary  was used for separation (Polymicro Technologies). Prior to installation, capillaries were pretreated with 0.1 M NaOH for 12 h, distilled water for 1 h, 0.1 M HCl for 6 h and water again for 12 h. The samples were hydrodynamically injected and separated at 25°C at a constant voltage of 12 kV. The running buffer was 100 mM Borate/100 mM SDS buffer at pH 7.7. Before each run, the capillary was rinsed with 0.1 M NaOH, distilled water, and then the running buffer for 2 min under 20 psi pressure. Data was collected and analyzed using 32 Karat Software .2. PZ-HPV-7, RWPE1, WPE1-NA22, WPE1-NB14, WPE1-NB11 and WPE1-NB26 cell lines were cultured in Keratinocyte Serum Free Medium (K-SFM) supplied with 0.05 mg/mL bovine pituitary extract (BPE) and 5 ng/mL epidermal growth factor (EGF) , and 1% penicillin/streptomycin . CWR22Rv1 cells were cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum  and 1% P/S. DU145 cells were cultured in Eagle’s Minimum Essential Medium  supplemented with 10% HI-FBS and 1% P/S. PC3 cells were cultured in F-12K medium (ATCC) supplemented with 10% HI-FBS and 1% P/S. Cell lysates were prepared as follows: confluent cells were first trypsinized and collected into 15 mL tubes in the presence of FBS-containing buffer or medium and then washed (3X) with ice cold DPBS buffer. The cells were lysed using M-PER mammalian protein extraction reagent mixed with 5/100 (v/v) Halt protease and phosphatase inhibitor cocktail EDTA-free (Thermo Fisher Scientific) for 10 min at 4°C. Cell debris was removed by centrifuging samples at 14,000 x g for 10 min at 4°C. The total protein concentrations were determined using Dc Protein assay (Bio-Rad).All cell lines were acquired from ATCC and grown in a humidified incubator at 37°C with 5% CO2O, 0.5 µL of 1.14 mM substrate stock solution, 4 µL of 10X Src kinase reaction buffer , 0.6 µL of 100 ng/µL active Src kinase (Millipore), 4 µL of 10 mM ATP. Final concentration of the assay solution was 8 mM MOPS, 0.2 mM EDTA, 4 mM MgCl2, 1.5 ng/µL Src kinase, 14.25 µM substrate, and 1 mM ATP. Assays were initiated by adding ATP. At each time point, 2 µL of the reaction mixture was removed and diluted with 8 µL 0.1 M HCl to stop the reaction. Cell lysate phosphorylation assays were performed similarly as described above using the following conditions: 2.5 µg/µL cell lysate, 6.75 mM Tris (pH 7.5), 1 mM ATP, 4 mM MgCl2, 57 µM substrate. Assays were initiated by adding substrate. Total protein content was used as the internal normalization of all cell extracts.The pure enzyme phosphorylation assays were performed with a total volume of 40 µL from the following stock solutions: 30.9 µL of H2 humidified incubator at 37°C. Single cell capillary electrophoresis was performed using a custom-made CE-LIF system, as described previously 2, 1 mM CaCl2, and 10 mM HEPES, pH 7.4, 25°C) for 4 min. Microinjection of DU145 cells was performed using an Eppendorf Transjector 5246 and InjectMan system. 114 µM peptide 1 and 90X phosphatase inhibitor cocktail mixture  was microinjected into a single cell . For cells not treated with Src inhibitor, the ECB flow was kept on until 20 s after microinjection. For inhibitor-treated cells, the ECB flow was halted and replaced with 1 mL of inhibitor-containing ECB for 5 min. The ECB flow was turned on  after microinjection to remove any residual peptides from the extracellular environment. The microinjected cell was incubated for 2 min prior to lysis by a focused Nd:YAG laser Custom made cell chambers were prepared by utilizing polydimethyl siloxane  to glue a silicon O-ring to a glass coverslip (Fisher). The day prior to use, DU145 cells in suspension were diluted in 500 µL of complete media, plated on the chamber, and incubated in a 5% CODifferences between non-cancer cells  and aggressive cancer cells  were compared using the 2-tailed Student’s t-test, and a P-value <0.05 was considered statistically significant. Simple regression test with introduced indicator variable as 4,3,2,1,0 was performed to study the linear trend among RWPE1 series of cell lines, and a P-value <0.05 was considered statistically significant. Correlations between specific Src activity  and phosphorylation status of Src  were compared using Pearson’s correlation. A correlation coefficient r>0.9 was considered very highly correlated; 0.7<r<0.9 was considered highly correlated; 0.5<r<0.7 was considered moderately correlated.Figure S1Phosphorylation kinetics of peptide 1 by lysates from nine prostate cell lines.(TIF)Click here for additional data file.Figure S2(a) Western blots of Src kinase from prostate cell line lysates and (b) validation of the antiSrc antibody.(TIF)Click here for additional data file.Figure S3Normalized Src activity (where 1.0 is activity in the absence of inhibitor) in DU145 lysates versus log (a) [saracatinib] and (b) [imatinib] (µM).(TIF)Click here for additional data file.Figure S4Single cell analysis of Src kinase activity in DU145 cells by CE. (a) Cells exposed to standard media, (c) standard media and saracatinib/AZD0530 (1 µM), and (e) standard media and imatinib (1 µM). All cells were subsequently microinjected with Src kinase sensor 1, incubated for 2 min, lysed via sonication, the lysate loaded onto the capillary via electrokinetic injection, and electrophoresed. Electropherograms (a), (c), and (e) are representative of results where n = 5 for each experiment. The small peak at 160 s is a component present in the media. Peptide 1 is the large peak observed approximately 100 s prior to that of the corresponding phosphopeptide. Plot (b) is the control for (a): lysate from cells were exposed to standard media spiked with synthetically prepared phosphopeptide 2; Plot (d) is the control for (c): lysate from cells exposed to standard media spiked with peptide 1 and phosphopeptide 2; Plot (f) is the control for (e): lysate from cells exposed to standard media spiked with peptide 1 and phosphopeptide 2. Controls were run either immediately prior to or following the corresponding experiments in (a), (c), and (e).(TIF)Click here for additional data file.Figure S5Total Src content and phosphorylation status in prostate cell lines.(TIF)Click here for additional data file.Figure S6Src pY527 status in the RWPE1-derived cell lines with increasing invasive ability plotted along the x-axis.(TIF)Click here for additional data file.Table S1Stability of the Src sensor in prostate cell lysates.(PDF)Click here for additional data file.


In August 2010 an iceberg four times the size of Manhattan broke off Greenland’s northwestern coast and began drifting out to the sea. At nearly 100 square miles, this was the largest iceberg to appear in Arctic waters since 1962 and a fresh indicator that Greenland’s frozen landscape is undergoing significant changes.1Originally part of the much larger Petermann glacier, which flows down from Greenland’s interior into a coastal fjord, the iceberg detached for unknown reasons. It could be that ordinary glacial dynamics were at work, and indeed, Petermann glacier “calves” (breaks off) icebergs routinely, although generally not ones so large as this. But the unusual size of the iceberg also might signal a response to global warming, and that’s what worries scientists.Accelerated melting of the glaciers and other frozen masses that make up Earth’s cryosphere has become a widespread phenomenon. Apart from what’s happening in Greenland, coastal glaciers also are thinning off West Antarctica, where an enormous ice sheet containing enough water to raise sea level by more than 20 feet has attracted growing concern. Meanwhile, a vast percentage of the world’s land-based glaciers also are in retreat, according to Richard Armstrong, a senior research scientist at the National Snow and Ice Data Center (NSIDC) at the University of Colorado, Boulder.“There’s a lot of regional variation, so there are some exceptions to the trend,” Armstrong says. “But in general, the Earth is warming, and glaciers are shrinking in most areas.” Likewise, the human health implications of the world’s changing ice cover vary regionally.Glacial retreat is often described as global warming’s most visually compelling manifestation. But the influence of climate on the cryosphere doesn’t lend itself to simple generalizations. Glaciers, ice sheets, and seasonal snowpack are part of an exceedingly complex climate system with many unknowns.Beata Csatho, an associate professor of geophysics at the State University of New York at Buffalo, says glaciology is just now emerging from an observational phase dominated by data collection. Scientists have only now begun to model these data to project future trends, she says. But uncertainty about how the cryosphere responds to climate change can be frustrating to those in the public who want definitive answers to either support or reject assumptions about global warming. That puts glaciologists in an uncomfortable position, pushed to supply conclusions prematurely and besieged by misconceptions.Skeptics who cite one or two advancing glaciers as evidence that humans don’t have an impact on climate are likely overstating their case, says Jeffrey Kargel, a University of Arizona glaciologist. The near-unanimous scientific consensus—including the opinion even of most climate change skeptics—is that anthropogenic greenhouse gas emissions have likely accelerated an ongoing warming trend, although by how much isn’t clear.4Some scientists are concerned that if warming continues, the global meltdown could cause catastrophic effects. Sea level is already rising at an accelerated rate—a global average of 0.1 inch per year between 1993 and 2003, according to the United Nations Intergovernmental Panel on Climate Change (IPCC), nearly twice the average annual rise over the longer period of 1961 to 2003.The IPCC has projected a rise in sea level of anywhere from 7 inches to 2 feet by the end of this century.The fastest warming rates are in the Arctic, where temperatures are increasing at twice the global average.9According to Walt Meier, a research scientist at the NSIDC, high Arctic warming rates can be explained by a reflective phenomenon known as albedo: melting sea ice exposes dark ocean surfaces, which absorb sunlight instead of reflecting it back into space. Oceans retain that warmth and then transfer it back to the atmosphere. “And that sets up a vicious cycle,” Meier explains. “More melting, followed by more ocean warming, followed by more warming of the air.”The consequences are clearly evident in satellite imagery that shows Arctic sea ice decreasing by about 9% per decade since 1979. In September of that year, Arctic sea ice covered slightly more than 3 million square miles, an area roughly equal to that of the lower 48 states, Meier says. By September 2010, the sea ice had dwindled to roughly 1.8 million square miles.10Meanwhile, climate change appears to exert a mysterious influence on how Arctic winds and ocean currents interact, says Hamish Pritchard, a scientist with the British Antarctic Survey in Cambridge, UK. And as Arctic winds change in direction and speed, he says, they’re somehow drawing warmer ocean currents closer to Greenland’s coast, where glaciers that buttress the island’s massive ice sheet spill into the sea.Glaciers that gain as much mass from snow as they lose from calving icebergs are said to be in equilibrium. But if calving dominates over the accumulation of new mass, which Pritchard says is the case in Greenland now, then seawater can intrude where coastal glaciers attach themselves to land. “Ice always melts when it’s in contact with seawater, and that erodes the glacier’s frontal regions,” Pritchard says. Eric Rignot, an earth scientist at the University of California, Irvine, recently documented that Greenland’s coastal glaciers are melting 100 times faster at their floating end points than they are on land.11At the same time, warming air is degrading the island’s ice sheet from above. Around 10 years ago, scientists discovered that “moulins,” or narrow channels in the ice sheet, were draining unexpectedly large amounts of summer meltwater from the surface of the ice toward the underlying ground. And as that water accumulates below the ice sheet, sections of the ice lift off the ground and start flowing rapidly toward the coast, explains Gordon Hamilton, an associate research professor at the University of Maine, Orono.The IPCC did not consider this “moulin effect” when, in its 2007 assessment report, it predicted that sea level might rise by as much as 2 feet by 2099.Summer meltwater isn’t currently so much an issue in Antarctica, which is so far south that the majority of its ice sheet stays frozen all year.  Separated from West Antarctica by the Trans-Antarctic Mountains, the East Antarctic ice sheet is the largest ice mass on the planet, contains enough water to raise sea level by almost 200 feet. But East Antarctica’s ice sheet rests on an enormous landmass above present-day sea level and is generally considered stable, according to Claire Parkinson, a climatologist with the National Aeronautics and Space Administration.The West Antarctic ice sheet, however, rests on a landmass grounded roughly a mile below sea level, meaning that if the ice sheet wasn’t there, it would be replaced by a deep ocean. And that makes it vulnerable to encroaching seawater that could speed its disintegration.Both of West Antarctica’s biggest ice shelves—namely, the Ross ice shelf to the west and the Ronne-Filchner ice shelf to the east—appear stable at this point, Parkinson says. Yet according to Pritchard, glaciers along West Antarctica’s midpoint are thinning at an accelerated pace: the Pine Island glacier is thinning by up to 18 feet per year, the neighboring Smith glacier by 27 feet per year, and the Thwaites glacier by 12 feet per year.13Other nearby glaciers also are thinning from what Ted Scambos, a glaciologist with the NSIDC, says is an influx of deep, warm ocean water pushed toward the coast by changing wind patterns. That dynamic, he says, results in part from the Southern Hemisphere’s ozone hole, which allows for regional warming in the upper atmosphere that, in turn, influences local air currents.14According to Csatho, ice shelf losses in the West Antarctic are partly offset by strong gains from snowfall in some inland areas, so the degree to which the ice sheet might be imperiled isn’t clear. “But if the ice shelves do collapse, then portions of the ice sheet are at greatly increased danger of flowing into the ocean,” Parkinson adds.Scientists first considered this possibility in 1968, when the late John Mercer, a professor at The Ohio State University, predicted that global warming caused in part by industrial pollution might destroy West Antarctica’s ice shelves and allow its ice sheet to flow toward the coast.10The collapsing Larsen ice shelves had no effect on sea level, given that they were already floating and displacing water, according to Scambos. What’s significant is that when the ice shelves disappeared, land-based glaciers behind them began moving faster toward the coast, just as Mercer predicted. Scambos says there isn’t enough land ice on the Antarctic Peninsula to raise sea level appreciably. The Thwaites, Pine Island, and Smith glaciers are a different story, however, given that the West Antarctic ice sheet—with its capacity for a more catastrophic rise in sea level—lies behind them.No one knows how high the global sea level will rise in coming decades or how many people will be affected, although small island nations and coastal areas are expected to bear the brunt of the impact.18Excessive glacial runoff can pool in downstream areas and create lakes that appear and then disappear from one year to the next. And when these lakes breach, the resulting GLOFs endanger local lives and infrastructure. “In March 2006, an avalanche fell from the Quelccaya ice cap into one of these lakes and created a mini-tsunami that breached a dam and flooded the entire valley below,” says Lonnie Thompson, a glaciologist and professor at The Ohio State University. The Quelccaya ice cap of Peru is the largest tropical ice mass in the world.Should the Andean glaciers disappear altogether, Thompson adds, Peru’s population could face an opposite problem: significant water shortages. Indeed, the “zero-degree isotherm,” or the altitude at which snow begins to melt, has climbed to higher elevations at earlier time points in spring, reflecting a steadily warming climate.That’s already happening now in Peru, where many populations rely on meltwater from Andean glaciers for domestic and industrial water needs.Similarly, communities in the Southwestern United States rely on meltwater from the Sierra Nevada snowpack, which forms every year in the mountains that define California’s eastern border. The Sierra Nevada snowpack begins to accumulate in October, peaks around April, and then melts entirely. When rainfall decreases in summer, meltwater makes up the shortfall, draining into reservoirs and contributing a significant fraction to water supplies throughout the Western states.21According to research by Sarah Kapnick, a PhD candidate in atmospheric and oceanic sciences at the University of California, Los Angeles, the Sierra Nevada snowpack has been trending toward an earlier peak by about 0.6 day per decade since 1930, such that it starts melting almost a week sooner now than it did 80 years ago.Apart from the southwestern United States and Peru, it’s not clear where retreating ice masses could affect local water supplies. It is said that 1.3 billion people in Southern Asia might face a risk, given widespread glacial retreat in the Eastern Himalayas.3However, in March 2010, Alford and Armstrong completed a study for the World Bank that suggests the IPCC likely overstated its conclusion.Regional variation makes it hard to predict how water supplies originating in the Himalayas will respond to climate change. Even as glaciers retreat in its eastern ranges, those in the west—given that they reside at higher altitudes where temperatures don’t fall below freezing—are stable or advancing. “The wild card is precipitation,” says Armstrong. “Most of the climate models are pretty consistent on temperature increases with rising greenhouse gas [levels], but they fall apart when it comes to precipitation—some show more and others less with climate change depending on location. And if you get more precipitation, it would fall as snow above the freezing level, and you could wind up with increasing glacier mass at higher elevations.”Armstrong’s point illustrates an essential problem: although climate change has global consequences, it manifests in different ways depending on regional features. And that complexity leaves interpretations of its cause and future impacts open to debate, even to attack.But look at the global system as a whole, Scambos emphasizes, and a fundamental truth emerges. “Virtually every glacial region on Earth is in retreat, as are [ice masses] on both of the poles,” he says. And that gives us a consistent picture that hangs together well—this is happening at the same time that the oceans, land, and air are also warming.”


We also demonstrated by coimmunoprecipitation and in vitro binding assays that Nak1 and Orb6 physically interact, and that the Nak1 C-terminal region is required forNak1/Orb6 complex formation in vivo. However, results from in vitro kinase assays did not show phosphorylation of recombinant Orb6 by HA-Nak1, suggesting that Orb6 activation may not involve direct phosphorylation by Nak1. To investigate the role of Orb6 phosphorylation and activity, we substituted Ala at the ATP-binding and conserved phosphorylation sites. Overexpression of kinase-dead HA-Orb6K122A in wild-type cells resulted in a loss of cell polarity, suggesting that it has a dominant-negative effect, and it failed to rescue the polarity defect of nak1 or orb6 ts mutants. Recombinant GST-Orb6S291A did not autophosphorylate in vitro suggesting that Ser291 is the primary autophosphorylation site. HA-Orb6S291A overexpression only partially rescued the orb6 polarity defect and failed to rescue the nak1 defect, suggesting that autophosphorylation is important for Orb6 function. GST-Orb6T456A autophosphorylated in vitro, indicating that the conserved phosphorylation site at Thr456 is not essential for kinase activity. However, HA-Orb6T456A overexpression had similar effects as overexpressing kinase-dead HA-Orb6K122A, suggesting that Thr456 is essential for Orb6 function in vivo. Also, we found that both phosphorylation site mutations impaired the ability of Myc-Nak1 to coimmunoprecipitate with HA-Orb6. Together, our results suggest a model whereby autophosphorylation of Ser291 and phosphorylation of Thr456 by an upstream kinase promote Nak1/Orb6 complex formation and Orb6 activation.The Ndr-related Orb6 kinase is a key regulator of polarized cell growth in fission yeast, however the mechanism of Orb6 activation is unclear. Activation of other Ndr kinases involves both autophosphorylation and phosphorylation by an upstream kinase. Previous reports suggest that the Nak1 kinase functions upstream from Orb6. Supporting this model, we show that HA-Orb6 overexpression partially restored cell polarity in  The levels of HA-proteins expressed in these strains were similar as detected by western blots  but not to GST (lane 5), indicating that Nak1 and Orb6 interact directly in vitro.It was previously reported that Orb6 and Nak1 interact in a yeast two-hybrid test n assays . We foun1–629 and HA-Nak1554–652, but not with HA-Nak11–562, HA-Nak11–585 or HA-Nak11–607  were expressed in E. coli. and purified  are required to facilitate Nak1 phosphorylation of Orb6.In order to separate the signals from phosphorylated HA-Nak1 and Orb6, we also cleaved the GST moiety from GST-Orb6 prior to performing the kinase assay. Interestingly, we found that purified Orb6 lacking GST no longer autophosphorylated , lane 1,S291A and GST-Orb6T456A. We also constructed and purified a kinase-dead GST-Orb6K122A containing an Ala substitution at the invariant catalytic lysine residue in the ATP binding site that is critical for activity of all protein kinases in vitro kinase assays to determine whether these purified recombinant mutant proteins were active. We found that only GST-Orb6 and GST-Orb6T456A were autophosphorylated, while phosphorylation of GST-Orb6K122A and GST-Orb6S291A was not detected  was obtained from Dr. David Beach; the orb3 (h+ orb3–167 leu1–32 ade-M216) and orb6 (h- orb6-25 leu1–32 ade6-M210) ts strains were obtained from Dr. Paul Nurse The 1–562, pRep3XC-HANak11–585, pRep3XC-HANak11–607, and pRep3XC-HANak11–629 were constructed by inserting a PCR-amplified genomic DNA fragments encoding the corresponding regions of Nak1 into the SpeI/NotI sites of pRep3XC-HA. pRep3X-HANak1554–652 was constructed by inserting a PCR-amplified genomic DNA fragment encoding the corresponding region of Nak1 into the SpeI/BamHI site of pRep3X-HA. pET33b-His6-HA-Nak1 was constructed by inserting HA-Nak1 into the BamHI/SalI site of pET33b. pGEX-5X-1-Nak1 was constructed by inserting a PCR-amplified cDNA fragment encoding Nak1 into the BglII/NotI sites of pGEX-5X-1. pGEX-5X-1-Orb6 and pGEX-5X-1-Orb6 301–469 were constructed by inserting PCR-amplified cDNA fragments encoding the corresponding regions of Orb6 into the BglII/NotI sites of pGEX-5X-1. pGEX-6p-1-Orb6 was constructed by inserting a PCR-amplified cDNA fragment encoding Orb6 into the BamHI/NotI site of pGEX-6p-1. Point mutations in pGEX-5X-1-Orb6 were made using the QuickChange Lightning Site-Directed Mutagenesis Kit (Stratagene) to produce pGEX-5X-1-Orb6K122A, pGEX-5X-1-Orb6S291A, and pGEX-5X-1-Orb6T456A. Similarly, point mutations in pRep3X-HAOrb6 were made to produce pRep3X-HAOrb6K122A, pRep3X-HAOrb6S291A, and pRep3X-HAOrb6T456A.Procedures for DNA cloning and analysis have been previously described 6-HA-Nak1 was expressed in E. coli BL21 cells and purified by binding to NiNTA agarose beads (Qiagen) and eluting with NiNTA elution buffer . The eluted protein was concentrated and imidazole was removed using a Vivaspin 6 ultrafiltration device .HisE. coli BL21 from pGEX-5X-1 or pGEX-6p-1 and purified by binding to Glutathione Sepharose 4B beads  and eluting with GST elution buffer . The eluted protein was concentrated using a Vivaspin 6 ultrafiltration device . GST was removed from purified recombinant GST-Orb6 following cleavage with PreScission Protease  at 4°C for 12 h.GST fusion proteins were expressed in 6-HANak1 for 2 h. The beads were washed 3 times with wash buffer , and were divided evenly into two parts. The samples were resuspended in SDS-PAGE sample buffer and boiled for 5 min, separated on SDS-PAGE gels, and then stained with Coomassie Blue or analyzed by western blots using anti-HA (12CA5) antibody.GST-Orb6 bound to Glutathione Sepharose 4B beads were incubated with BL21 cell lysate (2 mg protein) containing Hisin vitro kinase assays were performed as previously described 600  = 0.8. Cells were collected by centrifugation and resuspended in lysis buffer . Cells were vortexed with glass beads, and crude lysates were cleared by centrifugation at 14,000 rpm in a microfuge for 15 min. Relative protein concentrations were determined by Bio-Rad protein assay. Cell lysates were pre-cleared with protein A sepharose slurry for 20 min, and then incubated with 12CA5 (anti-HA) antibody and protein A sepharose for 2 hours at 4°C. Immunoprecipitates were centrifuged at 1,000 rpm in a microfuge, and washed three times with 750 μl yeast lysis buffer; samples were divided evenly upon the last wash. Half of the samples were analyzed by western blots with 12CA5 antibody to detect HA-epitope tagged proteins. The other half of the immunoprecipitates were resuspended in kinase buffer  and incubated at 30°C for 25 minutes. To remove HA-Nak1 following kinase reactions, the reactions were spun at 1000 rpm in a microfuge for 1 min, and the sepharose beads were removed. Samples boiled in Laemmli sample buffer for 5 min and resolved by SDS-PAGE; gels were stained with Commassie blue and visualized by autoradiography.Immunoprecipitation and


NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.CtIP plays an important role in homologous recombination (HR)–mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome ( DNA double-strand break (DSB) repair plays a critical role in the maintenance of genome stability, and mutations in key regulators in the DSB repair pathway are often associated with cancers and human diseases exhibiting chromosomal aberrations. CtIP is an essential DNA repair factor participating in homologous recombination (HR)–mediated DSB repair. In this study, we found that phosphorylation of CtIP by cell-cycle-dependent kinases CDKs modulates CtIP interaction with Nbs1, a component of the Mre11-Rad50-Nbs1 complex that is associated with Nijmegen breakage syndrome (NBS). We also showed that CtIP is phosphorylated by ataxia-telangiectasia gene product ATM, a kinase critical for the activation of DNA damage response pathway. Importantly, we found that CDK-mediated phosphorylation of CtIP is a prerequisite for CtIP to be phosphorylated by ATM upon DNA damage. These findings reveal a novel mechanism of how cell-cycle-dependent activities regulate HR in response to DNA damage, which significantly helps in understanding the regulation of DSB repair in mammalian cells. This study also provides new insight as to how the DNA damage checkpoint and repair proteins that are associated with human diseases participate in the maintenance of genome stability to prevent cancer. DNA double-strand breaks (DSBs) are often generated during normal cellular processes such as the development of immune systems and meiotic recombination, and they can also be induced by DNA damaging agents DSBs can be repaired by Ku-dependent non-homologous end joining (NHEJ) and homologous recombination (HR)-mediated DSB repair The Mre11/Rad50/Nbs1 (MRN) complex binds to DSB ends and plays important roles in facilitating ATM activation and promoting end resection to initiate HR-mediated DSB repair Various studies suggest that CtIP and its homologues are important for regulating end resection during the cell cycle In this study, we identified a new cluster of CDK sites situated in the middle of CtIP and demonstrated that phosphorylation of these CDK sites is a prerequisite for ATM to phosphorylate CtIP upon DNA damage. We also showed that ATM-mediated phosphorylation of CtIP, mainly through the newly identified conserved site T859, is essential for promoting end resection and HR, suggesting a mechanism of how CDK-mediated phosphorylation activates damage-induced HR. We further showed that phosphorylation of these identified CDK sites on CtIP induces associations of CtIP with the FHA/BRCT domains of Nbs1. Interestingly, this CDK-dependent interaction of CtIP with the FHA/BRCT domains of Nbs1 facilitates ATM to phosphorylate CtIP, thereby coupling CDK-phosphorylation with ATM-mediated phosphorylation of CtIP to promote end resection and HR.In vitro kinase assays showed that purified CtIP can be phosphorylated by ATM  or camptothecin (CPT) treatment  almost completely abolished damage-induced phosphorylation as revealed by damage-induced phosphorylation shift on SDS-PAGE , suggestonserved . In vitrhis site . Collectin vivo. Indeed, mutating a middle cluster of seven CDK sites  almost completely abolished the CtIP shift (Treating cell lysates with phosphatase or mutating 12 putative CDK sites (SP/TP sites) increased CtIP mobility on SDS-PAGE to a similar level -GST fragment containing the FHA/BRCT domains with GST tagged at the C-terminus, but not the corresponding FHA/BRCT mutants Nbs1-(1–335)-R28A/K160M-GST and Nbs1-(1–335)-RRHK-GST, bind CtIP efficiently , top. FuWe also found that Nbs1-(1–335), but not Nbs1-(1–335)-RRHK interacts with the middle region of CtIP [CtIP-(200–600)], and mutating all CDK sites (7A+S327A) in this CtIP-(200–600) fragment abolished the interactions  top. On To further analyze CDK-phosphorylation-mediated interaction of CtIP with the FHA/BRCT domains of Nbs1, we mutated different CDK sites in the CtIP-(200–600) fragment. While the CtIP- (200–600)-S549A/S568A mutant still binds to the Nbs1 FHA/BRCT domains [Nbs1-(1–335)-GST], the CtIP-(200–600) fragment containing the 7A-CDK or 5A-CDK mutation fails to bind , top, suin vitro ATM kinase assay  as well as ATM-mediated phosphorylation to regulate CtIP function in DSB repair supports the notion that CtIP is a key regulator for cell cycle-dependent selection of DSB repair pathways. The regulated interaction of CtIP and Nbs1 to modulate ATM-mediated phosphorylation of CtIP also provides new information as to how these proteins function coordinately with each other to promote appropriate DSB repair pathways during the cell cycle to avoid genome instability.2. Insect cell line Sf21 was cultured in Grace's insect medium (Gibco) supplemented with 10% fetal bovine serum; Sf9 insect cell line was cultured in Sf-900II SFM medium (Gibco). T98G cells were synchronized via serum starvation by culturing cells in DMEM containing 0.1% FBS for 48 hours, followed by release into complete medium containing 10% FBS Human U2OS, T98G, HeLa and 293T cells were cultured at 37°C in Dulbecco's modified eagle's medium with 10% fetal bovine serum (FBS) in the presence of antibiotics and 5% COMonoclonal anti-CtIP antibody was generously provided by R. Bear . The kinase reaction was conducted at 30°C for 30 min. Phosphorylated proteins were separated by SDS-PAGE and visualized using a PhophorImage scanner . To test the effects of Nbs1 WT and mutants on CtIP phosphorylation by ATM in vitro, indicated amounts of CtIP and Nbs1 proteins were pre-incubated for 15 min on ice and then combined with Flag-ATM immunoprecipitates and γ-32P-ATP to perform the ATM kinase reaction.GST-fused CtIP C-terminal region (residues 750–897) was expressed in BL21 and affinity-purified with glutathione-Sepharose 4B . CtIP wild-type and indicated mutants were expressed in Sf9 insect cells and purified by Ni-NTA and GST tandem affinity purification. C-terminal His-tagged Nbs1 wild-type and indicated mutant were expressed in Sf9 insect cells and purified by Ni-NTA column followed by MonoQ column. ATM Mass spectrometry was described before Proteins were dissolved in 60 µL of 100 mM Tris-HCl, pH 8.5 containing 8 M urea. The protein was reduced by adding 500 mM tris (2-carboxyethyl) phosphine to a final conc. of 5 mM, followed by carboxyamidomethylation of cysteines with 500 mM iodoacetamide . Protein was prepared for analysis of phosphorylation as described For TiO2 enrichment of phosphopeptide, a TiO2 column was made by pressure-slurry packing TiO2  into fused-silica capillary (250 µm i.d.). The column was washed with buffer A [water/acetonitrile/formic acid ] and buffer B [water/acetonitrile/formic acid ], then eluted using 250 mM ammonium bicarbonate onto an analytical column, which was then inserted into an Agilent 1200 quaternary HPLC pump for mass spectrometry analysis.Data-dependent tandem mass spectrometry (MS/MS) analysis was performed with a LTQ-Orbitrap mass spectrometer (ThermoFisher). Full MS and tandem mass spectra were extracted and searched against an EBI-IPI human protein database . A decoy database containing reversed sequences ProLuCID searches were performed on an Intel Xeon 80-processor cluster (search tolerance set to 3 Da). The Cysteine mass was modified by +57.02146 Da to account for carboxyamidomethylation, and Serine, Threonine and Tyrosine were modified by +79.9663 Da for phosphorylation. Since no enzymatic cleavage conditions were imposed, the search included all candidates within the mass tolerance window, regardless of tryptic status Figure S132P] ATP in the presence or absence of ATM kinase (immunoprecipitated from 293T by anti-Flag M2 beads). The radiolabeled CtIP was visualized following SDS-PAGE. Coomassie blue staining shows input of purified CtIP. B. Schematic representation of the EGFP-based HR repair assay substrate, EGFP-HR, as previously described A. CtIP is phosphorylated by ATM. Purified CtIP protein (1 µg) was incubated with [γ-(TIF)Click here for additional data file.Figure S2T98G cells were synchronized in G0 by serum starvation, followed by release into complete media for 10 hr or 20 hr to obtain G1- or S-phase population cells, respectively. T98G cells, asynchronous (Asyn) or synchronized in G1 (10 hr) or S (20 hr), were treated with or without IR (10 Gy) at indicated time points after releasing from G0. Western blot analysis was performed using anti-CtIP antibody. The cell cycle profiles were determined by fluorescence activated cell sorted (FACS) analysis of propidium iodide stained cells.(TIF)Click here for additional data file.Figure S3Left: Schematic representation of the FHA and BRCT domains on Nbs1 and the Nbs1 mutants generated, including N-terminus (1–335) and C-terminus (336-end) truncation mutants, and/or point mutations in the FHA and BRCT domains. Interaction domain, i.d. Right: Purified Nbs1 WT and indicated mutant proteins were analyzed by SDS-PAGE gel with Coomassie blue staining. B. Purified Nbs1(1–335) or Nbs1(1–335)-RRHK (A. 35)-RRHK  were inc(TIF)Click here for additional data file.Figure S4Top: Schematic representation of full-length CtIP with twelve putative CDK consensus sites (SP/TP) indicated, and Flag-tagged N-terminal, middle and C-terminal CtIP fragments . Bottom: Sf21 insect cells were co-infected with baculoviruses expressing Nbs1-1–335-GST and indicated Flag-CtIP fragments with or without indicated CDK-site mutations. GST pull-down experiments were carried out, and immunoblotting was performed using anti-Flag M2 antibody. B. The C-terminus of Nbs1 (336-end) interacts with the C-terminus of CtIP (462-end). Top: Schematic representation of CtIP full-length and GST-tagged CtIP fragments . Bottom: Sf21 insect cells were co-infected with baculoviruses expressing Flag-Nbs1 336-end, and GST-CtIP 1–461 or GST-CtIP 462-end fragments. GST pull-down experiments were carried out, and immunoblotting was performed using anti-Flag M2 antibody. C. CtIP phospho-mimic mutants 8E-ATM  and T859E were generated and co-expressed with Flag-tagged Nbs1 WT (full length) or 336-end in Sf21 insect cells by baculovirus infection, followed by GST pull-down or anti-M2 IP experiments and immunoblotting. Top, Both CtIP-WT and 8E-ATM mutant interact with full-length Nbs1. Bottom, T859E mutation does not affect the interaction between CtIP and Nbs1 C-terminus.A. The FHA/BRCT domains of Nbs1 (1–335) interact with the middle region of CtIP (200–600) in a CDK-phosphorylation dependent manner. (TIF)Click here for additional data file.Figure S5Left, Three serine site mutations, S233, S276, and S347, abolished the interaction between CtIP 200–600 with Nbs1 FHA domain mutant (Nbs1 1–335 R28A). Right, Two threonine site mutations, T245 and T315, reduced the interactions between CtIP 200–600 and Nbs1 BRCT domain mutant (Nbs1 1–335 K160M). B. Phospho-binding sites in the Nbs1 FHA/BRCT domains are important for its interaction with CtIP-(200–600) fragment. Sf21 insect cells were co-infected with baculoviruses co-expressing Nbs1-1–335-GST WT or indicated mutants with Flag-CtIP 200–600 fragment. GST pull-down experiments were carried out, and immunoblotting was performed using anti-Flag M2 antibody.A. Indicated Nbs1 and CtIP variants were co-expressed in Sf21 insect cells by baculovirus infection, followed by GST pull-down experiments and immunoblotting. (TIF)Click here for additional data file.Figure S6A. Overexpression of MDC1 does not affect CtIP hyper-phosphorylation by ATM. U2OS cells expressing vector control or HA-tagged MDC1 were treated with or without IR , lysed, and immunoblotting was performed with indicated antibodies. B. Schematic drawing of the EGFP-MMEJ repair assay substrate, as previously described (TIF)Click here for additional data file.Table S1Phosphorylated peptides of endogenous CtIP (from HeLa cells) were identified by mass spectrometry. All recovered peptide sequences are listed, with phosphorylated residues labeled with (p).(PDF)Click here for additional data file.Table S2Summary of phosphorylated peptides recovered by mass spectrometry analysis performed using HeLa cells expressing endogenous CtIP and SF9 insect cells expressing recombinant human CtIP (hCtIP). A plus (+) sign indicates that peptides were recovered from indicated, putative SP/TP sites, and a minus (-) sign indicates that peptides were not recovered from indicated site(s).(PDF)Click here for additional data file.


We report a case of a very fast and intriguing decrease in IgE concentrations after exclusion from the diet of any CM lysate in an unusual clinical presentation of cow's milk allergy in an infant. Analysis of IgE kinetics after allergen elimination suggests rapid cessation of IgE biosynthesis and a short IgE half-life. An unusual clinical presentation of cow's milk allergy (CMA) in an infant gave us the opportunity to observe a very fast and intriguing decrease of IgE concentrations after allergen withdrawal, raising new questions about IgE production and metabolism in newborns and infants. A 17-day-old neonate, born to atopic parents at full term, was hospitalized in the children hospital of Tours (France) for poor feeding and increasing diarrhoea for 4 days, associated with a severe metabolic acidosis, after a symptom-free interval of 2 weeks. The diarrhoea ceased soon after admission. Erythema and a pustular rash of the face as well as a “gloves and socks-” type skin rash were noted at 18 days of age, leading to the finding of very high concentrations of total IgE (1298 kIU/L) and IgE specific to cow's milk (CM)  . First-sThe IgE detected in the newborn had been self-produced since IgE did not cross the placenta and his mother had no CM-specific IgE (not shown). This IgE immune response thus probably reflected a rapid maturation of IgE+ B cells into plasmablasts, as recently evidenced in mice . No lessC(t)) = ln (Cn) − ke · t, where C(t) is the IgE concentration over time (kIU/L), Cn is the estimated IgE concentration just after CM withdrawal, ke (day−1) is the first-order elimination constant, and t (day) is time. The ke parameter was estimated using log-linear regression. The elimination half-life (T1/2 ) was calculated as T1/2 = ln (2)/ke and was 1.7 days in this infant. The combined kinetics of IgE and omalizumab, an anti-IgE humanized monoclonal antibody, were previously described in adults using PK-PD modelling [T1/2 values (CI901/2T), which was (1.5–3.8) days. Although low, the T1/2 of IgE of our infant was within this CI901/2T and thus has to be considered as “normal.” However, to calculate this elimination kinetics, it was postulated that IgE production ceased after CM withdrawal, which is not necessarily the case. It is therefore possible that the true IgE T1/2 of this patient may be shorter than 1.7 days.Pharmacokinetic modelling techniques were used to describe the elimination kinetics of total IgE in this infant. The kinetics model was ln (odelling . The parNevertheless, our findings suggest that the IgE production stopped rapidly—if not immediately—after allergen elimination, suggesting that a very limited IgE plasmablast life span combined with the very short IgE half-life. Whether this hypothetical IgE plasmablast short life span is restricted to IgE or whether it is due a more general immaturity of lymphoid niches in the newborn, as suggested in murine models , warrant