Even though Ser814 is within a consensus site for casein kinase II (CK2), we showed that CK2 is not involved in the phosphorylation of TRPC6 and does not modify its activity. In summary, we identified a new basal phosphorylation site (Ser814) on TRPC6 and showed that CK2 is not responsible for the phosphorylation of this site.TRPC are nonselective cation channels involved in calcium entry. Their regulation by phosphorylation has been shown to modulate their routing and activity. TRPC6 activity increases following phosphorylation by Fyn, and is inhibited by protein kinase G and protein kinase C. A previous study by our group showed that TRPC6 is phosphorylated under unstimulated conditions in a human embryonic kidney cells line (HEK293). To investigate the mechanism responsible for this phosphorylation, we used a MS/MS approach combined with metabolic labeling and showed that the serine at position 814 is phosphorylated in unstimulated cells. The mutation of Ser Drosophila melanogaster. Up to 28 mammalian isoforms of dTRP have been cloned so far and have been distributed into six subfamilies  2+ entry as long as the stimulation is maintained. TRPC6 is one of these Ca2+ entry channels Calcium homeostasis is crucial for every cell type, and its tight regulation allows intracellular calcium to be a widely used second messenger 69, thus decreasing its channel activity 768448448 decreases its activity and that TRPC6 is phosphorylated under basal conditions 814 was phosphorylated and contributed to 50% of the basal phosphorylation state of TRPC6. Surprisingly, mutant TRPC6S814A displayed an activity similar to that of wild-type TRPC6. As Ser814 is within a consensus sequence for phosphorylation by CK2, we used two CK2 inhibitors to investigate the possible involvement of this kinase. However, the inhibition of CK2 did not modify the phosphorylation state or activity of TRPC6. Thus, we have identified a new phosphorylation site on TRPC6.Post-translational modification of TRPCs has been shown to influence their activity and routing. Following EGFR activation, TRPC6 is phosphorylated by Fyn, a Src family protein tyrosine kinase. Phosphorylation by Fyn allows optimal activation of TRPC6 814, located in the C-terminus, was phosphorylated with an Ascore of 57.75. An Ascore of 20 or more means that the phosphorylation is on that particular residue with a probability of 99%, and not on another serine, threonine, or tyrosine that may be present on the peptide 814.HEK293 cells stably expressing TRPC6 (HEK293 T6.11) were solubilized, and TRPC6 was immunoprecipitated using an anti-hemagglutinin (HA) antibody and size fractionated by SDS-PAGE. The gel was stained with colloidal Brilliant Blue, and the bands corresponding to TRPC6  were exc814 was phosphorylated under unstimulated conditions, we substituted it for an alanine. HEK293 cells were transiently transfected with the HA-tagged TRPC6 or TRPC6S814A mutant and metabolically labeled with inorganic 32P. HA-tagged proteins were then immunoprecipitated with an anti-HA antibody and size fractionated by SDS-PAGE. The phosphorylation states of the proteins were revealed by autoradiography. Under basal conditions, the level of phosphorylation of mutant TRPC6S814A corresponded to 52.8±28.0% of that of WT TRPC6 . Once the [Ca2+]i had returned to the basal level (2 min after the addition of CCh), the extracellular medium was replaced with medium containing 1.8 mM CaCl2. 2+, CCh-induced Ca2+ release was similar in TRPC6-, TRPC6S814A-, and mock-transfected cells. When 1.8 mM CaCl2 was added to TRPC6- and TRPC6S814A-transfected cells, Ca2+ entry raised the [Ca2+]i to a plateau level of approximately 250 nM and 260 nM, respectively . Expression of TRPC6S814A gave similar results, with an increase of CCh-induced net Ca2+ entry reaching 131±17% (n  =  6). The substitution of Ser814 for Ala did not modify the level of expression of TRPC6 nor its amount at the plasma membrane  814 of TRPC6 under basal conditions, we used two selective CK2 inhibitors, DMAT  and TBCA ((E)-3-acrylic acid). T6.11 cells were metabolically labeled with inorganic 32P for 4 h in the presence of either 10 µM DMAT or 10 µM TBCA. TRPC6 was then immunoprecipitated with an anti-HA antibody and size fractionated by SDS-PAGE, and its phosphorylation state was revealed by autoradiography. An analysis of the amino acid sequence surrounding Ser2+ signaling. In T6.11 cells, incubation for 4 h with DMAT and TBCA potentiated CCh-induced Ca2+ release by approximately 30%. However, after calcium restoration, DMAT and TBCA did not significantly alter CCh-induced Ca2+ entry -induced Ca2+ entry is mostly through TRPC6 2+ release (data not shown) or AVP-induced Ca2+ entry . Further studies are needed to verify whether one of these residues is phosphorylated under unstimulated conditions.Basal phosphorylation of TRPC6 has been previously observed in resting platelets and in TRPC6 stably expressed in HEK293 cells Drosophila TRP is phosphorylated under unstimulated (dark) conditions 721 and Ser936 are highly phosphorylated in the dark, and that Ser936 is dephosphorylated in the presence of light, a phenomenon that requires the activation of the phototransduction cascade. Our study showed that Ser814 of TRPC6 is phosphorylated under unstimulated conditions. However, the mutation of Ser814 into Ala did not significantly modify the activity, the level of expression, nor the trafficking of TRPC6. Ser814 is localized in a highly hydrophilic region within the C-terminus of TRPC6, between the proline rich motif (amino acids 759-770), which binds FKBP12 814 might play a role in the interaction of those proteins with TRPC6 or the folding and trafficking of the protein, which could be masked in recombinant cell model. To demonstrate the significance of this phosphorylation event in a non-recombinant cell model, we need to identify the kinase involved in the phosphorylation of Ser814. A sequence analysis of TRPC6 revealed that Ser814 is within a consensus site for phosphorylation by CK2 (S/T-X-X-D/E). Furthermore, CK2 favors an acidic environment as indicated by the results of Meggio et al., who showed that any acidic residue in positions −2 to +7 favors phosphorylation by CK2 814 constitute a favorable acidic environment for CK2. CK2 is also known to exhibit constitutive activity, making it a potential kinase for the basal phosphorylation of Ser814. Moreover, the subcellular location and functioning of TRPP2 is modulated by the phosphorylation by CK2 S814A. We also verified whether CK2 could indirectly regulate Ca2+ signaling in TRPC6-expressing cells. It has previously been shown that the calcium-sensitive potassium channel KCa2.2 loses Ca2+ sensitivity following calmodulin phosphorylation by CK2 2+ entry into HEK293 cells stably expressing TRPC6 (T6.11) despite the fact that it does enhance CCh-induced Ca2+ release. Similarly, in A7r5 cells expressing high levels of TRPC6, the inhibition of CK2 with DMAT did not affect AVP-induced Ca2+ release or Ca2+ entry. We thus showed that CK2 does not modulate the activity of TRPC6.A recent study, which used an MS/MS approach similar to ours, revealed that 814 under unstimulated conditions and to identify the kinase responsible for this phosphorylation.In summary, we showed that the serine in position 814 of TRPC6 is phosphorylated under basal conditions in HEK293 cells, and that it accounts for one of two distinct basal phosphorylation sites. Our results further showed that CK2 is not the kinase responsible for these phosphorylations, and that CK2 does not directly or indirectly modify TRPC6 activity. Further studies are needed to demonstrate the significance of the phosphorylation of Ser32P-orthophosphoric acid were from Perkin–Elmer Life Sciences . All primers and oligonucleotides were from Integrated DNA Technologies . Phusion High-Fidelity DNA polymerase was from Finnzymes . Restriction enzymes, PNGase F, and T4 DNA ligase were from New England Biolabs . Unless otherwise stated, all other reagents were from Sigma  or Laboratoire MAT .Cell culture media, serum, Hepes, trypsin, Opti-MEM I, LipofectAMINE 2000, and Zero Blunt Topo PCR cloning kits were purchased from Invitrogen . Phosphate-free culture media and G418 were from Wisent . NP-40 was from Roche . HEK293 and A7r5 cell lines were from ATCC . DMAT, TBCA, CCh, AVP, phosphatase cocktail inhibitor set #2, and fura 2/AM were from Calbiochem . Rabbit polyclonal and mouse monoclonal anti-HA-specific antibodies were from Covance . Peroxidase-conjugated donkey anti-rabbit antibodies, peroxidase-conjugated sheep anti-mouse antibodies, protein A-sepharose CL-4B, and Biomax MR films were from GE Healthcare . Western Lightning Chemiluminescence Reagent Plus, 0.2 µm nitrocellulose membranes, and et al.et al.Excised acrylamide bands containing immunoprecipitated and separated TRPC6 were sent to the Proteomics Platform of the Eastern Quebec Genomics Center  for MS/MS analysis. Proteins were extracted from gels, placed in 96-well plates, and washed with water. Tryptic digestion was performed using a MassPrep liquid handling robot  according to the manufacturer's specifications and the protocol of Shevchenko Peptide samples were separated by online reversed-phase nanoscale capillary liquid chromatography and analyzed by electrospray mass spectrometry. The experiments were performed using a Thermo Surveyor MS pump connected to a LTQ linear ion trap mass spectrometer  equipped with a nanoelectrospray ion source (ThermoFisher). The peptides were separated on a PicoFrit column  with a 2-50% linear gradient of solvent B  for 30 min at a flow rate of 200 nL/min (obtained by flow-splitting). Mass spectra were acquired using a data dependent acquisition mode using Xcalibur software version 2.0. Each full scan mass spectrum (400 to 2000 m/z) was followed by collision-induced dissociation of the seven most intense ions. The dynamic exclusion (30 s exclusion duration) function was enabled, and the relative collisional fragmentation energy was set at 35%. All MS/MS samples were analyzed using Mascot  set up to search for the sequence of mouse TRPC6 assuming trypsin as the digestion enzyme. Mascot was searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 2.0 Da. The iodoacetamide derivative of cysteine was specified as a fixed modification, and the oxidation of methionine and the phosphorylation of serine, threonine, and tyrosine were specified as variable modifications. Four missed cleavages were allowed.Scaffold  was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they exceeded 95% probability as specified by the Peptide Prophet algorithm Standard molecular biology techniques were used to isolate, analyze, and clone DNA 2. T6.11 cells (HEK293 stably transfected with mouse TRPC6) 5 HEK293T cells diluted in 1.5 ml of culture medium without antibiotics were added to each well. The cells were incubated for 16 h at 37°C in a humidified atmosphere containing 5% CO2. Twenty-four hours after the transfection, the cells from one well were transferred into a 60-mm Petri dish for metabolic labeling assays or deposited on three poly-L-lysine-treated coverslips for [Ca2+]i measurements.HEK293T cells and A7r5 vascular myocytes were maintained at subconfluence in DMEM supplemented with 10% fetal bovine serum, 50 U/ml of penicillin, and 50 µg/ml of streptomycin at 37°C in a humidified atmosphere containing 5% CO32P-inorganic phosphate. The cells were then washed twice on ice with ice-cold PBS-EDTA  prior to being lysed.Stably or transiently transfected cells grown in 60 mm Petri dishes were washed once with phosphate-free DMEM and incubated for 4 h in phosphate-free DMEM supplemented with 250 µCi/ml of The cells were lysed with 1 ml of ice-cold lysis buffer  supplemented with a phosphatase inhibitor cocktail for 30 min on ice with gentle agitation. They were then scraped from the surface of the Petri dish and centrifuged at 15 000×g for 15 min at 4°C. The supernatant was collected and immunoprecipitated with 50 µl of protein A-sepharose beads (50% slurry) and anti-HA rabbit antibody (1∶1000) for 2 h at 4°C. The samples were then centrifuged for 1 min at 4°C at 800×g and washed twice with 500 µl of ice-cold lysis buffer. Immunoprecipitated proteins were dissolved in 40 µl of 2X Laemmli buffer and boiled for 5 min before being separated on 8% SDS-polyacrylamide gels. The gels were then either stained, dried, and exposed to a film for autoradiography, or the protein bands were transferred onto a 0.2 µm nitrocellulose membrane  for immunoblotting. For MS/MS analyses, stained bands corresponding to TRPC6 were excised.The nitrocellulose membranes to which the whole cell lysates and immunoprecipitated proteins had been transferred were stained with Ponceau S (0.1% in 5% acetic acid) to visualize the marker proteins, destained in TBST  and blocked in TBST containing 5% (w/v) nonfat dry milk for either 1 h at room temperature or overnight at 4°C. The membranes were then washed and incubated in TBST for 2.5 h at room temperature or overnight at 4°C with specific primary antibodies (mouse anti-HA 1∶1000). After three washes with TBST, the membranes were incubated for 1.5 h at room temperature with TBST containing 1∶10 000 peroxidase-conjugated sheep anti-mouse IgG. The blots were washed three times with TBST, and the immune complexes were detected with a Western Lightning Chemiluminescence Reagent Plus kit using the manufacturer's protocol.et al. 2+]i. Briefly, T6.11 4, 10 mM glucose, 1.8 mM CaCl2, 20 mM Hepes, pH 7.4) and loaded with fura 2/AM (1.5 µM in HBSS) for 20 min at room temperature in the dark. After washing and de-esterifying in fresh HBSS for 20 min at room temperature, the coverslips were inserted in a circular open-bottom chamber and placed on the stage of a Zeiss Axovert microscope fitted with an Attofluor Digital Imaging and Photometry System . Isolated fura 2-loaded cells were selected and their [Ca2+]i were measured by fluorescence videomicroscopy at room temperature using alternating excitation wavelengths of 334 and 380 nm (10 nm bandpass filters). Emitted fluorescence was monitored through a 510 nm dichroic mirror and a 520 nm long pass filter set. Free [Ca2+]i was calculated from the 334/380 fluorescence ratio using the method of Grynkiewicz et al. 2+-free HBSS was supplemented with 0.5 mM EGTA to chelate any remaining extracellular Ca2+. For the transient transfections, cells were co-transfected with cDNA encoding the M5 muscarinic receptor, and only those responding to CCh were analyzed. [Ca2+]i values were recorded every 3 s.We used the method described by Zhu


Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. Throughout the planet, environmental temperatures can reach low to freezing levels. Organisms indigenous to these habitats are presented with potential desiccation, which can lead to potentially detrimental challenges such as decreased enzymatic rates, freezing, and aggregation of endogenous proteins , 2. BesiThe temperature at which water freezes varies based on solution homogeneity . Pure waCellular cryodamage incurred from freezing is dependent on freezing rate and ice crystal location . For intOne line of defense against cold temperature stress is the production of small molecules that can act as cryoprotectants , 7. CommAn alternate strategy of cryoprotection is the production of antifreeze and ice nucleation proteins , 8. BothAcinetobacter or Bacillus. On the other hand, antifreeze proteins (AFPs) are a class of proteins characterized by the ability to bind to embryonic ice crystal and inhibit further growth , 7229, 7Antifreeze proteins have been proposed to inhibit ice growth through an adsorption inhibition mechanism based on the Kelvin effect . AccordiHowever, the adsorption inhibition mechanism has one major assumption: irreversible binding of antifreeze proteins . The proHowever, Pertaya and colleagues  argue thα-helices to large ordered β-solenoids [The efficient inhibition of ice crystal growth requires a large variety of ice binding surfaces complementary to the many ice crystal planes. This diversity in binding can provide ideal coverage of the ice crystal . Adaptinolenoids , 77.α -helix or extensive hydrogen bonding network in the protein core to retain rigidity [There are some similarities amongst antifreeze proteins. One similarity is a flat, hydrophobic ice binding surface that is a common antifreeze protein identifier. With this surface, antifreeze proteins may even be globular . In orderigidity , 29.A second common feature amongst antifreeze proteins is the presence of amino acid tandem repeats in the protein sequence. These repeats are non-specific but are structured so that ice binding residues are aligned on one side to create an ice binding face , 29, 76.With these similarities in mind, Doxey et al.  were ablMpAFP from Marinomonas primoryensis [MpAFP consists of five distinct domains, of which two (Regions II and IV) contain repetitive sequences. Region II has 120 perfectly conserved 104 amino acid tandem repeats. Region IV is a smaller repeat region comprised of 13 tandem repeats of 19 amino acids each and was the domain confirmed with antifreeze activity.Currently, the most well characterized bacterial antifreeze protein structure is oryensis . MpAFP cβ-roll [β-roll loop each, these tandem repeats form a calcium binding turn along the length of the domain when X-Gly-Thr-Gly-Asn-Asp is aligned , and a C terminal domain . These pPseudomonas syringae was destabilized by ozone treatment, the resultant ice nucleation activity was lowered from −2.8°C to −7.3°C [P. syringae with lipases, detergents, and organic solvents. However, ice nucleation activity could be recovered with phospholipid supplements. Yu and colleagues [Pantoea ananatis ice nucleation protein was expressed in Escherichia coli missing FabH, a protein responsible for fatty acid elongation, there was reduced ice nucleation activity. This activity loss was also recoverable by overexpressing FabH.The relatively hydrophobic and globular N-terminal domain, comprising 15% of the protein, is hypothesized to bind to lipids, polysaccharides, and other ice nucleation proteins . Interaco −7.3°C . A similo −7.3°C  treated lleagues  also demInvestigating the N-terminal domain specifically in InaQ, Li and colleagues  were ablPseudomonas syringae, the consensus is Ala-Gln-Glu-Gly-Ser-Asn-Leu-Thr-Ala-Gly-Tyr-Gly-Ser-Thr-Gly-Thr-Ala-Gly-Ala-Asp-Ser-Ser-Leu-Ile-Ala-Gly-Tyr-Gly-Ser-Thr-Gln-Thr-Ser-Gly-Ser-Glu-Ser-Ser-Leu-Thr-Ala-Gly-Tyr-Gly-Ser-Thr-Gln-Thr. This fragment can be broken into three mid-fidelity fragments of 16 residues each. In PbINP from Pseudomonas borealis, the consensus of these fragments is Gly-Tyr-Gly-Ser-Thr-X-Thr-Ala-X-X-X-Ser-X-Leu-X-Ala [Xanthomonas campestris, Kumaki and colleagues [β-turns. These 16 residue fragments can be further broken down into two low fidelity octapeptides, which are speculated to influence the bends in CRD folding.As the bulk of the protein, the central repeating domain is proposed to be the site of ice interaction . There aeu-X-Ala . When thlleagues  found thβ-conformation with alternating β-strands and random coils linked by interpeptide bonds. They speculated that stacking of β-structures in a hairpin manner produced a rigid structure with alternating patterns of rectangular and trapezoidal structures. Unlimited aggregation was believed to occur by interdigitization of similar sides of another ice nucleation protein . More recently, Graether and Jia [β-helix using a Thr-X-Thr motif for ice binding similar to antifreeze proteins. An ice nucleation protein from X. campestris was speculated to form a β helix structure based on 16-residue loops allowing the alignment of Thr-X-Thr motifs [Two decades ago, Kajava and Lindow  proposed and Jia  argue thr motifs . Howeverr motifs , 42.PbINP as a right handed β-helical structure that is stabilized by inward facing Ser and Gly ladders. The protein dimerizes via desolvation of the aromatic side chains from an exposed Tyr ladder on each monomer (Further supporting Graether and Jia's  hypothes monomer . DimerizIn the last 15% of the protein, the function of the globular C-terminal domain remains undetermined and unexplored . Li et aIce binding depends on the ability to recognize and interact with ice crystal planes. Typically, ice crystal controlling proteins are comprised of a flat, hydrophobic ice binding surface , 72, 77.Currently, a detailed mechanism for adsorption inhibition is continuously evolving. Initially, hydrogen binding between the complementary ice binding surface and ice crystal planes was hypothesized to arrest ice growth . HoweverA secondary hypothesis relies on entropy, van der Waals forces, and hydrophobic interactions for ice binding. It has been speculated that non-anchored clathrate waters within the troughs of ice binding surface are released immediately prior to adsorption , 72. To An alternate hypothesis proposes that anchored clathrate waters influence the hydration layers of the ice crystal controlling proteins . These lIce nucleation activity does not utilize the adsorption inhibition mechanism. However, Garnham and colleagues  speculatPseudomonas putida GR12-2 remained consistent following entry into the stationary phase, a growth stage associated with stress and cell maintenance. Similarly, ice nucleation proteins have also been reported to be expressed at high levels under conditions of cold temperature [Pseudomonas syringae occurred from a combination of cold temperature and nutrient starvation, which is indicative of the stationary phase. However in another case, Chen and coworkers [Pseudomonas fluorescens MACK-4 showed optimal expression following cold temperature growth with sorbitol, mannose or starch, and peptone. This suggests that ice crystal controlling protein expression may be dependent on the bacterium studied, with cold temperature being a common inducer. Understandably, cold expression is required as some ice crystal controlling proteins have been reported to be thermolabile, becoming inoperable at regular bacterial growth temperatures [Knowledge of the regulation of ice crystal controlling protein expression is limited. Typically, these proteins are induced under cold stress and can be expressed seasonally , 7, 27. perature , 88. In perature  shows theratures .However, this temperature-based induction is not very well understood. One model proposes that ice crystal controlling protein induction occurs as a response to upregulation of cryoprotective genes by a cold sensor . A cold Regulation of ice crystal controlling proteins also occurs at the posttranslational level. N-terminal acetylation and/or C-terminal amidation have been found for some ice crystal controlling proteins . In otheAntifreeze and ice nucleation protein production is a common strategy for the survival of various organisms in cold environments. The diversity between organisms suggests that due to convergent evolution the antifreeze protein expressed in a particular system may be unique to that organism or environment. Regardless, the ice crystal controlling nature of these proteins has been exploited in many biotechnical applications over the last two decades.The most common application for antifreeze protein is the enhancement of cryopreservation of biological materials. Antifreeze protein treatments protect against freezing damage to cell membranes and their associated proteins by preventing large ice crystals or spicules development , 91. In Recently, antifreeze proteins have been considered for non-preservation based applications including use as hydrocarbon hydrate inhibitors and biopolymers , 98. MosPseudomonas syringae, have been used in the recreation and food industries. These environmentally safe and efficient proteins have replaced inorganic equivalents such as silver iodide [Ice nucleation proteins, mainly from r iodide . Able tor iodide .ipdC and iaaM genes [Outside of the industry, ice nucleation proteins have research applications as well. Exploiting the cell surface anchoring capabilities, recombinant proteins linked to ice nucleation proteins have been expressed as fusion proteins to be displayed on the cell surface of the expression host . Only liaM genes . TranscraM genes .Enterobacter cloacae expressing an ina gene from Erwinia ananas was shown to increase the freezing temperature of corn borer and cotton bollworm following ingestion [Instead of purified proteins, viable ice nucleation active bacteria are being considered for agricultural use as biocontrol agents for insect pests. Transgenic ngestion . Tang anngestion  report sBoth ice crystal controlling proteins are prevalent globally in a wide variety of psychrophilic bacteria. With a focus on freeze tolerance, bacterial antifreeze proteins are known for a low level of non-colligative freezing point depression with greater emphasis on ice recrystallization inhibition. Superior ice recrystallization inhibition is utilized for improved survivability against freeze-thaw stress common to temperate, non-Antarctic environments. On the other hand, bacterial ice nucleation proteins induce ice crystal formation at high subzero temperatures and can influence precipitation in the atmosphere. For both antifreeze and ice nucleation proteins, only a small number of bacterial sources of these enzymes have been studied in any detail and very few of these bacterial enzymes have been isolated and characterized at the protein structure level. However, the suggested mechanism for both protein classes features the same ice crystal interactions, using an ordered ice-like hydration layer, with protein functions differentiated based on protein size.


The activity of the protein kinase Akt is frequently dysregulated in cancer and is an important factor in the growth and survival of tumour cells. Akt activation involves the phosphorylation of two residues: threonine 308 (Thr308) in the activation loop and serine 473 (Ser473) in the C-terminal hydrophobic motif. Phosphorylation of Ser473 has been extensively studied in tumour samples as a correlate for Akt activity, yet the phosphorylation of Thr308 or of downstream Akt substrates is rarely assessed.The phosphorylation status of Thr308 and Ser473 was compared with that of three separate Akt substrates – PRAS40, TSC2 and TBC1D4 – in fresh frozen samples of early-stage human non-small cell lung cancer (NSCLC).Akt Thr308 phosphorylation correlated with the phosphorylation of each Akt substrate tested, whereas Akt Ser473 phosphorylation did not correlate with the phosphorylation of any of the substrates examined.The phosphorylation of Thr308 is a more reliable biomarker for the protein kinase activity of Akt in tumour samples than Ser473. Any evaluation of the link between Akt phosphorylation or activity in tumour samples and the prediction or prognosis of disease should, therefore, focus on measuring the phosphorylation of Akt on Thr308 and/or at least one downstream Akt substrate, rather than Akt Ser473 phosphorylation alone. The activation of Akt involves the phosphorylation of two residues: threonine 308 (Thr308) in the activation loop of the kinase by the protein kinase PDK1, and serine 473 (Ser473) in the hydrophobic motif by the mTORC2 complex (α/β), tuberous sclerosis complex-2 (TSC2), Foxo-family transcription factors, Bad, Par-4, PRAS40 and TBC1D4 . This leads to the production of phosphoinositidetrisphosphate Pd TBC1D4 . It is t3 phosphatase, PTEN, and mutations in or haploinsufficiency of PTEN are frequently observed in human tumours  or PI3-kinase, as well as Akt itself  phosphorylation of Akt on Ser473 has in some studies been found to correlate with poor prognosis . Donkey horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG antibodies were purchased from Jackson ImmunoResearch Laboratories .Rabbit polyclonal pAkt (Thr308), pAkt (Ser473), pTBC1D4 (Thr642), pPRAS40 (Thr246) and pTSC (Ser939) antibodies were purchased from Cell Signalling Technology . Mouse anti-FPatients with suspected lung cancer were identified from an elective thoracic surgery list at the Bristol Royal Infirmary, Bristol, UK. A Research Nurse contacted study subjects regarding participation and informed consent was given to use their tumour and matched normal lung tissue. The study was approved by the local research ethics committee .The research nurse was present at thoracic surgery to collect the tissue, which was surplus to diagnostic requirements. This was separated into individual samples of tumour and normal tissues, which were immediately snap frozen in liquid nitrogen. A pathologist recorded the histology and stage of the tumour, and made an assessment of the percentage of tumour cells present in each tumour sample. Only those NSCLC tumour samples comprising at least 90% of tumour cells were included further in the study. All samples were stored at −80 °C until required for analysis.The cohort had an average age of 64.5 years, 71% were male, 10% were non-smokers, 10% had accumulated 1–10 pack years, 27% 11–30 pack years and 53% >30 pack years (one pack year being defined as 20 manufactured cigarettes smoked per day for 1 year). Three patients had previously been exposed to asbestos. Following surgery, all tumours were confirmed to be stage I/IIa and histology showed that 53% were adenocarcinomas, 40% squamous cell carcinomas and 3% bronchiolalveloar carcinomas.M Tris pH 7.5, 120 mM NaCl, 1% (v/v) Nonidet-P40, 40 mMβ-glycerophosphate, 1 mM benzamidine, 1 mM EDTA, 50 mM NaF, 10 mM Na4P2O7, 10 μg ml–1 pepstatin, 10 μg ml–1 antipain, 10 μg ml–1 leupeptin, 5 mM Na3VO4 and 2 mM phenylmethylsulphonyl fluoride). Samples were pulsed for 10 s followed by a 30-s incubation on ice; a procedure repeated three times. Tissue lysates were rotated end-over-end at 4 °C for 30 min, and rested on ice for 15 min before centrifugation at 16 000 g for 15 min to remove insoluble material. Protein concentration was determined by BCA assay . Tissue lysates were stored at −80 °C.Frozen tissue samples were homogenised using a Polytron homogeniser in 3 ml ice-cold extraction buffer (50 mμg protein) were solubilised by boiling in 4 × Laemmli sample buffer and separated by SDS–PAGE using 4-12% gradient gels . Proteins were transferred to polyvinylidene difluoride membranes . Membranes were blocked using 5% (w/v) bovine serum albumin in TBS-T  Tween-20) for 1 h, washed five times in TBS-T and then incubated with primary antibody (1 μg ml–1) for 1 h in TBS-T containing 5% w/v bovine serum albumin. The membranes were again washed five times in TBS-T and then incubated with the appropriate secondary antibodies diluted in TBS-T for 1 h. Immunoblots were visualised using the enhanced chemiluminescence (ECL) detection system .Lysates (15 P<0.05). Changes in phosphorylation in tumour samples relative to normal samples were coded with the values 0 (signifying an increase observed at P⩽0.05), 1 (decrease at P⩽0.05) or 2  in the tumour tissue relative to normal. The Spearman's distribution-free rank correlation test was applied to the data to test pair-wise correlations between the phosphorylation of Akt on Thr308 or Ser473 and the phosphorylation of the three Akt substrates PRAS40, TSC2 and TBC1D4. Quantitatively similar results were obtained when analysing the data using the Pearson's correlation coefficient test (data not shown).Three separate samples of tumour tissue and three separate samples of normal tissue from an individual patient were subjected to western blotting and ECL detection. Normal and tumour protein samples from individual patients were separated on different gels and therefore the absolute levels of phosphorylation between patients were not compared. The extent of phosphorylation of each protein was quantified by densitometric scanning of the western blot . Data are expressed as mean±s.e.m., and the strength of evidence for the difference in phosphorylation between the normal and tumour samples was determined by Kruskal–Wallis rank order test , TSC2 (on Ser939) and TBC1D4 (on Thr642).P<0.05; Kruskal–Wallis test) when compared with normal tissue. The magnitude of the change varied, but in four tumours this was in excess of a ten-fold increase. Akt Thr308 phosphorylation was decreased in 8 out of 29 (28%) of tumours (P<0.05); again the magnitude varied but in three tumours this was greater than a ten-fold decrease. In the remaining nine tumours, Akt Thr308 phosphorylation was apparently unchanged .Akt phosphorylation on Thr308 was increased in 12 out of 29 (41%) of tumours (at P<0.05), decreased in 14 out of 29 tumours (48% P<0.05) and unchanged in the remaining 9 tumours, when compared with normal tissue.A similar analysis of the data for Akt phosphorylation on Ser473 showed that it was increased in 6 out of 29 tumours  or decreased (grey) at P>0.05), Ser473 phosphorylation was also unchanged in five but was decreased in the remaining four cases (group III tumours).In seven of the eight cases where Thr308 phosphorylation was decreased, Ser473 phosphorylation was decreased in parallel and these are termed group II tumours. In the nine cases where Thr308 phosphorylation was unchanged  in just 18 out of the 29 cases studied. Given the complex interplay between these sites with respect to regulation of kinase activity, this raised the question of which of Akt Thr308 or Ser473 phosphorylation correlates best with Akt activity.When active, Akt phosphorylates numerous downstream substrates that bring about the pleiotropic biological effects of this kinase. We undertook a detailed examination of the phosphorylation of three well-characterised Akt substrates in the tumour samples: PRAS40 (on Thr246), TSC2 (on Ser939) and TBC1D4 (on Thr642). PRAS40 and TSC2 are both important regulatory components of the mTOR complex. TBC1D4 is a Rab GTPase-activating protein that plays a role in the regulation of glucose metabolism.The data for the phosphorylation of all three substrates are provided in In the nine group Ia tumours, where Akt Thr308 phosphorylation was increased, the phosphorylation of at least one of the three Akt substrates was also increased  and 4A iIn group II tumours, reduced Akt phosphorylation on Thr308 was accompanied by reduced phosphorylation of at least one of the three Akt substrates in all except two tumours  but did not correlate with phosphorylation of Ser473 in the hydrophobic motif (P⩾0.05 in each case).The data in In some tumours, phosphorylation of a substrate was observed to increase under conditions where the phosphorylation of Akt and one or both of PRAS40 and TSC2 were unchanged or decreased. This was most specifically the case for TBC1D4 phosphorylation on Thr642 (hence the lower correlation with Akt Thr308 phosphorylation compared with PRAS40 and TSC2). TBC1D4 Thr642 has also been reported to be a substrate for p90 ribosomal S6 kinase 1 and serum- and glucocorticoid-induced protein kinase 1  also activates Akt but only by approximately ten-fold , presumably as a result of a major conformational change in the enzyme that allows substrate binding and phosphorylation  of the tumours we examined. We did not examine the upstream defects responsible for the activation of Akt; however, we did find that EGFR tyrosine phosphorylation was significantly elevated in 6 of these 12 tumours, suggesting that changes in EGFR activity might have been responsible (data not shown). Clearly, however, other mechanisms will also play a role such as increased activity of the insulin-like growth factor receptor or cMet and PTEN mutation status.It is of interest that Akt Thr308 phosphorylation and activity were reduced in a significant number of tumours (group II; Our observations have important consequences for the future use of Akt as a molecular biomarker in predicting the efficacy of targeted therapy against the PI3-kinase/Akt pathway and its regulators. We propose that any evaluation of the link between Akt phosphorylation in tumour samples and the prediction or prognosis of disease should focus on the phosphorylation of Thr308 and/or at least one downstream Akt substrate, rather than Ser473 phosphorylation alone, in order to more reliably determine the activity of the kinase and thus better guide disease prognosis and treatment. Furthermore, although our current results pertain to NSCLC, they almost certainly have relevance to other tumour types in which this kinase plays a role in tumourigenesis.


These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or “DSB homeostasis”, might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable  Meiosis is a specialized cell division that underpins sexual reproduction. It begins with a diploid cell carrying both parental copies of each chromosome, and ends with four haploid cells, each containing only one copy. An essential feature of meiosis is meiotic recombination, during which the programmed generation of DNA double-strand-breaks (DSBs) is followed by the production of crossover(s) between two parental homologs, which facilitates their correct distribution to daughter nuclei. Failure to generate DSBs leads to errors in homolog disjunction, which produces inviable gametes. Although DSBs are essential for meiosis, each break represents a potentially lethal damage; as such, its formation must be tightly regulated. The evolutionarily conserved ATM/ATR family proteins were implicated in this control; nevertheless, the mechanism by which such control could be implemented remains elusive. Here we demonstrate that Tel1/Mec1 down-regulate meiotic DSB formation by phosphorylating Rec114, an essential component of the Spo11 complex. We also observed that Rec114 activity can be further down-regulated by its removal from chromosomes and subsequent degradation during later stages in meiosis. Evidence presented here provides an insight into the ways in which the number of meiotic DSBs might be maintained at developmentally programmed level. Saccharomyces cerevisiae, where the process is best understood, Spo11 activity requires nine additional proteins, five of which are meiosis specific , and four that are expressed during both meiosis and vegetative growth  In most sexually reproducing organisms, meiotic recombination is initiated by programmed catalysis of DNA double strand breaks (DSBs) by Spo11, an evolutionarily conserved type II topoisomerase-like transesterase Drosophila, it is about 25 Meiotic DSBs are essential for meiosis; nevertheless, each break represents a potentially lethal or mutagenic DNA lesion that must be repaired before the first meiotic division (MI). As such, Spo11 catalysis is tightly regulated at the temporal, spatial, and quantitative levels. For instance, the catalysis does not normally take place until the locus has undergone replication Drosophila and budding yeast homologs, tefu+ and TEL1, respectively, in down-regulating meiotic DSB formation Maintaining the number of meiotic DSBs at the developmentally programmed level would require both positive and negative means of regulating break formation. Although much is known about the genetic requirements for DSB formation Here we present evidence that Rec114, an evolutionarily conserved Spo11-accessory protein and an essential component of the meiotic DSB-machinery S/T]Q cluster domains (SCDs), defined as a region where three or more SQ or TQ motifs are found within a tract of 100 residues or less Budding yeast Tel1 and Mec1, like their mammalian counterparts, ATM and ATR, are serine/threonine kinases REC114, REC114-13xMYC , Hop1, and Zip1 ormation .TEL1/MEC1. To this end, we assessed Rec114 migration patterns in a rad24Δ tel1Δ strain. In a rad24Δ tel1Δ strain, the Tel1/Mec1 signaling is down-regulated to a level comparable to that in mec1Δ tel1Δ cells kept viable by a suppressor mutation, sml1Δ; however, rad24Δ tel1Δ cells do not exhibit the severe meiotic progression defect observed in the latter rad24Δ tel1Δ background , or com1Δ/sae2Δ backgrounds, Spo11 remains covalently bound to the break ends, preventing their further processing. Accumulation of unprocessed meiotic DSBs in these mutants triggers a TEL1-dependent checkpoint response MEC1-mediated checkpoint response rad50S or dmc1Δ background . We found that Rec114 mobility shift was abolished in a rec114-8A dmc1Δ strain (To further address the role(s) of Tel1/Mec1 in Rec114 mobility shift, we examined its migration pattern in a strain expressing a Δ strain , indicatin vivo phosphorylation of Rec114 at a specific residue(s) during normal meiosis, we generated phospho-specific antibodies against three of the eight ATM/ATR consensus sites in Rec114. T175 and S187 were chosen based on their biological relevance  or to a phospho-mimetic aspartic acid (D) residue, respectively. Spore viability of rec114-8A diploids was comparable to that of REC114 in all genetic backgrounds tested  pch2Δ) 8D is more abundant than Rec114 , and also because replacing as few as two (T175 and S187) of the eight consensus sites with a phosphomimetic residue confers a rec114-8D like phenotype with respect to haploinsufficiency and synthetic interaction with spo11-hypomorphic alleles (in vivo phosphorylation sites (To investigate function(s) of Tel1/Mec1 phosphorylation of Rec114, the effect of mutating the S or T residues of the eight Tel1/Mec1 consensus sites was examined. We began the analysis with two s tested . rec114- alleles . Notablyon sites .rec114-phosphomimetic and spo11-hypomorphic alleles, which are known to confer sublethal reductions in crossover (CO) levels rec114-8D on CO-levels at the well characterized HIS4-LEU2 artificial meiotic recombination hotspot /Southern analysis  in CO homeostasis  alleles  may resurec114-8D, down-regulates DSB formation. If so, the absence of the phosphorylation in rec114-8A should lead to an increase in DSB levels, assuming that no other mechanism was acting redundantly. Indeed, a substantial increase could be observed for break sites near YCL064C or YCR048W on ChrIII  limiting break formation, and that it/they is/are yet to be discovered.The dramatic effect of rec114-8D on break level was notably attenuated in a dmc1Δ background compared to rad50S or com1Δ/sae2Δ . Spo11-myc ChIP was performed without the use of formaldehyde (FA) cross-linking to enrich for Spo11 proteins that had remained covalently bound to the break ends upon DNA-cleavage. To ensure the highest degree of comparability between the three REC114/rec114 allele backgrounds, the experiments were performed strictly in parallel for all steps from culturing to the final analysis. The resulting profiles of covalently bound Spo11 in the three backgrounds reproduced the published DSB hotspot profiles rec114-8A is higher than in wild type, while Spo11 in rec114-8D is strongly reduced  on CHIP assay (here on referred to as ChIP-chip), which confers greater resolution and offers easier normalization than a Southern blot based analysis e.g. . In cons reduced .rec114-8D relative to rec114-8A, then the ratio of the Spo11 profiles of rec114-8A over rec114-8D, (hereon referred to as 8A/8D), should define DSB sites. In fact, the correlation between DSB hotspots and the 8A/8D peaks should be greater than that of not-normalized profiles. Indeed, profiles of these ratios identify near 100% of the published DSB hotspots  that excludes that there is significant 8D>8A at DSB sites (data not shown). Even for the smaller difference between WT and 8A, WT/8A produces a clear anti-correlation  also produced a significant, although somewhat weaker, correlation , and all but one overlapped with local Rec1148A maximum in the DSB cluster (e.g. YCR047C) and its neighboring axis site as a function of time, we observed that the extent of increase at the DSB site  should map to DSB sites. Analysis shows that the majority of DSB-sites coincide with 8A/8D peaks . Indeed, comparison of the 500 strongest peaks and 500 hottest hotspots revealed a highly significant correlation (−37). Interestingly, 8A/WT and WT/8D peaks also exhibit significant correlations with DSB sites  suggesting the relation: 8A>WT>8D at DSB sites. Inversion of the DSB anti-correlated 8D profile also lead to the observed positive correlation of WT/8D (−7) and WT/8D ratios (p<.04), lending solid statistical support to the interpretation Rec1148A>Rec114>Rec1148D at the 500 strongest DSB hotspots. Selecting just 100 strongest sites produced similar significances, while selecting more hotspots (3600) results in loss of significance, as the effect of 8A becomes insignificant compared to the effect of 1/8D for weak hotspots . Some meiotic DSBs persist in an r to SPM , while i 5 hours . Thus, tRec1148D , suggestRec1148D  might beAll Spo11-accessory proteins examined to date, including Rec114, are recruited to the chromosomes before the initiation of meiotic recombination, and remain chromosome-associated until Zip1 dependent homolog synapsis takes place ndt80Δ background to exclude any influence by the NDT80 dependent Rec114 degradation (above).In early meiotic prophase, there is little overlap between Rec114 and Zip1; at later stages, Rec114 foci become less abundant and dimmer in synapsed chromosome regions but remain bright in unsynapsed regions of the same nucleus ndt80Δ background behaved as reported 8A -foci decline more rapidly than Rec114, while that of Rec1148D containing nuclei remain abundant until at least 6 hours in SPM   , ii, in NDT80 dependent turnover, suggesting the existence of a feedback system that couples the steady state DSB levels to post-leptotene regulation of Rec114 activity.Rec114 is an evolutionarily conserved essential component of the meiotic DSB-machinery 8D and a subtler increase in function in the rad50S background for Rec1148A. Why are these effects not symmetric? One trivial explanation could be that introducing eight aspartic acid residues into Rec1148D may render the protein partially non-functional, aside from mimicking phosphorylation. However, Rec1148D is a stable protein that binds well to chromatin, excluding general protein stability-, nuclear import- or chromatin-binding defect for Rec1148D. Furthermore, since Rec1148D behaves similar to Rec1142D  in terms of reduction of DSBs, inferred based on reduced spore viability in a spo11-HA background (8D or Rec1142D), while other effects might prevent the observation of a strong increase in break levels under constitutive “on” conditions (e.g. Rec1148A).Three independent studies have implicated a role of the ATM kinases in down-regulating Spo11 catalysis ckground , and tworad50S background. Increased DSB formation in Rec1148A, even if only moderate, identifies Rec114 as a rate limiting target of negative feedback at least in the com1Δ/sae2Δ or rad50s background. On the other hand DSB formation is strongly impeded in Rec1148D (or Rec1142D), suggesting that phosphorylation affects a critical function of Rec114. Importantly, phosphomimicking Rec1148D shows a reduced interaction with DSB-hotspots suggesting a plausible mechanism explaining its reduced activity.Several models e.g.  propose Budding yeast Rec114 physically interacts with Mei4 and Mer2, two other components of the DSB-machinery, to form a complex referred to as RMM (Rec114-Mei4-Mer2) During normal meiosis, steady state levels of meiotic DSBs decrease to the background level as cells proceed beyond pachytene with complete cessation of DSBs depending on Ndt80 rad50S, dmc1Δ or zip1Δ background), Tel1/Mec1 prevent Ndt80 activation by hyper-phosphorylating Hop1/Mek1 NDT80 dependent Rec114 turnover in rec114 phospho-mutants might be under Tel1/Mec1 regulation  signaling of successful completion of homolog synapsis and meiotic DSB repair should precede irreversible global inactivation of the DSB machinery. We present evidence for two feedback based mechanisms of such regulation: synapsis dependent removal and Ndt80 dependent degradation of Rec114. Linking the progression of synapsis to down regulation of DSB formation conveniently ensures that enough DSBs have been formed to guarantee successful homology search. However, synapsis alone does not appear to lead to irreversible inactivation of the DSB machinery. This ultimate decision is instead linked to the exit from pachytene, when the activation of Ndt80 provides some guarantee that early prophase events have successfully completed.rec114-8D mutant, would delay RMM inactivation, likely prolonging its active lifespan and raising the level of DSBs eventually produced. Indeed, Rec1148D remained longer and in greater abundance at chromosome axes than Rec114 or Rec1148A. DSB homeostasis could also account for the apparent ‘catching up’ of break levels in a rec114-8D dmc1Δ strain  might be required to activate the synapsis and/or Ndt80 based feedback loops (Evidence here and elsewhere indicates that cells have the means to prevent excessive DSB formation via negative feedback Δ strain . For insck loops .spo11-hypomorphs) with a low rate of DSB formation that was up to now solely attributed to CO homeostasis. But clearly, postponing the inactivation of the DSB machinery in response to problems in synapsis and break repair helps to provide more DSBs, on which CO homeostasis can act to ensure correct chromosome segregation.DSB homeostasis may contribute significantly to the relatively mild effect on spore viability of mutants  was removed to generate pJC15, an integration plasmid without an epitope tag. Specific [S/T]Q to AQ or DQ mutations were introduced into either pNS2 or pJC15 utilizing the QuickChange Multi Site-Directed Mutagenesis kit (Stratagene). The entire open reading frame (ORF) of each allele was sequenced to ensure that the allele did not contain any incidental mutation(s). Each rec114 allele was introduced into a rec114Δ::KanMX4 haploid strain (RCY336/337), where the endogenous REC114 gene was replaced by a kanamycin resistant gene. Transformants were identified based on their ability to grow on hygromycin plates but not on kanamycin. Southern blot and PCR analyses were performed on candidate colonies to confirm integration of a single copy of a specific rec114-HygroMX4 allele at the endogenous locus, replacing the rec114Δ::KanMX4 allele. Correct rec114 haploid transformants of each allele were taken through standard yeast genetics manipulation to generate corresponding rec114 homozygous diploid strains suitable for meiotic analyses.The myc13 tag from a spo11 hypomorphic alleles .Three of the eight S/T[Q] consensus sites in Rec114, T175, S187 and S256, were selected for generation of phospho-specific antibodies. T175 and S187 were chosen based on the fact that replacing these residues with a non-phosphorylatable alanine (A) confers haploinsufficiency and synthetic interaction with  alleles . S256 wa alleles . Specifits tel1Δ sml1Δmec1-4 meiosis, where the culture was kept at 23°C and shifted to 30°C 2 hours after transferring into sporulation medium (SPM).Induction of synchronous meiosis is carried out according to the established protocols GST-REC114 and GST-rec114-8A plasmid-construction and protein expression were carried out as described 8A. The mixtures were incubated at 30°C for 25 minutes and subjected to electrophoresis on SDS gels. Gels were transferred onto a nitrocellulose membrane and subjected to Western blot analyses using anti-Rec114 or phospho-specific antibodies.Whole-cell extracts (WCE) were prepared from cell suspensions in 20% trichloroacetic acid (TCA) by agitation with glass beads. Precipitated proteins were solubilized in SDS-PAGE sample buffer, and appropriate dilutions were analyzed by SDS-PAGE and Western blotting. Antibodies for Western blotting were mouse monoclonal anti-myc , rabbit polyclonal anti-Rec114 (1∶1000), anti-Phospho-Rec114-S187, anti-Phospho-Rec114- T175, anti-Phospho-Rec114- S265 , goat anti-mouse IgG conjugated to horseradish peroxidase , and donkey anti-rat IgG conjugated to horseradish peroxidase .Southern blot analysis following Pulse Field Gel electrophoresis (PFGE) using DNA prepared in agarose plugs or standard agarose gel electrophoresis were performed as described Rec114 and Spo11-myc chromatin immunoprecipitation (ChIP), quantitative PCR (qPCR), and microarrays hybridization/analysis were performed as described Surface spread meiotic chromosomes were prepared as described http://www.langsrud.com/fisher.htm).For comparing proportions (e.g. matching versus non-matching peaks) significance values were computed using Fisher's exact test Click here for additional data file.Figure S2HIS4-LEU2 hotspots. A. (i) Representative image of a Southern analysis of HIS2-LEU2 artificial recombination hotspot. Relevant DNA fragments are as described in Mimicking Rec114 phosphorylation leads to a modest reduction in DSB levels at (PDF)Click here for additional data file.Figure S38A profiles. A. Spo11-myc profile of a rec114-8A rad50S strain normalized (divided) by Spo11-myc profile of a rec114-8D rad50S strain . Red bars represent Spo11-oligo counts per hotspot cluster rec114-8A normalized (divided) by Rec114 profile of rec114-8D  and REC114 normalized by rec114-8D . Red bars represent Spo11-oligo counts per hotspot cluster 8A/Rec1148D and Rec114/Rec1148D and DSBs. C. At axis sites defined by peaks of the axis protein Hop1 DSB sites match peaks of Spo11-myc and Rec114(PDF)Click here for additional data file.Figure S48A profiles. A. The cumulative fraction of peaks of a specified profile is plotted against the distance from the nearest DSB cluster (in kb). Results of comparison between 3600 DSB sites Genome wide correlation between DSB hotspots and peaks of Spo11-myc and Rec114(PDF)Click here for additional data file.Figure S58A versus Rec1148A at DSB hotspots. Rec114 ChIPchip profiles of REC114 (green), rec114-8A (red), and rec114-8D (blue) are shown for selected regions in ChrVI (A), ChrIX (B), and ChrXI (C). * denotes strong DSB hotspots where Rec114 signal is highest in rec114-8A followed by REC114 and then by rec114-8D (upper panel). Ratio between signals from each mutant over wild type (lower panel) shows that majority of DSB hotspots are loci showing 8A>WT>8D quantitative relationship, while nearby axis shows 8D>WT>8A. For very weak hotspots, however, it is difficult to discern the 8A>WT>8D relationship as the positive effect of rec114-8A becomes insignificant compared to the negative effect of rec114-8D.Additional examples of the mirror-like behavior of Rec114(PDF)Click here for additional data file.Figure S6ndt80 background. A. Representative images of REC114 ndt80Δ cells at t = 6 hours showing persistent Rec114 foci in Zip1 free regions or a PC (stars). B. Fraction of cells showing a polycomplex (PC) in REC114 NDT80 (black columns) or REC114 ndt80Δ (white columns) cells as a function of time. C. Fraction of cells showing a PC at t = 6 hours in the indicated strain background. Majority of PCs contained both Zip1 and Rec114 signals (yellow).Rec114 foci persist in the (PDF)Click here for additional data file.Table S1S. cerevisiae strains used in this study. All strains are MATa/MATα diploids homozygous unless specified. For HIS4LEU2 hotspot recombination assay diploid strains JCY1193 (REC114), JCY1195 (rec114-8AQ) and JCY1197 (rec114-8DQ) were derived from NHY187  and NHY285 .(PDF)Click here for additional data file.Text S1Supplementary methods.(DOCX)Click here for additional data file.


Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS. Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and  A large part of respired O2 in metazoan organisms is utilized by mitochondria in processes directly linked to oxidative phosphorylation. Not surprisingly, mitochondria undergo a time dependent progressive damage during cellular hypoxia and myocardial ischemia/reperfusion. The mitochondrial electron transport chain is one of the principal targets of hypoxia induced oxidative damage [Hypoxic environment characterized by low Oe damage . While ce damage . The mece damage . Complexe damage . Complexe damage . Our pree damage ,10.in vitro by PKA [Protein kinases play an important role in the modulation of oxidative phosphorylation under different physiological and environmental conditions . Phosphoo by PKA . Notablyo by PKA . Similaro by PKA . This pho by PKA . On the o by PKA . 2 species (ROS) in an in vitro reconstituted system [In our previous studies we showed that PKA plays a major role in both hypoxia and ischemia-reperfusion mediated injury to the electron transport chain . Our resd system . Furtherd system . The phod system .While phosphorylation of CcO subunits during hypoxia or myocardial ischemia-reperfusion has been shown to be important in causing mitochondrial and cellular injury, so far there has been no direct evidence to show the role of PKA and its mitochondrial activation under hypoxia. Additionally, since reports from various groups show that subunit phosphorylation may result in both inhibition and activation of CcO under different pathophysiological conditions, it was imperative to investigate the distinctive nature of the kinase activated under hypoxia and myocardial ischemia. In this paper we show an increased sequestration of PKAα subunit in mitochondrial matrix compartment under hypoxia with an accompanying increase in mitochondrial PKA activity by a process that is linked to ROS formation. We propose that the increased mitochondrial matrix PKA under hypoxia and myocardial ischemia mediates the loss of CcO subunits and activity. Finally, we show that replacement of wild type Vb subunit of CcO with phosphorylation resistant mutant reduces hypoxia-induced degradation and loss of activity. 2) or normoxic (21% O2) conditions for indicated durations. For hypoxia, growth medium was pre-equilibrated at 1% O2 in hypoxia glove box  overnight before adding to the cells. Cells were maintained under hypoxia for specified lengths of time. Harvesting and fractionation of cells at the end of hypoxia were performed at normoxic conditions. H89, MPI, KH7, N-Acetyl Cysteine (NAC) and Mito-CP were added at indicated concentrations to cells at the start of hypoxia inside the hypoxia chamber. Mito-CP is Mito-carboxy proxyl and is a mitochondria targeted nitroxide that mimics superoxide dismutase activity [RAW 264.7 macrophages  and H9C2 cardiomyocytes were maintained in DMEM  supplemented with 10% FBS. Cells at 70–80% confluence were grown under hypoxic . The separated proteins were transferred onto nitrocellulose membranes. Incubation with antibodies against CcO IVi1 , PKAα  and TOM20 (SantaCruz) and subsequently with secondary antibodies conjugated to either IR680 or IR800 dyes (Licor) were done simultaneously for both normoxic and hypoxic protein blots under identical conditions. To maintain uniform exposure, the blots were scanned in the Odyssey Licor system  at an intensity setting of 3.5 for 800nm and 2.5 for 680nm. The figure is a representative blot from 3 separate experiments.Isolated mitochondria (100 µg) were resuspended in H-medium , and incubated with increasing concentrations of digitonin  in the presence of trypsin 15 µg. Reactions were carried out for 20 min on ice, after which, 150 µg soybean trypsin inhibitor was added to stop trypsin action. Samples were then centrifuged at 20,000 tag at its C-terminus. Thirty six hours after transfection, the cells were maintained under either normoxia or hypoxia (1% O2) for 12h. At the end of hypoxia, the cells were fixed with cold methanol for 10min and rapidly washed with ice cold PBS. Antibodies against FLAG and CcO subunit I , were used to stain the cells. Alexa 488 and 594 conjugated antibodies  were used as secondary antibodies.COS-7 cells grown on coverslips were transfected with cDNA coding for PKAα fused to a Myc-DDK 3, 1.2mM NaH2PO4, 1.2mM MgSO4, 1mM CaCl2, and 10mM dextrose, pH 7.4, at 37°C in a Langendorff coronary perfusion system. Hearts were submerged into a heat-jacketed organ bath set at 37°C. After 20 minutes of equilibration, global ischemia was initiated by stopping coronary perfusion. Hearts were subjected to a 30-minute global ischemia and a 120-minute full reperfusion. For different treatments, the modulating chemical was included in the perfusion medium at the following concentrations: H89 (1µM) (n=6), MitoQ (0.1 µM) (n=3) and Mito CP (1µM) (n=3). At the end of the reperfusion period, hearts were sliced into 1-mm-thick transverse sections and incubated in triphenyltetrazolium chloride solution  at 37°C for 15 minutes as reported previously [All the mouse work was carried out in compliance with the institutional guidelines and regulatory standards of University of Pennsylvania. This study was approved by the Institutional Animal Care and Use Committee  of University of Pennsylvania. Swiss albino mice (25-30 g) were heparinized (2000 U/kg IP) and then anesthetized with avertin (100 mg/kg IP). The hearts were rapidly removed and perfused with an oxygenated Krebs-Henseleit solution containing 120mM NaCl, 5.8mM KCl, 25mM NaHCOeviously . InfarctPKA assay was performed using a kit as described by the manufacturer . The assay uses Kemptide, a highly specific PKA substrate. To account for PKA independent phosphorylation, for each assay a parallel reaction was carried out in presence of PKI, a specific PKA inhibitor. The final PKA activity was obtained by subtracting residual activity in presence of PKI from total activity. Sample extracts for the assay were prepared by lysing either whole cells or isolated mitochondria in lysis buffer containing 50mM Tris-HCl, 1% NP-40, 150mM NaCl and 1mM EDTA. The kinase reaction was carried out for 30 min at 30°C in a total volume of 60 µl and 10 µg of protein as enzyme source. After the reaction, the assay mix was spotted on P80 phospho cellulose paper, washed and counted in a Beckman model LS5000TA liquid scintillation counter.For SDS/PAGE analysis, either 40 μg of mitochondrial extract or 80 μg of whole cell extract was separated on a 12% denaturing polyacrylamide gel and transferred to nitrocellulose membranes .Blue native PAGE was carried out essentially as described previously . Briefly2, 2 mM NaCN, 2.5 mg/ml BSA, 13 mM NADH, 65 μM ubiquinone and 2 μg/ml antimycin A) and measuring the decrease in absorbance at 340 nm due to NADH oxidation. Complex II–III activity (succinate–cytochrome c reductase) was measured by incubating 25 μg of freeze-thawed mitochondrial extract in 1 ml of assay medium  and measuring the increase in absorbance at 550 nm due to cytochrome c reduction. CcO activity was measured by incubating 1 μg of freeze-thawed mitochondrial extract in 1 ml of assay medium  and measuring the decrease in absorbance at 550 nm due to cytochrome c oxidation. Mitochondrial Aconitase activity was assayed as described before [2, and 30 mM sodium citrate. The reaction was followed at 340nm and the result represented as µmols of NADPH formed. Assays for complex I, complex II–III and CcO were performed as described by Birch-Machin and Turnbull  using a d before . BrieflyThe XF24 Extracellular Flux Analyzer  was used to measure mitochondrial respiration to assess the effect of hypoxia on the electron transport chain complexes. Maximum mitochondrial respiratory capacity is an indicator of the integrity and functionality of the respiratory chain complexes. It was measured by uncoupling mitochondrial respiration by treating cells with 2,3-dinitrophenol (100µM). RAW 264.7 cells were maintained under normoxia or under hypoxia for 12h as explained above with or without the inhibitors. After hypoxia, the compounds were washed off with fresh medium and the cells were incubated for 3h in XF24 medium under normoxia. Finally, the cells were seeded in XF24 microplates  and centrifuged at 1300 rpm for 8 min at room temperature. Both basal respiration and uncoupled respiration were measured in normoxic conditions. At the end of measurement, total protein was estimated from each well of the plate by BCA method  and used to normalize the oxygen consumption rate values. Maximum Oxygen consumption rates were calculated by subtracting oxygen consumption rate measured after addition of Rotenone and Antimycin (1µM each) from uncoupled respiration measured after addition of 2,3-dinitrophenol. Control and ischemic hearts were frozen in liquid nitrogen immediately after the experiment and stored at −70 °C until EPR measurements were performed. The X-band EPR of cardiac tissues were recorded at liquid helium temperatures on a Bruker E500 ELEXYS spectrometer with 100 kHz field modulation, equipped with an Oxford Instrument ESR-9 helium flow cryostat and a DM-0101 cavity. For EPR measurements, frozen heart tissues from control and ischemia-reperfusion experiments were minced to appropriate sizes under liquid nitrogen, and then transferred to a 4 mm quartz EPR tube . The heart tissue weight in each EPR experiment was about 0.3 g. The volume of the tissue sample in the EPR tube was always greater than the working zone of the cavity in each EPR experiment. The only variable was the packing factor . However, this tissue packing factor was not perceived to be a major problem, as reloading of tissues in the same EPR tube (with a different packing) resulted in EPR spectra with a 5% variation in signal intensity. Spectrometer conditions were as follows: microwave frequency, 9.635 GHz; modulation frequency, 100 kHz; modulation amplitude, 10 G; receiver gain, 85 dB; time constant, 0.01 s; conversion time, 0.08 s; sweep time, 83.9 s. EPR spectra were obtained over the temperature range 4-50 K using an incident microwave power of 5 mW and modulation amplitude of 10 G. The spectrometer was calibrated with the radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) exhibiting an EPR signal centered at g = 2.0036.ROS generation by normoxic and hypoxic cells was measured by Dichlorofluorescein fluorescence. Briefly, RAW 264.7 cells were maintained under normoxia or hypoxia in presence of N-Acetyl Cysteine or Mito-CP. At the end of hypoxia, cells were counted and plated in 96 well plates in phosphate buffered saline. DCFDA (1µM) was added to each well and incubated for 15 min. DCF fluorescence was measured in a Chameleon plate reader  at Excitation 525nm and Emission 575nm. ROS generation in isolated mitochondria was measured using Amplex Red. Briefly, mitochondria (10µg) isolated from normoxic and hypoxic RAW 264.7 cells expressing wild type or S40A mutant CcO Vb was plated in 96 well plate in phosphate buffered saline containing 10mM Succinate. Amplex red (10 µM), 4U/ml Horseradish peroxidase and 40U/ml Superoxide dismutase were added and the reaction was incubated at 37°C for 15min. Fluorescence was measured in a Chameleon plate reader .cAMP was measured in both total homogenate and isolated mitochondria using Direct EIA cAMP kit according to manufacturer’s protocol . For each measurement 10µg protein was used. Values represent average of triplicate assays from two separate experiments.t tests for paired data p values <0.05 were considered statistically significant.Data from the cultured macrophages and isolated heart studies are presented as means ± S.D. Differences between paired variables were determined using two-tailed Student's RAW 264.7 macrophages present an excellent cell system for studying hypoxia mediated changes for several reasons. First, they respond to hypoxia readily and express markers of hypoxic stress at high level –35. Seco2) and PKA activity was measured in both isolated mitochondria and cytosol. As seen in Previous studies from our laboratory showed that PKA activity associated with mitochondria increased in cells subjected to hypoxia and hearts subjected to ischemia-reperfusion . In thisIt is known that PKAα and regulatory β subunits are anchored on AKAP proteins localized on the mitochondrial outer membrane, facing the cytosol –38 as weTo further ascertain the increased sequestration of PKAα in hypoxic mitochondria, intact mitochondria isolated from normoxic and hypoxic cells were permeabilized by incubating with increasing concentrations of digitonin. The reaction also included a small amount of trypsin to digest proteins that are released from mitochondria progressively with increasing digitonin concentrations, starting with the outer membrane to the inner membrane. At the end of the reaction samples were denatured and used for immunoblot analysis with antibodies for PKAα subunit. The CcO subunit IVi1 was used as a marker of inner mitochondrial membrane and TOM20 as marker for the outer mitochondrial membrane. To confirm the altered distribution of PKAα subunit during hypoxia, we ectopically expressed tagged PKAα subunit in COS-7 cells and followed its subcellular distribution under normoxia and hypoxia through immunofluorescence microscopy. The confocal microscopy patterns in Since hypoxia did not result in any increase in cAMP level either in cytosol or mitochondria and activation of PKA was unaffected by adenylate cyclase inhibitor, KH7, we investigated alternative mechanisms of activation of PKA. Oxidative stress and ROS has been implicated in the cAMP independent activation of PKA –44. We tRAW 264.7 cells were treated with PKA modulators for the duration of growth under hypoxia or normoxia. The effect of PKA inhibition during hypoxia on mitochondrial respiratory capacity was assessed using Seahorse respirometer. Our previous studies showed that under both hypoxia and alcohol toxicity CcO subunit Vb was one of the three subunits reduced as a function of time ,46. To aIn order to extend our findings on the role of PKA in hypoxia in cell culture model to hypoxic conditions found in pathologies, we tested the effect of PKA modulators on myocardial ischemia-reperfusion injury. The infarct size resulting from temporary global ischemia and 2 hours of reperfusion was measured in mouse hearts perfused with PKA modulators. In untreated ischemia-reperfused hearts, the infarct size amounted to 61.7 % of the total area at risk . The heaActivities of respiratory complexes and aconitase were measured in the heart tissues. + of aconitase. Control hearts, on the other hand, did not show significant damage to metal centers. Importantly, preconditioning with H89 or Mito-Q significantly reduces the formation of oxidized aconitase in ischemia-reperfused hearts. Aconitase is an important marker of oxidative stress that has been shown to be oxidatively damaged under mitochondrial stress . OxidatiProtein kinases have long been shown to play roles in various aspects of mitochondrial biogenesis and function ,48,49. PPKA signaling in hypoxia has been studied by our lab and others in different model systems. In bone cells and bovine aortic endothelial cells, apparent PKA activity was shown to rapidly increase with decreasing oxygen concentration . In PC-1Our previous studies have shown that CcO subunits I, IVi1 and Vb are phosphorylated under hypoxia or myocardial ischemia-reperfusion . FurtherIncreased mitochondrial PKAα levels and activity were reported in rabbit hearts subjected to ischemia-reperfusion injury . In thisThe increase in mitochondrial PKAα subunit level under hypoxia is prevented by the antioxidants N-acetyl cysteine and Mito-CP, which suggests a direct role of ROS in the activation . PreviouPreviously we identified the ischemia/reperfusion induced phosphorylation site to Ser40 of CcO Vb subunit . In thisHighly coupled mitochondria with steep trans membrane potential have been shown to generate high levels of ROS mostly through catalytic activities of complex I and reverse electron transfer ,67. StudIn summary, we describe a novel mechanism of mitochondrial PKA activation under hypoxia and ischemia/reperfusion. This PKA is distinctly different from the relatively low levels of basal PKA activity reported under normoxic conditions for the following reasons: 1. the stress induced PKA is modulated by ROS, 2. its target sites are distinctly different from sites reported under normoxic conditions, 3. The PKA activity induced under hypoxic condition is not inhibited by KH7, an inhibitor of soluble adenylate kinase, 4. Phosphorylation of CcO by stress induced PKA induces subunit degradation and CcO dysfunction rather than activation of CcO reported under normoxic conditions.Figure S1Full images of immunoblots presented in Figure 2A. The experimental details are given in materials and methods and Figure 2A.(PPTX)Click here for additional data file.Figure S2Full images of immunoblots presented in  NS=Non specific. The experimental details are given in materials and methods and (PPTX)Click here for additional data file.


Vibrio cholerae protein VCA0965 contains an AGDEF active site and is presumed to be an inactive DGC.Diguanylate cyclases (DGCs) regulate biofilm formation and motility in bacteria by synthesizing the second messenger cyclic di-GMP (c-di-GMP) in response to environmental stimuli. DGC enzymatic activity is believed to be dependent on the presence of a GG(D/E)EF active site motif, however approximately 25% of known DGCs contain a degenerate active site. The V. cholerae causes a 3-fold reduction in flagellar-based motility. Additionally, an RXXD allosteric inhibition mutant of VCA0965 strongly inhibited motility and stimulated biofilm formation. This activity was lost when the active site of VCA0965 was mutated to AGDAF, suggesting that VCA0965 synthesizes c-di-GMP. In support of this, ectopic expression of VCA0965 and VCA0965 containing a mutation in its RXXD motif significantly increased the intracellular c-di-GMP levels in V. cholerae and Escherichia coli. Furthermore, we found that purified VCA0965 was able to synthesize c-di-GMP in vitro. Systematic mutation of the first amino acid in the AGDEF motif of VCA0965 revealed that glycine, methionine, and histidine also produced an active DGC capable of inhibiting motility and increasing the intracellular concentration of c-di-GMP in V. cholerae.Ectopic expression of VCA0965 in Based on these results, we conclude that VCA0965 is capable of c-di-GMP synthesis and that the first amino acid of the GG(D/E)EF motif is more tolerant of substitutions than currently appreciated. Vibrio cholerae, high levels of c-di-GMP induce biofilm formation, reduce swimming motility, and inhibit colonization in a murine infection model [Cyclic di-GMP (c-di-GMP) is a bacterial second messenger that is widely utilized by bacteria, with more than 80% of sequenced bacteria predicted to use this signal ,2. C-di-on model ,10. Theson model ,11-13 anon model ,15. Intron model  and degron model ,17.DGCs are characterized by the presence of a conserved GGDEF domain composed of approximately 200 amino acids . These dC. crescentus, PelD from Pseudomonas aeruginosa, and CdgA from Bdellovibrio bacteriovorus which bind c-di-GMP to regulate cell cycle progression, biofilm formation, and predation, respectively [P. aeruginosa and SgmT from Myxococcus xanthus[V. cholerae requires its degenerate GGDEF domain, but not c-di-GMP, to retain its PDE activity, and the highly degenerate GGDEF domain of YybT from Bacillus subtilis exhibited ATPase activity [Pectobacterium atrosepticum DGC ECA3270 retains DGC activity despite having a degenerate SGDEF active site motif [One explanation for the high frequency of degenerate DGCs is that these proteins in some cases act as c-di-GMP receptors by binding to c-di-GMP at either their degenerate active site or their RXXD inhibition site. These degenerate DGCs respond to c-di-GMP in several ways. C-di-GMP may mediate participation of the DGC in a regulatory cascade, as does PopA from ectively -22. Binds xanthus,24. Addiactivity ,25. In ote motif . Intereste motif .V. cholerae DGC VCA0965. We examined the ability of all 40 V. cholerae DGCs to inhibit motility in semisolid agar, and we determined that VCA0965 was active in this assay. This result was surprising as VCA0965 is a DGC that encodes a degenerate AGDEF active site. Rather than functioning as a receptor for c-di-GMP, our results suggest that VCA0965, despite its degenerate active site motif, is capable of c-di-GMP synthesis.Here we investigate the degenerate E. coli and V. cholerae strain C6706str2 were grown in LB at 35°C with shaking at 220 rpm unless otherwise indicated. LB was supplemented with 100 μg mL-1 kanamycin as needed to maintain plasmids. Plasmids were induced with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) unless otherwise specified.Strains used in this study are listed in Table -1 kanamycin and 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) as appropriate, were allowed to solidify for 1 to 4 hours prior to use. Plates were inoculated from overnight cultures by stabbing through the agar with a pipette tip, and left undisturbed at room temperature for 1 hour before inversion and incubation at 37°C for 20 hours. Motile bacteria were digitally imaged using an Alpha Innotech Red Gel Imager with white light and an 800 ms exposure time. Images were processed to calculate colony area by determining differential pixel density using ImageJ software [2. The initial motility screen  were prepared immediately before each assay from LB supplemented with 0.35% agar. Plates, supplemented with 100 μg mLsoftware . A stickV. cholerae cultures. Wells were supplemented with 100 μg mL-1 kanamycin and 0.1 mM IPTG as needed. Plates were incubated at 37°C with gentle aeration for 7 hours before washing in PBS, fixing with 95% ethanol and staining with 0.41% crystal violet in 10% ethanol. Crystal violet was eluted in 160 μL of 95% ethanol. The optical density at 600 nm (OD600) was determined using a Spectra Max M5 spectrometer (Molecular Devices). To avoid saturation, the elutions were also diluted 1 to 10 and the resultant OD600 was measured. If the OD600 of the undiluted elution exceeded 1, then the OD600 was calculated from the 1 to 10 dilution. Flow cytometry cultures were prepared by diluting an overnight culture 1:100 in LB. Cultures were supplemented with kanamycin and IPTG as needed and grown at 35°C and 220 rpm for 4 hours. Flow cytometry analysis was performed as described [MBEC plates were prepared by inoculating 150 μL of LB/well with a 1 to 1000 dilution from overnight escribed .V. cholerae 1:1000 into LB and grown to an OD600 between 0.6 and 1.0. Cultures were supplemented with kanamycin and IPTG as needed. Samples were extracted and analyzed by liquid chromatography coupled with tandem mass spectromentry (LC-MS/MS) as previously described [Cultures were prepared by diluting an overnight culture of escribed  using anE. coli JM109(DE3) (Promega) containing the pVCA0965(180–396) vector was prepared and used to inoculate LB broth supplemented by 100 μg mL-1 kanamycin. The culture was grown at 37°C with shaking until an OD600 between 0.7-0.8 was reached. The cells were then induced by addition of IPTG to a concentration of 1 mM. The induced cells were incubated between 16 and 18 hours at 16°C with shaking. Following induction, cells were collected via centrifugation in a Sorvall RC-5B Superspeed Centrifuge for 10 minutes at 2,678 g and 4°C. The pellet was then resuspended in lysis buffer (25 mM Tris-Cl (pH 8.0), 500 mM NaCl, 5 mM 2-mercaptoethanol, 20 μg/mL DNase, one tablet Roche protease inhibitor kit) and lysed using a M-110P processor (Microfluidcs) 3 times at 20,000 psi. Novagen Ni-NTA His-bind resin was equilibrated in binding buffer (25 mM Tris-Cl (pH 8.2), 500 mM NaCl, 20 mM imidazole, 5 mM 2-mercaptoethanol) and added to the lysate. Resin and lysate were incubated for 60 minutes at 4°C with constant end over end rotation. After incubation, lysate and resin were applied to a column and allowed to flow to pack. The resin was washed three times with small amounts of binding buffer. The protein was then step eluted by the addition of imidazole elution buffer (25 mM Tris-Cl (pH 8.2), 500 mM NaCl, 20 mM imidazole, 5 mM 2-mercaptoethanol) with 50 mM, 100 mM, 200 mM, or 300 mM imidazole. VCA0965 was determined to elute in the 200 mM and 300 mM imidazole fractions. These fractions were concentrated on Amicon Ultra Centrifugal Units by centrifugation at 16,000 g for 20 minutes at 4°C. Concentrated fractions were washed once with dialysis buffer (30 mM Tris-Cl (pH 7.6), 100 mM NaCl) and then stored at −80°C in 20% glycerol.An overnight culture of 2) for 24 hours at room temperature. Samples were then boiled for five minutes until precipitate formed and centrifuged at 15,871 g for 10 min. to remove precipitated proteins. The concentration of c-di-GMP was determined using LC-MS/MS as described above.Purified proteins were diluted 1/25 and mixed with 0.015 mM GTP in reaction buffer  and/or its RXXD allosteric inhibition site (referred to as the I-site) ,20,21. T cholerae . We observed that overexpression of VCA0965 and VCA0965-I caused a 4- and 15-fold increase, respectively, in the intracellular concentration of c-di-GMP compared to a vector control .The above findings that VCA0965 and/or its I-site mutant were able to repress motility and induce biofilm formation only with an intact AGDEF active site suggested that VCA0965 was capable of influencing intracellular c-di-GMP levels. To test this hypothesis, we measured the impact of VCA0965, VCA0965-I, VCA0965-A and VCA0965-AI overexpression on intracellular c-di-GMP levels in V. cholerae and E. coli. To further confirm that VCA0965 is an active DGC, we purified VCA0965 and assessed its DGC activity in vitro. Due to the fact that VCA0965 is predicted to contain five transmembrane domains that span the inner membrane, it was necessary for purification purposes to construct a truncated version of VCA0965 composed exclusively of the cytoplasmic portion of the protein containing amino acids 180 to 396. VCA0965(180–396) was incubated with 12.5 µΜ GTP and synthesized c-di-GMP was quantified using LC-MS/MS. Both VCA0965(180–396) and the positive control DGC, WspR(R242A) [It remained possible that VCA0965 modulated c-di-GMP levels by interacting with other components in the cell, although these components would have to be conserved between R(R242A) , produceR(R242A) . The WspV. cholerae reduced motility . To answer this question, we constructed mutants of VCA0965 containing all 19 amino acids in the first position of the AGDEF active site and determined if expression of these proteins in V. cholerae. Indeed, all three of these VCA0965 mutants significantly increased c-di-GMP when induced compared to the uninduced controls; the histidine and methionine mutations were statistically significant (p < 0.05 using a one-tailed Student’s t-test) while the glycine substitution bordered on statistical significance with a p-value of 0.067 EF active site is overrepresented in this genus EF active site. Moreover, this analysis shows that most of the GGDEFs encoding the AG(D/E)EF active site we identified in Table The N-terminal domains of GGDEF proteins typically encode conserved domains that are thought to bind environmental signaling cues to control the associated enzyme domain . HMM-Proos Table . This suV. cholerae, and the DGC domain of VCA0965 is capable of in vitro c-di-GMP synthesis. To our knowledge, this is the first description of an active DGC containing a naturally encoded AGDEF active site motif and only the second description of an active non-canonical DGC; the only other example being the SGDEF containing ECA3270 from Pectobacterium atrosepticum[Here we show that VCA0965, which encodes a degenerate AGDEF active site, is an active DGC that synthesizes c-di-GMP. Overexpression of VCA0965 represses motility by increasing c-di-GMP levels in osepticum. ECA3270Retention of activity in ECA3270 and VCA0965 is surprising when the steric constraints of the active site are considered. Within the active site, the GGDEF motif is located in a hairpin . C-di-GMin silico bioinformatic analysis of the AGDEF motif found that AG(D/E)EFs account for approximately 1.6 percent of DGCs, making this motif fairly common and suggesting that AGDEFs may have a variant structure that allows them to retain activity. How this variance impacts function remains to be addressed.Contrary to what might be expected based on the structure of the DGC domain, degeneracy at the first amino acid is quite common among DGCs. 23.7% of all DGCs are degenerate at one or more amino acids in the GGDEF motif . Of theset. al.[et. al. who observed that some DGCs were able to induce biofilm formation at 12 µΜ c-di-GMP, the levels we determined are produced when VCA0965 is induced EF motif should be considered flexible in regards to DGC activity. In combination with the finding for ECA3270, a more accurate description of a functional active site should be (G/S/A)G(D/E)EF. However, whether enzymes which naturally encode a GG(D/E)EF will retain DGC activity upon substitution of the first glycine to serine or alanine remains to be determined. It is possible that intragenic epistatic interactions are necessary for these alterations of the first amino acid to remain functional. Additional non-canonical active site motifs should be assayed to determine if enzymatic activity of these DGCs is widespread. Furthermore, structural analysis of ECA3270 and VCA0965 can highlight how variance at the first amino acid of the GGDEF motif can accommodate DGC activity.Vibrio cholerae DGC, VCA0965, is able to synthesize c-di-GMP to control motility and biofilm formation. Moreover, we found that addition of three other amino acids, glycine, methionine, and histidine, maintained DGC activity. Our results illustrate that the first residue of the GG(D/E)EF active site in DGCs is more flexible to substitutions than previously appreciated. As many DGCs encode non-canonical amino acids at this position, caution must be used when determining the activity of these enzymes. Whether or not substitution of non-canonical amino acids at the first position of the active site has important functional consequences on DGC activity remains to be explored.The research presented here shows that the AGDEF containing DGC: Diguanylate cyclase; PDE: Phosphodiesterase; c-di-GMP: Cyclic di-GMP.The authors declare no competing interests.JLH, GBS and BJK collected and analyzed the data and JLH and CMW wrote the manuscript. All authors read and approved the final manuscript.


E. coli. This rOPCML tumor suppressor protein therapeutic caused growth inhibition by apoptosis in 6/7 ovarian cancer cell lines tested with no effect on OPCML expressing normal ovarian surface epithelium by an identical mechanism to the transfected normal protein. pERk and pAKT inhibition was seen with functional growth inhibition and increased apoptosis in rOPCML treated cells. rOPCML was then injected intraperitoneally twice weekly in two murine intraperitoneal models of ovarian cancer (nude mouse A2780 and SKOV3) and demonstrated profound inhibition of tumour weight, ascites volume and peritoneal dissemination compared with BSA control. Western analysis from rescued tumors confirmed that the same mechanism of specific RTK downregulation was also evident in vivo. In Summary, the OPCML tumour suppressor mediates its suppressor function by systems level negative regulation of at least 5 RTKs and a recombinant modified derivative is a potent tumor suppressor protein therapeutic in-vitro and in-vivo that recapitulates the in-vitro mechanism.OPCML, a GPI anchored tumor suppressor gene is inactivated by somatic methylation in multiple cancers. We previously identified this gene by LOH mapping and demonstrated that it was inactivated by somatic methylation in 80% of ovarian cancers. Restoring OPCML expression by stable transfection suppressed in-vitro growth and in-vivo tumorigenicity. We hypothesized that OPCML might abrogate growth signaling pathways. In SKOV-3 and PEO1, ovarian cancer cell lines with no expression of OPCML, we demonstrated that OPCML negatively regulates a specific repertoire of receptor tyrosine kinases (RTKs) EPHA2, FGFR1, FGFR3, HER2 and HER4, and reciprocally, OPCML siRNA and shRNA knockdown in normal ovarian surface epithelial cells up-regulates these same RTKs, with no effect on RTKs EPHA10, FGFR2, FGFR4, EGFR, HER3, VEGFR1 and VEGFR3. shRNA knockdown shows that loss of OPCML accelerates the growth of normal ovarian surface epithelial cells in vitro. Example immunoprecipitation experiments revealed that OPCML binds to EphA2, FGFR1 and HER2 extracellular domains with no such interaction to EGFR, thus OPCML binds directly to RTKS that it negatively regulates. Cotransfection of ECD less and ECD containing Her2/Neu shows that the functional tumor suppressor phenotype of OPCML is mediated via interaction with the ECD. We demonstrate that OPCML is located exclusively in the raft membrane fraction and sequesters RTKs that it binds to the raft fraction, leading to polyubiquitination and proteosomal degradation via a cav-1 endosomal mechanism resulting in systems depletion of this specific RTK repertoire, that does not occur with RTKs that OPCML does not bind. Pulse-chase experiments confirm rapid loss of HER2 in OPCML expressing cells and not in OPCML deficient cells. We demonstrate that OPCML abrogates EGF mediated phosphorylation of FGFR1, HER2 and EGFR and the downstream phosphosignaling of pErk and pAKT. A recombinant modified OPCML-like protein without a GPI anchor, signal peptide or glycosylation was constructed and expressed in