Trypanosoma cruzi is the causative agent of Chagaś disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored.The unicellular parasite T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin.Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.Herein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that  Trypanosoma cruzi with host cells, as well as their resulting signaling pathways, is fundamental to the understanding of the mammalian infection by the parasite. T. cruzi, the causative agent of Chagas` disease, is transmitted to humans by blood sucking triatomines (insect vector), ingestion of contaminated food or blood transfusion. The parasite multiplies as epimastigotes in the midgut of the insect digestive tract and differentiates to metacyclic trypomastigotes in the hindgut, being eliminated with the feces and urine after a blood meal. The excreted parasites can infect mammalian host cells through the bite wound or the mucous membranes. Inside cells, the parasites multiply as amastigotes and differentiate to trypomastigotes. The parasites are released in the circulation upon rupture of the host cells and may invade new cells or be ingested by the insect vector where they differentiate into epimastigotes The identification of molecules involved in the multiple interactions of T. cruzi genome suggests a major role of the protein phosphorylation cycle in signaling events T. cruzi epimastigotes, 6 are protein kinases or phosphatases T. bruceiLeishmaniaPost-translational modifications of proteins provide an efficient tool to regulate the activity of key proteins, with reversible phosphorylation being a frequent one. The large amount of kinases (190 protein kinase genes) and phosphatases (86 protein phosphatase genes) in the T. cruzi signaling during parasite invasion. A decrease in the amount of trypomastigote-induced host tyrosine phosphorylated proteins  was described T. cruziDespite the importance of protein post-translational modifications, much less is known about their role in T. cruzi trypomastigotes to elements of the extracellular matrix (ECM), such as fibronectin T. cruzi to survive in the mammalian host is partially due to its capacity to invade host cells and many molecules have been implicated in this step. Particularly, some belong to the subgroup II of the Gp85/Trans-sialidase superfamily, encoded by a high polymorphic supergene family composed of approximately 700 genes and equal number of pseudogenes T. cruzi invasion Adhesion of The importance of the cytoskeleton in signal transduction is increasingly being recognized. In trypanosomatids there is a characteristic sub-pellicular corset of microtubules linked to each other and to the plasma membrane, formed by α- and β-tubulin subunits. The flagellum contains a second microtubule organization composed by the conserved structure of “9+2″ microtubules (axoneme). This structure together with the paraflagellar rod, which runs alongside the axoneme, is linked to the cell body by other cytoskeletal elements T. cruzi trypomastigote cytoskeletal major proteins, as α- and β- tubulins and paraflagellar rod proteins, has been undertaken for the first time. Moreover, involvement of the ERK pathway is suggested.Thus, a thorough analysis of phosphorylation/dephosphorylation events in This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication 8523). The protocol was approved by the Animal Ethics Committee of the Instituto de Química, Universidade de São Paulo (Permit Number: 17/2011), which follows regulations issued by CONCEA , Brazil.Laminin-1 from Engelbreth-Holm-Swarm (EHS), Protease Inhibitor Cocktail, anti-mouse-IgG antibodies conjugated to Peroxidase, anti-phospho-Serine (pS) and anti- α-tubulin antibodies were purchased from SIGMA-ALDRICH; human plasma fibronectin from Gibco; DeStreak rehydration solution from GE Healthcare Life Science; PRO-Q Diamond Phosphoprotein Gel Stain, anti-phospho-Threonine (pT) and anti-phospho-Tyrosine (pY) antibodies from Invitrogen; and anti-phospho ERK antibody from Cell Signaling.Trypanosoma cruzi Y strain was maintained by infection of LLC-MK2 cells 3, pH 9.5 for 14 h at 4°C. After washing three times with PBS, 1×109 parasites in 1 ml of DMEM supplemented with 2% FBS were added to each well and incubated at 37°C in a 5% CO2 atmosphere. After the desired time, the parasites were collected and washed with PBS containing phosphatase  and protease inhibitors (Protease Inhibitor Cocktail plus 1 mM PMSF). The parasite pellet was kept at −80°C. Three independent experiments were performed for each treatment. The triplicates have been compared with each other by the Image Master Platinum 7 software.g for 7 min at 4°C. The supernatant was collected and proteins were quantified by Bradford DeStreak rehydration solution, containing phosphatase and protease inhibitors as described, was added to the frozen parasite pellet. Parasites were submitted to ultra sonic disruption for 15 min on ice followed by centrifugation at 20,000×T. cruzi. IEF was carried out by ramping 1000V for over 800Vh; ramping 8000V for over 11300Vh; holding at 8000V for 5400Vh; yielding a total of about 35000Vh, as suggested by the GE Healthcare instructions.The first dimension (IEF) was performed using Ettan IPGphor 3 System . 13 cm DryStrip (pH 4–7) immobiline strips were loaded with 450 µg of proteins from T. cruzi extracts with alkaline phosphatases (Thermo Scientific and Roche) eliminated most of the Pro Q recognized phosphorylated spots.For the second dimension, IPG strips were previously incubated with 1% DTT in the equilibrium buffer  for 15 min, followed by 15 min in equilibrium buffer supplemented with 2.5% iodoacetamide. The strips were washed with water and transferred to the top of 12% SDS-Polyacrylamde gel, overlaid with 0.5% agarose solution and submitted to electrophoresis. After running, the gels were stained with PRO-Q Diamond Phosphoprotein Gel Stain to identify phosphoproteins and then colloidal Coomassie was added for total protein staining. Gels were scanned with Typhoon Trio  and ImageScanner III  to identify phosphoproteins and total proteins, respectively. Gel analyses were carried out in ImageMaster 2D Platinum 7.0 . A standardized spot with 5% statistical significance test in ANOVA  was considered to determine significant changes in phosphorylation levels. Two experiments treating http://www.matrixscience.com). The parameters used were: enzyme, trypsin; allow up 1 missed cleavage; fixed modification, carbamidomethyl (C); variable modifications, oxidation (M), phosphorylation (S/T) and phosphorylation (Y); peptide tolerance, ±0.1Da; MS/MS; tolerance: ±0.1Da; and peptide charge, 1+2+ and 3+. The database Tcruzi2011, included in Tritryp database, was used to identify the proteins.The spots of interest were manually excised from the gel and treated as follows: (1) Destaining of the spots with 50% methanol-2.5% acetic acid; (2) Peptide reduction in 10 mM dithiothreitol (DTT) and peptide alkylation with 50 mM iodoacetamide; (3) Polypeptide enzymatic hydrolysis with 0.4 or 0.8 µg trypsin (PROMEGA), accordingly to the spot size, and (4) extraction of the peptides from the gel spots. For peptide sequencing, the mass spectrometer ESI-Quad-TOF coupled to UPLC system was used at Mass Spectrometry Laboratory of LNBio, CNPEM/ABTLuS . The mass spectra were processed through the program ProteinLynx V 2.1 (Waters) and analyzed with the MASCOT MS/MS Ion Search  for 30 min at 4°C, followed by centrifugation to remove the resin. The supernatants were incubated with 5 µL of anti-α-tubulin antibodies or 5 µL of anti-paraflagellar-rod protein monoclonal antibody (anti-PAR Mab) and 50 µL of Protein A-Sepharose for 14 h at 4°C. After extensive wash with Lysis Buffer, the resin was ressuspended in 50 µL of Laemmli Buffer  followed by SDS-PAGE and immunoblotting with a mixture of anti-pS, -pT, -pY, anti-α-tubulin antibodies or anti-PAR Mab. To raise Anti-PAR Mab, Balb/c mice were inoculated every other week with 50 µg of a trypomastigote extract, during 4 weeks. BALB/c female mice were housed under barrier conditions at the animal care facility at the Instituto de Química, Universidade de São Paulo . The hybridomes were obtained accordingly to the literature Extracts of 1x10×g for 40 min at 4°C. The supernatant (S) and the pellet (INS) containing soluble and polymerized tubulins and PAR, respectively, were separated and the pellet ressuspended in RIPA buffer , as described in reference The presence of phosphorylated ERK was detected in immunoblots of trypomastigote extracts as described above developed with anti-phospho ERK and anti-ERK antibodies. To analyze soluble and polymerized molecules, pellets of frozen parasites were ressuspended in homogenization buffer  and the nucleus stained with 20 µg/mL of 4',6-diamidino-2-phenylindole, dilactate (DAPI-Invitrogen). Images taken in a Nikon Eclipse E600 microscope were obtained under identical settings and were submitted to deconvolution with Huygens Essential image processing program. The number of modified parasites upon incubation with fibronectin and laminin was quantitated in 6 independent microscopic fields covering approximately 240 parasites.The immunoblotting and the immunofluorescence assays have been analyzed by the Student's t-test.Differences in the distribution of phosphorylated proteins in trypomastigotes incubated with fibronectin or laminin were detected by immunofluorescence, as compared to parasites incubated with BSA, when a mixture containing anti-pS, -pT and -pY antibodies was employed . The regp<0.05), when compared to BSA-incubated trypomastigotes. These spots were then removed, subjected to trypsin digestion followed by LC-MS/MS analysis. Proteins were identified using Tritryp, the genome database of Kinetoplastids.To analyze the modifications at the protein level, trypomastigotes were added to tissue cultured plates previously coated with laminin, fibronectin or BSA. At different periods of time , the parasites were collected and after washing, the whole-cell lysates were resolved by 2D-Electrophoresis and the phosphoproteins were detected by Pro-Q Diamond stain. The 2 h point was selected for further phosphoproteome analysis since no qualitative protein modifications were observed after this time (not shown). The phosphoproteome of fibronectin or laminin-incubated trypomastigotes  showed 5From the 50 phosphoproteome spots analyzed after incubation of trypomastigotes with fibronectin, 35 were dephosphorylated  and 15 had their phosphorylation levels increased (increase or phosphorylation emergency) , Fig. 2CUpon trypomastigote treatment with laminin, from 43 spots analyzed, 27 showed reduction in the phosphorylation level , while 16 spots were phosphorylated (increment or phosphorylation emergency) , Fig. 2CConsidering both treatments, 20 sequenced spots with distinct phosphorylation status corresponded to proteins with no annotated function. Only two (access # Q4DCU5 and Q4E246) are common to fibronectin and laminin-treated parasites  and S2. [169] inhibits translation initiation, a phenomenon related to the metacyclogenesis of T. cruziThe majority of the identified spots showing phosphorylation changes after incubation of trypomastigotes with either fibronectin or laminin are related to cytoskeletal proteins  , with hiT. bruceiChlamydomonasT. cruzi to laminin. Altogether, the data show the modification on the phosphorylation status of the cytoskeleton elements from T. cruzi in the adhesion to ECM elements.In addition to paraflagellar rod proteins and α- and β-tubulin, other proteins important for cytoskeleton and flagellar function were also modified at least in one of the experimental conditions, as for example flagellar radial spoke component, dyneins or I/6 protein. The latter, responsible for microtubules cross-linking Phosphorylation/dephosphorylation of cytoskeleton components in response to trypomastigotes adhesion to the extracellular matrix elements have been observed, implicating the involvement of tubulins and paraflagellar rod proteins in this phenomenon.274, D288, L289 and A306 changed by the amino acids T, E, V, R in β-Tubulin 1.9, respectively), was described in other proteomic analysis of T. cruziAnalyzing the 12 spots that match with α- or β- tubulin showing a variation in phosphorylation levels in response to incubation of trypomastigotes with fibronectin or laminin  and S2, in vivo proteolysis. The predominance of α-tubulin dephosphorylation in the adhesion step of trypomastigotes to fibronectin or laminin was confirmed by immunoprecipitation. Extracts of trypomastigotes previously incubated with fibronectin, laminin or BSA for 5 min, 30 min, 60 min or 120 min were immunoprecipitated with α-tubulin-antibodies, followed by the development of the immunoblotting with a mixture of anti-pS, pT, pY antibodies. Dephosphorylation of α-tubulin is clearly observed after 1 h incubation of trypomastigotes with laminin, reaching approximately 65% after 2 h (Dephosphorylation of α-tubulin (spot 111) and β-tubulin 1.9 (spot 113) has also been observed upon incubation of trypomastigotes with laminin. Other spots, though, identified as α- or β-tubulins showed a slight increase in their phosphorylation levels when incubated with fibronectin  or laminin . A considerable number of α- and β- tubulin spots with low molecular mass were also identified , a resulfter 2 h . Similarfter 2 h . Thus, bIn spite of the expressive dephosphorylation of α-tubulin, no changes in the morphological pattern of the cytoskeleton complex structure have been detected by immunofluorescence in parasites incubated for 2 h with laminin, fibronectin or BSA (control) (data not shown). This result strongly suggests that tubulin dephosphorylation is not correlated with microtubule remodeling.PAR proteins showed significant variation in phosphorylation levels after incubation of trypomastigotes with fibronectin or laminin. Good score values, percentage of coverage and number of peptides sequenced indicate a successful identification of PAR , S2, 2. A major PAR dephosphorylation after incubation of the parasites with laminin or fibronectin was corroborated by immunoprecipitation of the extracts of trypomastigotes incubated with fibronectin, laminin or BSA for 5 min, 30 min, 60 min or 120 min, with anti-PAR MAb followed by immunoblottings using a mixture of anti-pS, pT, pY and anti-PAR MAb . SimilarTo check whether reduction in PAR protein phosphorylation leads to any phenotypic change, p-formaldehyde fixed trypomastigotes previously incubated for 2 h with fibronectin, laminin or BSA (control) for 2 h were analyzed by immunofluorescence. No morphological differences could be detected in the distribution of PAR proteins by immunofluorescence microscopy upon adhesion, when compared to the control (data not shown). These findings together with those for α-tubulin strongly suggest that the parasite cytoskeleton dynamics is not affected by dephosphorylation events.T. cruziT. bruceiLeishmaniaT. cruzi trypomastigotes in response to fibronectin or laminin incubation in a time course experiment  was then evaluated.The mitogen activated kinase (MAPK) signal transduction pathway and, in particular ERK  signaling, play a fundamental role in diverse cellular functions and have been associated to fibronectin Reduction of ERK phosphorylation is clearly shown in the 60 and 120 min time points of trypomastigotes incubation with fibronectin, in contrast to the incubation with BSA (control) . HoweverIn short, dephosphorylation of tubulins, paraflagellar rod proteins and ERK is a common response to parasite adhesion to laminin or fibronectin. Notwithstanding, presently, there is no evidence to ascertain which of these events precedes the others.T. cruzi upon trypomastigotes adhesion to fibronectin or laminin. A clear evidence that adhesion to laminin or fibronectin leads to phosphorylation/dephosphorylation events on T. cruzi trypomastigotes was seen by immunofluorescence images of phosphoproteins . As predictable, PAR proteins and tubulins were identified in the proteome of all stages of T. cruzi (TcMAPK2) is present in all stages of the parasite, although in a more phosphorylated (active) form in trypomastigotes in which is localized along the flagellar contour T. cruzi response to adhesion to laminin and fibronectin suggesting that the downstream cascade should be further exploited.Due to the methodological approach employed, less abundant proteins or small differences in phosphorylation might have been missed. Other proteins that may have been modified in response to laminin or fibronectin and were not noticed in this report should then be expected. For example, one protein not detected in the phosphoproteomic analysis was ERK 1/2, known to be modified in response to ECM proteins in mammalian cells. In fact, using specific antibody to phosphorylated ERK1/2, dephosphorylation (inactivation) of that protein was observed. Interestingly, dephosphorylation of ERK was also observed in lung cancer cells after incubation with laminin T. cruzi. It should be noted that ser/thr kinase CK2 was described to be tightly associated with the parasite heterodimeric α/β tubulins and capable of phosphorylating α and β tubulins in vitroT. brucei β-tubulin (GLSVPELTQQMFDAK) 172 β-tubulin leads to defects in microtubule dynamics and cell division in yeast in vitro165 α6 tubulin by PKCα leads to microtubule elongation and motility in human breast cancer cells The phosphorylation sites of tubulin were not identified in T. cruzi, a strong association of protein phosphatase 2A (PP2A) with α-tubulin, myosin and actin has been reported, suggesting a role in trypomastigote transformation into amastigotes and flagellum shortening Much less is known about the role of phosphatases in microtubular function. For example, target mutagenesis of the Ppp1cc gene results in severe defect in spermatogenesis, with high phosphorylation of α- and β-tubulin isoforms, leading to the disorganization of the tubulin network T. cruzi epimastigotes T. bruceiAs happens with tubulins, the role of the phosphorylation/dephosphorylation cycle of paraflagellar rod proteins is unknown. PAR, a structure restricted to kinetoplastids, euglenoids and dinoflagellates, is composed mainly by PFR1 (PAR3) and PFR2 (PAR2) Many issues have to be addressed to understand completely the signal transduction upon adhesion of trypomastigotes to ECM. The role of phosphatases and kinases should be investigated as well as the modulation of the phosphorylation status of α-tubulin and PAR proteins as scaffold structures in the signaling process. ERK dephosphorylation and the ensuing effect in downstream elements should be more investigated. Since it is well established that trypomastigotes interact with ECM elements through members of the gp85 GPI-anchored surface glycoprotein family, as Tc85 or gp83 T. cruzi trypomastigotes to ECM elements, herein described for the first time, is at its inception. Phosphatases activation, cytoskeleton dephosphorylation, and ERK 1/2 inactivation may form a chain of events leading to the success of parasite invasion. In this context, the cytoskeletal proteins may directly participate in the signaling pathway that is activated during parasite attachment to the host cell.The knowledge on the modifications of microtubule and paraflagellar rod protein phosphorylation relative to adhesion of T. cruzi to invade cells.This novel hypothesis will be the focus of future investigations aiming at getting clues into the mechanism employed by Table S1(DOC)Click here for additional data file.Table S2(DOC)Click here for additional data file.


In order to quantify the relation between diminished KGDHC activity and the mitochondrial ATP generation, redox state, transmembrane potential, and generation of reactive oxygen species (ROS) by the respiratory chain (RC), we developed a detailed kinetic model. Model simulations revealed a threshold-like decline of the ATP production rate at about 60% inhibition of KGDHC accompanied by a significant increase of the mitochondrial membrane potential. By contrast, progressive inhibition of the enzyme aconitase had only little impact on these mitochondrial parameters. As KGDHC is susceptible to ROS-dependent inactivation, we also investigated the reduction state of those sites of the RC proposed to be involved in ROS production. The reduction state of all sites except one decreased with increasing degree of KGDHC inhibition suggesting an ROS-reducing effect of KGDHC inhibition. Our model underpins the important role of reduced KGDHC activity in the energetic breakdown of neuronal cells during development of neurodegenerative diseases.Reduced activity of brain  In Adiseases –7 and ardiseases . Variatidiseases , 10 may diseases , 11.The citric acid cycle is catalyzed by eight enzymes, among which KGDHC has the lowest activity . Thus, KThe molecular mechanisms underlying the age-dependent loss of brain KGDHC activity remain elusive. Inactivation of the enzyme complex by reactive oxygen species (ROS) is one possible explanation as KGDHC and the aconitase have been shown to be the main targets of ROS in the citric acid cycle .To further clarify the implications of reduced activities of the TCAC enzymes KGDHC and aconitase for the mitochondrial energy metabolism and the formation of ROS by the respiratory chain, we developed and applied a detailed kinetic model encompassing the TCAC, the respiratory chain (RC), translocation of adenine nucleotides between mitochondrial matrix and the cytosol, oxidative phosphorylation, and ion transport across the inner mitochondrial membrane. The submodel of the RC describes the electron transport as a multistep process whereby some of the intermediate redox sites allow electron transfer to molecular oxygen under formation of the superoxide anion (ROS).The reaction scheme of the kinetic model is shown in The kinetic model focuses on the mitochondrial-derived ATP production and is compartmentalized into cytosol and mitochondria. The model consists of 184 state variables describing the neuronal citric acid cycle, the respiratory chain, the oxidative phosphorylation, the mitochondrial ATP generation, the nucleotide exchange between the mitochondrial matrix and the cytosol, and the electrophysiological coupling between the pathways. Since more than 90 percent of the ATP produced in neuronal cells is derived from oxidative phosphorylation , we omitIn order to include the potential formation of ROS in our model, we developed a detailed submodel of the respiratory chain that takes into account the substructure of complexes I and III composed of several prosthetic groups and iron sulfur clusters see .The functional parts of complex I are the flavine mononucleotide (FMN), eight iron-sulfur clusters , and the docking site for ubiquinone. FMN accepts two electrons from NADH forming fully reduced flavine. The electrons are then successively transferred to the subsequent iron-sulfur clusters. When the first electron is moved, the flavin exists as a flavin radical. From the terminal iron-sulfur cluster N2, the electron is transferred to ubiquinone forming a bound semiubiquinone (SQ). A second electron transported from N2 to SQ generates ubiquinol that is released from complex I. This electron transfer takes place in the arm of complex I extending into the mitochondrial matrix, whereas the ubiquinone/ubiquinol conversion is located at the n-site of the inner mitochondria membrane. The transfer of one electron from N6b to N2 and from N2 to ubiquinone or semiubiquinone is linked with the export of one proton from the matrix into the cytosol. In the model, the electron state of complex I is represented as an array of integer numbers where the reduction of the flavinmononucleotide, each iron-sulfur cluster, and the existence of the bound semiubiquinone are encoded. All reactions are modeled as reversible mass action kinetics. Model simulations show that lumping together the complexes N1b, N4, N5, N6a, and N6b gives similar results as the full model but reduces the number of state variables from 1536 to 96.p), the low b-type heme (bL), the high b-type heme (bH), and the binding site for ubisemiquinone at n-site (SQn). The spatial arrangement of these redox carriers enables the transduction of two electrons from one molecule ubiquinol to two molecules of cytochrome c via the q-cycle mechanism. Ubiquinol diffuses from n- to p-site, reacts with oxidized Fe-S, thereby reducing it, and generates a bound semi-ubiquinon at p-site. The bound semi-ubiquinon reduces bL at p-site and free ubiquinon diffuses back to n-site. Only after release of ubiquinone from p-site, reduced Fe-S is able to transfer its electron to oxidized cytochrome c1, which passes it on to cytochrome c. The reduced bL  transfers its electron to oxidized bH. The reduced bH reacts with n-site ubiquinone to bound semi-ubiquinon. In a second round, reduced bH transfers its electron to bound semi-ubiquinon. In this way one electron is recycled in each turnaround of ubiquinol, and two protons are transferred from the mitochondrial matrix to the intermembrane space/cytosol per electron transferred to cytochrome c. Importantly, it is assumed that electrons are transferred from ubiquinol at p-side in a two-step process, first reducing the Fe-S and binding of SQp and afterward reducing bL, and release of Qp. As long as SQp is bound, Fe-S cannot transfer the electron to c1. This gives 48 state variable and 88 reactions for complex III. All electron transitions in complexes I and III are modeled as reversible mass action kinetics. For further details, see supplemental information in Supplementary Material available online at doi:10.1155/2012/757594.Each state of complex III is described as an array describing the reduction and binding states of its functional parts: cytochrome c1 (c1), the iron sulfur cluster (Fe-S), the binding site for ubisemiquinone at p-site  are specific for neuronal and brain tissue and based on extensive literature research. VThe model was implemented in MATLAB . The extended model of complex I of the respiratory chain was implemented in C++  and integrated with help of the ODE integration package CVODE (SUNDIALS). The developed source code can be provided on request.max values) such that measured intramitochondrial metabolite concentrations .p of complex III for all ATP demands, that is, KGDHC inhibition reduces ROS production in the respiratory chain by lowering the redox state of the mitochondrion. Since KGDHC itself is also an ROS producer and ROS production by this enzyme decreases with decreasing mitochondrial redox state [Since KGDHC is especially susceptible to ROS, we examined if the reduction of KGDHC activity has an effect on the occupation state of the confirmed relevant ROS producing sites . We founox state , endogenIt has to be noted that our model does not include the impairment of RC complexes by ROS as it has been observed in neurodegenerative diseases . A reduHere, we focused on the effects of KGDHC inhibition on the energy metabolism and showed that for a given degree of inhibition the impairment depends crucially on the functional state of the cell. For ROS production, KGDHC inhibition alone does reduce the ROS generation in the respiratory chain. Nevertheless, the detailed description of the RC in our model provided further arguments for the relative importance of ROS producing sites proposed in the literature.Taken together, our model helps to elucidate the causal chain of molecular events connecting reduced KGDHC activity to the energetic breakdown of neuronal cells during development of neurodegenerative diseases.The supplementary material contains the detailed kinetics of the model. It is subdivided into Oxidative phosphorylation, Electrophysiology containing the respiratory chain, and the citric acid cycle (CAC).Click here for additional data file.


One major goal in Neuroscience is the development of strategies promoting neural plasticity in the adult central nervous system, when functional recovery from brain disease and injury is limited. New evidence has underscored a pivotal role for cortical inhibitory circuitries in regulating plasticity both during development and in adulthood. This paper summarizes recent findings showing that the inhibition-excitation balance controls adult brain plasticity and is at the core of the pathogenesis of neurodevelopmental disorders like autism, Down syndrome, and Rett syndrome. The term “plasticity” refers to the ability of the nervous system to reorganize its connections functionally and structurally in response to changes in environmental experience, underlying the adaptive development of neuronal circuitry. The existence of time windows in early postnatal life  during which neural circuits display a heightened plasticity in response to external stimuli has been established for various brain regions subserving major behavioural functions . AftIn the last 50 years, great effort has been made to elucidate cellular and molecular mechanisms underlying the activation and regulation of critical periods in the brain. Unravelling these processes may potentially enable researchers to enhance plasticity in the adult brain. Moreover, a detailed knowledge of the events involved in the maturation and plasticity of neuronal circuitry would be a determinant in improving our understanding of the aetiology of developmental brain disorders. Although a complete picture in the field is still lacking, a large body of evidence has been accumulated see, , 10). In. In10]).By sculpting the pattern and timing of neuronal electrical activity, inhibitory GABAergic circuits are an ideal candidate for regulating the processes of experience-dependent synaptic modifications. Taking advantage of gene-targeting technology, this hypothesis has been directly tested by abolishing the expression of the 65 kD isoform of GABA-synthetic enzyme, hence reducing activity-dependent GABA synthesis and release at synaptic terminals. Mice that carry such a disruption of the GAD65 gene do not exhibit ocular dominance plasticity in response to monocular deprivation; only an enhancement of inhibition achieved with local delivery of diazepam enables a full rescue of ocular dominance plasticity in these mice . Converging results obtained with different experimental approaches have subsequently confirmed the key role of GABAergic inhibition in brain development and plasticity e.g., –17). It . It 17])A antagonist) directly into the visual cortex reactivates ocular dominance plasticity in response to monocular deprivation in adult rats [One of the major challenges in neuroscience is the development of strategies aimed at promoting nervous system plasticity in adulthood, when recovery from injury and functional rehabilitation are severely hampered. Recently, new evidence has challenged the classic dogma that ocular dominance plasticity is a physiological phenomenon exclusively restricted to the early postnatal development and pointed to a reduction of intracortical inhibition levels as a crucial step for the restoration of plasticity processes in the adult brain. The most direct demonstration that GABAergic inhibition limits plasticity in the adult visual cortex derives from a recent study reporting that pharmacological reduction of intracortical inhibition obtained through the infusion of either MPA (an inhibitor of GABA synthesis) or picrotoxin  representation resulting from transient exposure to sound stimuli . StrikinThus, reduction of GABAergic inhibition may emerge as a common feature of the strategies that successfully reopen a period of stimulus exposure-based plasticity in the adult brain , 28, 35.During the critical period, the high susceptibility of neuronal connections to experience-dependent changes is essential for a proper maturation of normal sensory functions. This high potential for plasticity, however, may also favour the emergence of developmental pathological states when an anomalous perturbation of sensory-driven activity takes place. A paradigmatic case is that of amblyopia, a widely diffused and still untreatable pathology of the visual system affecting 2–4% of the total world population . AmblyopIt is currently accepted that, due to a lack of sufficient residual plasticity within the brain, the reinstatement of visual functions in amblyopic subjects is possible only if corrective treatment is started early in development. The classic amblyopia therapy consists in patching or penalizing the preferred eye, thus forcing the brain to use the visual input carried by the weaker amblyopic eye . HoweverThe mechanisms underlying vision improvements in adult amblyopic patients remain to be elucidated, since the activation of cortical plasticity may occur at several different levels of the visual system and through a variety of neural processes. A number of studies, however, suggested that an impairment of the balance between excitation and inhibition could affect visual cortex development and that cortical overinhibition could underlie the degradation of spatial vision abilities –48. AccoEarly studies in animal models of amblyopia reported that the administration of anti-inhibitory compounds  leads to a substantial restoration of binocularity in the visual cortex , 53. RecWhile the physiological maturation of GABAergic connections is essential for a tight control of developmental cortical plasticity and for promoting the acquisition of mature sensory abilities, it is currently accepted that abnormal levels of inhibition achieved during development can cause pathological states of severe brain disability , 57, 58.Rett syndrome is a progressive developmental disorder characterised by mental retardation and severe dysfunction in motor coordination skills , predomiDetailed electrophysiological analysis of these animal models showed a reduction of neuronal activity in cortical and hippocampal neurons due to a shift in the balance between cortical excitation and inhibition in favour of inhibition , 68 and Since the gene encoding BDNF is under MeCP2 regulation  and the Down syndrome is caused by tri-plication of chromosome 21 (Chr21) and is the most common genetic cause of mental retardation . People A large number of studies have shown that the cognitive impairment displayed by Ts65Dn mice is mainly related to excessive levels of inhibition in temporal lobe circuitry, causing a failure of long-term synaptic plasticity in the hippocampus –100. TheOne of the major challenging tasks in the field of Down syndrome therapy is unravelling dosage-sensitive genes whose dysfunction, due to the presence of an extra copy of chromosome 21, might be responsible for the main functional and morphological defects. A recent study by Chakrabarti et al.  has showDespite the increasing knowledge concerning the molecular mechanisms underlying Down syndrome, a suitable treatment for this disorder is still lacking. Since environmental enrichment is particularly effective in reducing GABAergic inhibition , it may in vivo investigation of structural brain morphology in people with autism. Several regions have been reported to be enlarged or reduced relative to controls, but a large consensus on these results is currently missing .The aetiological mechanisms of autism are at present poorly defined. Despite a likely contribution of environmental causes, genes play a crucial role in the onset of this pathology with concordance between monozygotic twins reaching 90%, as compared with less than 10% for dizygotic twins and siblings , 111. OnConverging results have pointed to an increased excitation/inhibition ratio in sensory, mnemonic, social, and emotional systems as a core mechanism underlying neurological and behavioural deficits observed in autistic patients . ConsistOn the cellular level, it has been shown that in a valproic acid rat model of autism, the amygdala is hyperreactive to electrical stimulation and displays enhanced synaptic plasticity as well as defective inhibitory transmission . MoreoveSince autism is a developmental disorder, the imbalance in the ratio of excitation versus inhibition could result from abnormal processes during neural circuit maturation. Indeed, defects in synaptogenesis and synaptic refinement have been suggested to be a leading cause of autism, and mutations of genes that normally control the patterning of synaptic maturation of specific neuronal subpopulations have been shown to segregate with the pathological phenotype , 128. AmIt should be pointed out, however, that the exact role of excitation/inhibition balance in autism is still debated. Indeed, an increased inhibitory synaptic transmission and a decreased glutamatergic excitation have been also reported in different transgenic mouse models of autism , 134.Altogether the results reviewed here show how dramatic can be the influence exerted by inhibitory transmission on brain plasticity. Not only are these findings crucial to our knowledge about the molecular mechanisms underlying the expression and regulation of plasticity processes, but they also have strong implications for the treatment of neurological disorders related to an aberrant development of GABAergic circuits. The possibility of rescuing a normal phenotype in animal models of these pathologies by manipulating levels of intracortical inhibition draws attention on the GABAergic system as an eligible candidate for the development of new therapeutic strategies.


Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by invitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation.Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the  PPP family STPs share a limited number of conserved catalytic subunits. However, the diverse physiological roles are accomplished by a large number of regulatory domains. The PPM family comprises PP2c and mitochondrial pyruvate dehydrogenase phosphatase that are Mg2+-dependent. Data on the biological function of PPM family STPs is scarce. PTPs share the common catalytic motif, Cys-X5-Arg Drosophila genome Reversible protein phosphorylation regulates a multitude of biological functions in eukaryotes. Levels of protein phosphorylation are controlled by kinases that catalyze the phosphorylation of proteins at serine, threonine, and tyrosine residues, and phosphatases that remove phosphoryl groups from proteins. Protein phosphorylation mediates protein-protein interaction, regulates enzyme activity, controls subcellular localization, influences protein stability, and regulates the activity of ion channels. Eukaryotic protein kinases comprise a single protein superfamily sharing a common catalytic structure. They can be subdivided into eight different families  according to their structural and functional properties Drosophila photoreceptor cells is a G protein-mediated, phospholipase Cβ-dependent signaling pathway that is activated through absorption of a photon by rhodopsin and terminates in the opening of the cation channels TRP and TRPL. Known kinases and phosphatases of proteins of the Drosophila phototransduction cascade comprise eye protein kinase C (eye-PKC) in vitroDrosophila PP2A encoded by the mts locus has been indirectly shown to dephosphorylate INAD 32P]ATP and head extracts from heterozygous mts mutant flies resulted in considerably elevated INAD phosphorylation levels as compared to incubation with head extracts from wild type control flies mts mutation had no obvious effect on TRP phosphorylation. A heterozygous mts mutation rescued the prolonged deactivation phenotype of the visual response in P209inaC flies lacking eye-PKC Drosophila rhodopsin becomes phosphorylated upon light activation and this phosphorylation is removed when the receptor is reconverted to its inactivated state gprk1 gene rdgC) locus encodes the corresponding phosphatase required for rhodopsin dephosphorylation rdgC mutant fly exhibits severe light-dependent retinal degeneration and defective deactivation kinetics of the visual response. Expression of a truncated rhodopsin protein lacking the putative phosphorylation site targeted by RDGC in an rdgc mutant background rescued both the retinal degeneration and the slow deactivation phenotype The visual transduction cascade in in vivo. Here, we present a detailed analysis of the dependence of the TRP phosphorylation sites on the phototransduction cascade and on light. Using two phosphospecific antibodies, we determined the occupancy of two phosphorylation sites to be at most 56% (Thr849) and 34% (Thr864) in the light, and we present the results of a candidate screen to identify kinases and phosphatases involved in regulating the phosphorylation state of the Drosophila TRP protein. We provide in vivo evidence that eye-PKC is one but not the only protein kinase involved in TRP phosphorylation.Recently, we reported the identification of 21 TRP phosphorylation sites Drosophila melanogaster were used: w Oregon R, yw;;P343trpw;;P365trp17yw;;ninaEw;1Gαqw,P24norpAw;P209inaCw;+;D621Gtrp,P343trpw;306rdgC849 and Thr864 in different mutants of the phototransduction cascade using phosphospecific antibodies, flies were dark-adapted for 12–18 h and were then illuminated for 1 h or vice versa before they were subjected to Western blot analysis. To screen candidate mutants for an alteration in TRP phosphorylation at Thr849 and Thr864, flies were light- or dark-adapted for 12–18 h and were then subjected to the opposite light condition for 1 h prior to Western blot analysis. Dark-kept flies were dissected under dim red light , and flies kept in white light were dissected under this light condition.The following strains and mutants of P24norpA, and P365trp/+. Immunoprecipitation was carried out as described before using a polyclonal α-TRP antibody Flies used for immunoprecipitation were wild type, P24norpA, P365trp/+), six independent experiments were carried out. Immunoprecipitated TRP from three of these experiments was digested with trypsin and TRP from the other three experiments was digested with chymotrypsin. For each experiment, two technical replicates were carried out. Thermo raw files of the acquired nano-LC-MS/MS runs were processed and analyzed using Progenesis LC-MS software . The LC-MS runs in each dataset were automatically aligned using one LC-MS run as reference. Two-dimensional maps (m/z versus retention time) of each run were generated, aligned to the reference run, and used to find a common set of feature outlines . Peptide abundance values were subsequently calculated as the sum of the peak areas within the isotope boundaries of the feature. Abundance values for each feature were normalized to account for different amounts of sample or run-to-run variations. The normalization process implemented in the Progenesis LC-MS software is based on the assumption that most features show no change in abundance in a given experiment. For each feature, the software calculates a quantitative abundance ratio between the run being normalized and the reference run. The normalization factor for each run is then calculated from the distribution of the log of the abundance ratios using a recursive median approach. Further details about the calculation of peptide abundances and the normalization process in the Progenesis LC-MS software can be found at the company’s website (http://www.nonlinear.com/support/progenesis/lc-ms/faq/). Before quantification, the number of features was reduced to signals with a charge state of +2, +3, +4, and +5. TRP phosphopeptides were identified by means of a Mascot generic file created by the Progenesis LC-MS software using the same search parameters as described previously In-gel digestion was performed as previously described 2-CGRKK(pT)QKGD-CONH2, comprising pT849, and NH2-CARKN(pT)FASD-CONH2, comprising pT864, were synthesized and injected into rabbits (Pineda antibody service). Affinity purification was carried out as described previously In order to produce antisera, two phosphopeptides, NHImmunocytochemistry was carried out as described previously 1D gel electrophoresis and protein transfer onto nitrocellulose membranes (Roth) was carried out as described previously, with modifications http://flyatlas.org) were purchased from Bloomington Stock Center. Flies were light- or dark-adapted for 12–18 h and were then switched to the opposite light condition for 1 h before heads were subjected to Western blot analysis using the two phosphospecific antibodies.Candidate mutants in kinases and phosphatases with an eye mRNA signal of greater or equal to 70 according to the flyatlas gene expression database  were used. Flies were light- or dark-adapted over night and TRP extracted from fly heads was purified by immunoprecipitation and SDS PAGE. The coomassie-stained TRP band was in-gel digested with either trypsin or chymotrypsin and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). Identification and quantification of phosphopeptides from wild type flies yielded similar results as before P24norpA flies differed markedly from light-adapted wild type flies  in the light. In the P365trp/+ flies, the retrieval rate of phosphopeptides from tryptic and chymotryptic digests was lower than in the other flies analyzed. Overall, TRP phosphorylation in dark-adapted P365trp/+ flies differed markedly from TRP phosphorylation in dark-adapted wild type flies. TRP phosphorylation in dark-adapted P365trp/+ flies rather resembled the situation found in light-adapted wild type flies. Taken together, these results suggest that phosphorylation and dephosphorylation of most light-dependent TRP phosphorylation sites depend on phospholipase Cβ and on TRP ion channel activity.Previously, we used a phosphospecific antibody directed against TRP phosphorylated at Ser936 and showed that dephosphorylation of Ser936 depended on the phototransduction cascade. Ser936 was the only site that was dephosphorylated in the light and became phosphorylated in the dark According to predicted kinase consensus sequences and due to our findings showing that some sites become phosphorylated in the light while Ser936 even shows enhanced phosphorylation in the dark, it is likely that different kinases and phosphatases are involved in TRP phosphorylation. For further analysis, we focused on two sites, Thr849 and Thr864, that displayed a highly significant difference between light- and dark-adapted flies with much stronger phosphorylation in the light. Thr849 was predicted to be a protein kinase C phosphorylation site and Thr864 to be a protein kinase G phosphorylation site (NetPhosK phosphorylation site prediction tool). For a detailed analysis of TRP phosphorylation at Thr849 and Thr864, and for performing a screen to identify kinase(s) and phosphatase(s) of these sites, we generated two phosphospecific antibodies, α-pT849 and α-pT864, that specifically detected TRP phosphorylated at Thr849 or at Thr864, respectively. The specificity of the antibodies for the phosphorylated form of these sites was confirmed by dot blot analyses of the phosphopeptides used to generate the antibodies and the corresponding non-phosphorylated peptides . We alsoP343trp fly heads such that the amount of TRP was different while the total protein amount loaded on the gel was constant (3 heads). In the analyzed range (TRP from 3 to 0.375 heads), we observed good correlation  between the amount of TRP applied and the obtained Western blot signals  were used. Phosphorylation of both Thr849 and Thr864 was considerably increased in dark-adapted P365trp/+ flies as compared to wild types. Taken together, we suggest that Ca2+ is the trigger for phosphorylation of both Thr849 and Thr864 because on the one hand Ca2+ influx into the photoreceptor cells is sufficient to evoke TRP phosphorylation but on the other hand a completely functional phototransduction cascade is not capable of triggering phosphorylation when TRP is impermeable for ions.In order to demonstrate that the phosphospecific antibodies can be used to assess the phosphorylation of Thr849 and Thr864 quantitatively, we tested the linearity of the obtained signal intensities with the two phosphospecific antibodies by Western blot analysis using protein extracts containing different amounts of the TRP protein. To ensure equal concentrations of all other proteins, we mixed extracts from wild type fly heads with extracts from  signals . We then signals  and to de sample . In illue sample . The ninvice versa, already one min after changing the light conditions we detected significant changes in the phosphorylation state. However, phosphorylation and dephosphorylation kinetics were considerably faster for the Thr864 phosphorylation site as compared to Thr849.To investigate the time course of Thr849 and Thr864 phosphorylation and dephosphorylation, wild type flies were dark-adapted or illuminated for 12–18 h and were then subjected to the opposite light condition for different periods of time . For botinput/α-TRPinput)×(α-TRPIP/α-pTRPIP)×100%, where α-pTRPinput and α-TRPinput are the signal intensities obtained with the phosphospecific and the generic TRP antibodies in the extracts before immunoprecipitation, and α-pTRPIP and α-TRPIP are the signal intensities from the immunoprecipitates. Assuming that all TRP molecules immunoprecipitated with the phosphospecific antibodies were phosphorylated, this analysis revealed that in the light, at most 56% (+/−13.8%) TRP molecules were phosphorylated at Thr849 and at most 34% (+/−19.6%) at Thr864  in this initial screen were tested again two more times. Out of the 85 tested mutants, eight displayed altered phosphorylation of Thr849 and Thr864. The rescreen revealed significant effects on phosphorylation in four mutants in case of Thr849 and four mutants in case of Thr864 . As expected, the amount of TRP detected with the generic TRP antibody was much lower (about 7fold lower for α-Thr849 as well as α-Thr864) in these immunoprecipitates as compared to immunoprecipitates obtained from light-adapted flies. This difference in the amount of immunoprecipitated TRP correlates well with the difference in phosphorylation at Thr849 and Thr864 that was determined by quantitative Western blot analysis to be 5.4fold higher for Thr849 and 3.9fold higher for Thr864 in the light. It is important to note that TRP is likely immunoprecipitated as tetramers. Thus, using phosphospecific antibodies to pull down phosphorylated TRP may result in coprecipitation of non-phosphorylated TRP. This would lead to an overestimation of phosphate occupancy. Additionally, some uncertainty remains with respect to the signal of the two phosphospecific antibodies on Western blots with extracts from dark-adapted flies. These signals could result from residual phosphorylation at Thr849 and Thr864 in the dark or from cross-reactivity of the phosphospecific antibodies with non-phosphorylated TRP. In the latter case, the phosphate occupancy of Thr849 and Thr864 would be slightly overestimated. Even then, since TRP channels are tetramers and phosphorylation of one subunit may already affect channel function, these phosphorylations could potentially regulate all TRP channels present in the photoreceptor.P365trp/+ mutant, elevated intracellular Ca2+ levels might enhance phosphorylation of Thr849 and Thr864. However, phosphorylation of Thr849 was stronger in light- than in dark-adapted P365trp/+ flies . We therefore conclude that phosphorylation of Thr849 not only depends on the phototransduction cascade and Ca2+ but also on eye-PKC. The current finding that TRP phosphorylation at Thr849 depends on eye-PKC seems to conflict with our previous finding that TRP was still light-dependently phosphorylated in the eye-PKC null mutant Drosophila, PKC53E pkc53e null mutant exhibited reduced phosphorylation of TRP at Thr849. We therefore conclude that in vivo, eye-PKC and PKC53E act redundantly in phosphorylating TRP at Thr849 and possibly at other sites that are embedded within PKC consensus sequences. Non-canonical PKCs that were tested in our screen  were not found to affect phosphorylation at Thr849. Enhanced phosphorylation at Thr849 in the light was observed in two of the tested mutants, rolled and snf1a. The rolled (rl) gene encodes a mitogen-activated protein kinase rolled mutant on TRP phosphorylation. The 1rl allele used for the screen is a hypomorphic loss-of-function allele. The snf1a gene encodes an AMP-activated protein kinase that has been implicated in the cellular energy sensing pathway Our candidate screen for kinases and phosphatases revealed that phosphorylation of Thr849 was significantly reduced in the eye-PKC mutant casein kinase 1α, licorne, tao-1 and mppe. However, in neither of these mutants light-dependent phosphorylation of Thr864 was completely abolished, ruling out the possibility that one of these kinases is uniquely responsible for phosphorylation of this site. Drosophila casein kinase 1α (CK1α) is a negative regulator of the wingless (Wg) pathway. It phosphorylates the Armadillo protein and thereby stimulates its degradation ck1α mutant we used in our screen harbors a transposable element disrupting the ck1α gene. Licorne is a MAP kinase kinase www.flybase.org). TAO-1 is a protein serine/threonine kinase of the STE family that controls microtubule dynamics, animal behavior, brain development, and epithelial tissue growth tao-1 is strongly expressed in the eye and ovary. The tao-1 mutant used in our screen harbors a transposable element in the 5′ region of the tao-1 gene.The TRP phosphorylation site Thr864 showed reduced phosphorylation in light-adapted flies in four of the tested mutants, 2+/Zn2+-dependent metallophosphoesterase that desphosphorylates and thereby activates α-Man-II mppe mutant fly exhibits incomplete deglycosylation of Rh1. Subcellular localization and signaling of Rh1 are not affected in this mutant, but incomplete deglycolsylation causes morphological and functional defects in aged flies. In our screen, the mppe mutant exhibited a reduced phosphorylation of Thr864. Since MPPE is a protein phosphatase, loss of MPPE activity should result in a higher degree of phosphorylation if MPPE would directly dephosphorylate the respective site. We therefore conclude that MPPE is indirectly involved in Thr864 phosphorylation, for example by activating a kinase through dephosphorylation or by affecting the glycosylation state of proteins involved in TRP phosphorylation.MPPE is a Mn2+.Our data suggest that regulation of most TRP phosphorylation sites depends on the phototransduction cascade. Although the TRP phosphorylation pattern is shaped by different kinases and phosphatases, most of them seem to be regulated by CaFigure S1Representative MS/MS spectra of newly identified TRP phosphorylation sites. Figures S1A, S1C, S1D, S1E, S1F, and S1G show fragmentation spectra and tables derived from tryptic TRP peptides and (DOCX)Click here for additional data file.Figure S2Validation of antibodies. A, To check for cross-reactivity with other phosphorylation sites or the non-phosphorylated peptide, 2.5 µg of phospho- and dephosphopeptides were applied onto nitrocellulose membranes and the membranes were then blocked and incubated with α-pT849 or α-pT864 antibodies and a secondary α-rabbit IgG conjugated to horse radish peroxidase. Enhanced chemiluminescence signals were recorded. Phosphopeptides were NH2-CGRKK(pT)QKGD-CONH2 containing phosphorylated Thr849 of TRP, NH2-CARKN(pT)FASD-CONH2 containing phosphorylated Thr864 of TRP, and NH2-CADEVpSLADD-CONH2 containing phosphorylated Ser936 of TRP. The dephosphopeptides were similar to the respective phosphopeptides except for the lack of the phosphoryl groups. B, To check linearity of the signal intensities obtained with the antibodies, different amounts of protein extracts from wild type heads were supplemented with protein extracts from P343trp null mutant heads to ensure equal overall protein content. Three head equivalents were loaded onto a gel and subjected to Western blot analysis using α-TRP antibody. After stripping, either α-pT849 or α-pT864 antibodies were used. These experimental conditions correspond to those used in the screen to identify kinases or phosphatases of TRP at T849 and T864. Error bars show SEM (N = 4).(TIF)Click here for additional data file.Figure S3Immunostaining of isolated ommatidia to investigate the subcellular localization of TRP in mutants used in the screen. Immunostaining of isolated ommatidia was carried out essentially as described (20). Briefly, flies were decapitated and heads were cut into halves. Eyes were dissected in 100 mM phosphate buffer, pH 7.2, using forceps. Fragments were pipetted through a 200 µl pipette tip several times and transferred to a lysine-coated cover slide and air-dried. Fixation was accomplished by a 10 min incubation with 2% paraformaldehyde in 1×PBS , pH 7.2, followed by a two 5 min washes with 1×PBS-S (PBS containing 0.1% (w/v) saponine). Ommatidia were permeabilized by incubation in cytoskeleton buffer  for 8 min at RT and washed three times with PBS-S for five min each. Incubation in primary antibody (α-TRP) was carried out in blocking solution  over night at 4°C. Ommatidia were washed three times with PBS-S and then incubated for 2–4 h in secondary antibody (α-mouse Cy5 (Dianova)) and AlexaFluor 546-coupled phalloidin (Invitrogen) in blocking solution at ambient temperature followed by a three-times-wash with aqua bidest. Ommatidia were mounted with Mowiol 4.88 and were examined with an AxioImager.Z1m microscope  with the ApoTome module (Zeiss) and recorded with an AxioCam MRm (Zeiss). Scale bar, 20 µm.(TIF)Click here for additional data file.Table S1Peptides used for quantification of TRP phosphorylation sites. Phosphorylated amino acids are underscored. p-values were calculated by an unpaired t-test using the normalized abundances from the two conditions given in each table. SD, standard deviation.(DOCX)Click here for additional data file.Table S2Listing of flies used for the candidate screen to identify kinases and phosphatases of TRP and detailed results.(DOCX)Click here for additional data file.


Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Gaq or Gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga13 siRNA, and by Ga13 minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis (U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632). PAR2-AP induced phosphorylation of MLC20, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to release catalytic subunit of PKA from its binding to IkBa and phosphorylation of RhoA at Ser188.We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated G Protease-activated receptors (PARs) comprise a family of G protein-coupled receptors with a unique activation mechanism involving proteolytic cleavage of the extracellular N-terminus domain of the receptor to expose a new built-in N-terminus part of the receptor that acts as a ligand . Molecular cloning studies have identified four PARs and these are activated by a large number of proteases PARs are located in several cell types and play an important role in many physiological functions. The gastrointestinal (GI) tract, of all the body systems, is exposed to the widest array of proteases in both normal situations and during diseases The role of PAR2 in GI motility is complex, and varies with species and tissue. In vivo studies have demonstrated that activation of PAR2 enhances GI transit q, Gi and G13, and stimulation of PI hydrolysis and RhoA/Rho kinase activity to induce muscle contraction. A novel cAMP-independent PKA pathway for feedback inhibition of PAR2-stimulated RhoA was demonstrated involving activation of NF-kB and release of PKA from its binding to IkBa complex leading to phosphorylation of RhoA at Ser188.The present study focused on characterizing expression of PAR2 and the signaling pathways to which these receptors are coupled in freshly dispersed and cultured smooth muscle cells of rabbit stomach. The small synthetic peptide SLIGRL, corresponding to tethered ligand sequences, was used to selectively activate PAR2 and to identify the signaling pathways activated by PAR2. Our results demonstrate that PAR2 are coupled to G125I]cAMP, [γ-32P]ATP, [32P]Pi, [35S]GTPγS, and [3H]myo-inositol were obtained from Perkin Elmer Life Sciences, Boston, MA; Collagenase CLS type II and soybean trypsin inhibitor were obtained from Worthington, Freehold, NJ; Western blotting, Dowex AG-1×8 resin (100–200 mesh in formate form), chromatography material and protein assay kit from Bio-Rad Laboratories, Hercules, CA; antibodies to Gaq, Gai1, Gai2, Gai3, Ga12, Ga13, Gas, phospho-specific MYPT1 (Thr696), MLC20 (Ser19) and CPI-17 (Thr38), Rho kinase, PKA catalytic subunit, IKK2(ser177/181), IkBa, and p65 subunit were from Santa Cruz Biotechnology, Santa Cruz, CA; myelin basic protein (MBP) was from Upstate Biotechnology; ML-9 was from Biomol Research Laboratories, Plymouth Meeting, PA; bisindolylmaleimide, Y27632, pertussis toxin, cAMP; Clostridium botulinum C3 exoenzyme, IKK-2 Inhibitor IV (IKK IV), MG-132, myristoylated PKI and U73122 were from Calbiochem, La Jolla, CA; RNAqueous™ kit was from Ambion, Austin, TX; Effectene Transfection Reagent, QIAEX®II Gel extraction Kit and QIAprep®Spin Miniprep Kit were from Germantown, MD; PCR reagents were from Applied Biosystems, Roche; SuperScript™ II Reverse Transcriptese and TOPO TA Cloning® Kit Dual Promoter were from Invitrogen, Grand Island, NY; EcoR I was from New England Bio Labs, Ipswich, MA; All other chemicals were from Sigma, St. Louis, MO.PAR2-activating peptide  was obtained from Bachem, Torrance, CA; ), IKK2 (IKK2[K44A]), IkB (IkBa[S32A/S36A]), and phosphorylation-site deficient RhoA (RhoA[S188A]) were subcloned into the multiple cloning site (EcoR I) of the eukaryotic expression vector pcDNA3. Recombinant plasmid DNAs were transiently transfected into the muscle in primary culture using Effectene Transfection Reagent (QIAGEN) for 48 h. Cells were co-transfected with 2 µg of pcDNA3 vector and 1 µg of pGreen Lantern-1 DNA. Transfection efficiency was monitored by the expression of the green fluorescent protein using FITC filters. Control cells were transfected with vector alone q, Ga12, and Ga13, and human Gai were amplified by PCR and verified by DNA sequencing as previously described i in random order was synthesized and ligated into pcDNA3.1(+) as a control minigene. All Ga minigene constructs used for transfection experiments were purified with an endotoxin-free maxiprep kit (Qiagen) following the manufacturing protocol.The cDNA sequences encoding the last COOH-terminal 11 amino acids of mouse Gaq/11 and Ga13 with lowest predicted off-target potential and 100% homology with the conserved sequences based on human, rat and mouse sequences were selected and obtained from Life Technologies . PAR-2 (ID#s453494): sense: 5′ CCUCCUCUCUGUCAUCUGGTT 3′, antisense: 5′ CCAGAUGACAGAGAGGAGGTC3′; Gaq/11 (ID# s5862): sense: 5′ GCAUCAUCGAGUACCCUUUTT 3′, antisense: 5′ AAAGGGUACUCGAUGAUGCCG 3′; and Gα13 (ID#20990): sense: 5′ GCAACGUGAUCAAAGGUAUTT 3′, antisense: 5′ AUACCUUUGAUCACGUUGCTG3′. siRNA transfection efficiency assays were performed with varying concentrations of siRNA (5–50 nM) and it was determined that 40 nM siRNA provided optimal transfection efficiency (data not shown). Non-specific siRNAs were used to determine the efficiency of siRNA. The siRNA transfection was performed using siRNA transfection reagent and siRNA transfection medium according to the manufacturer's instructions.The siRNAs  for PAR-2, Ga35S]GTPγS (5′-O-3-thiotriphosphate) using the method of Okamoto et al as described previously 6 cells/ml) were homogenized in 20 mM HPES medium (pH 7.4) containing 2 mM MgCl2, 1 mM EDTA and 2 mM DTT. After centrifugation at 30,000 g for 15 min, the crude membranes were solubilized for 60 min at 4 °C in 20 mM HEPES medium (pH 7.4) containing 2 mM EDTA, 240 mM NaCl, 0.5% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-pro-panesulfonate), 2 mM PMSF, 20 µg/ml aprotinin, and 20 µM leupetin. The membranes were incubated for 20 min at 37°C with 60 nM [35S]GTPgS in the presence or absence of PAR2-AP in a solution containing 10 mM HEPES (pH 7.4), 100 µM EDTA and 10 mM MgCl2. The reaction was terminated with 10 volumes of 100 mM of Tris-HCl medium (pH 8.0) containing 10 mM MgCl2, 10 mM NaCl and 10 µM GTP, and the mixture was placed in wells precoated with specific antibodies to Gaq, Gai1, Gai2, Gai3, Ga12, Ga13, and Gas. Coating with G protein antibodies (1∶1000) was done after the wells were first coated with anti-rabbit IgG (1∶1000) for 2 h on ice. After incubation for 2 h on ice, the wells were washed three times with phosphate buffer saline solution (PBS) containing 0.05% Tween-20 and the radioactivity from each well was counted by liquid scintillation. The amount of [35S]GTPgS bound to the activated Ga subunit was expressed as counts per minute (cpm) per milligram of protein.G proteins selectively activated by PAR2-AP were identified from the increase in Ga binding to the [6 cells/ml) were labelled with myo-[3H] inositol (15 µCi/ml) for 3 h at 31°C. Then cells were centrifuged at 350 g for 10 min to move excess [3H]inositol and resuspended in 10 ml of fresh medium. PAR2-AP was added to 0.5 ml of cell suspension and the mixture was incubated in a shaking water bath for 1 min. Cultured smooth muscle cells were labelled with [3H]myo-inositols (0.5 µCi/ml) for 24 h in inositol-free DMEM medium. The cultures were washed with phosphate-buffered saline (PBS) and treated with PAR2-AP for 1 min in HEPES medium (pH 7.4). The reaction was terminated by the addition of chloform:methanol:HCl (50∶100∶1 v/v/v). After chloroform (340 µl) and water (340 µl) were added, the samples were vortexed and the phase was separated by centrifugation at 1000 g for 15 min. The upper aqueous phase was applied to a column containing 1 ml of 1∶1 slurry of Dowex AG-1 X8 resin (100–200 mesh in formate form) and distilled water. Total inositol phosphates were eluted with 6 ml of 0.8 M ammonium formate-0.1 M formic acid. The eluates were collected into scintillation vials and counted in gel phase after addition of 10 ml of scintillant. The results were expressed as counts per minute per mg protein.Total inositol phosphates were measured by anion exchange chromatography using the method of Berridge et al 2, 10 mM EDTA, and 1 mM DTT. Twenty microliters of Rho kinase immunoprecipitates were added to the reaction mixture containing 100 mM Tris-HCl (pH 7.4), 1 M KCl, 50 mM MgCl2, 1 mM DTT, 1 mM ATP, and 10 µCi of [g-32P]ATP  along with 5 µg of myelin basic protein, followed by incubation for 15 min at 37 °C. Phosphorylation of myelin basic protein was absorbed onto phosphocellulose disks, and free radioactivity was removed by washing 3 times with 75 mM H3PO4. The amount of radioactivity on the disks was measured by liquid scintillation. The results are expressed as counts per milligram protein per minute.Rho kinase activity was measured by an immunokinase assay as previously described 125I]cAMP 6 cells/ml) of cell suspension was treated for 60 s with forskolin (10 µM) in the presence of 100 µM isobutylmethyl xanthine, either alone or in combination with PAR2-AP (1 µM). The reaction was terminated with cold 6% trichloroacetic acid (v/v) and vortexed vigorously. After centrifugation, the supernatants were extracted three times with water-saturated diethyl ether to remove the tricholoroacetic acid and the samples were then lyophilized and frozen at –20°C. The samples were reconstituted for radioimmunoassay in 50 µl of 50 mM sodium acetate (pH 6.2) and acetylated with triethylamine/acetic anhydride (2∶1 v/v) for 30 min. Cyclic AMP was measured in duplicates using 100 µl aliquots and the results were computed from a standard curve using the Prizm@. The results are expressed as pmol of cAMP/mg protein.Adenylyl cyclase activity was measured by the formation of cAMP in response to agonists by radioimmunoassay using [32P incorporated into each protein after immunoprecipitation with specific antibody to RhoA as previously described 32P] orthophosphate for 4 h. One milliliter of samples was incubated with PAR2-AP (1 µM) for 10 min and the reaction was terminated by rapid centrifugation. The pellet was homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5),150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 10 mM sodium pyrophosphate, and protease inhibitor cocktail (2 µl/ml). Cell lysates were separated by centrifugation at 13,000 g for 10 min at 4 °C, precleared with 40 µl of protein A-Sepharose, and incubated with RhoA antibody for 2 h at 4 °C and with 40 µl of protein A-Sepharose for another one hour. The immunoprecipitates were extracted with Laemmli sample buffer, boiled for 5 min, and separated by electrophoresis on SDS-PAGE. After transfer to nitrocellulose membranes [32P]RhoA was visualized by autoradiography.Protein phosphorylation was determined from the amount of 20, MYPT1, CPI-17, IKK2, and the p65 subunit of NF-kB was measured using phospho-specific antibodies and the degradation of IkB was measured using IkB antibody (1∶1000) as described previously 6 cell/ml) was treated with PAR2-AP (1 µM) and solubilized on ice for one hour in medium containing 20 mM Tri-HCl (pH 8.0), 1 mM DTT, 100 mM NaCl, 0.5% sodium dodecyl sulfate, 0.75% deoxycholate, 1 mM PMSF, 10 µg/ml of leupeptin and 100 µg/ml of aprotinin. The proteins were resolved by SDS/PAGE and electrophoretically transferred onto nitrocellulose membranes. The membranes were incubated for 12 h with phospho-specific antibodies to MLC20 (Ser19), MYPT1 (Thr696), CPI-17 (Thr38), IKK2(ser177/181), p65 subunit (Ser536), IkBa or NF-kB, and then for 1 h with horse-radish peroxidase-conjugated secondary antibody (1∶2000). The protein bands were identified by enhanced chemiluminescence reagent.Phosphorylation of MLC4 cells/ml was treated with 100 µl of medium containing various concentrations of PAR2-AP for different time periods (30 s to 10 min) and the reaction was terminated with 1% acrolein at a final concentration of 0.1%. The composition of the medium was 120 mM NaCl, 4 mM KCl, 2.6 mM KH2PO4, 0.6 mM MgCl2, 25 mM HEPES, 14 mM glucose, 2 mM CaCl2, and 2.1% Eagle's essential amino acid mixture. Concentration-response curves for PAR2-AP were constructed for the peak contraction, which occurred at 30 s after addition of PAR2-AP. The mean lengths of 50 muscle cells treated with PAR2-AP were measured by scanning micrometry and compared with the mean lengths of untreated cells. The contractile response was expressed as the percent decrease in mean cell length from control cell length.Contraction in freshly dispersed gastric circular smooth muscle cells was determined by scanning micrometry as previously described n experiments. P values were determined by an unpaired, two-tailed Student’s t test when comparing two samples, by one-way ANOVA with a Dunnett post hoc test when comparing more than two samples to a control, or by one-way ANOVA with a Tukey post hoc test when comparing multiple samples using GraphPad InStat software . Each experiment was done on cells obtained from different animals. A probability of p<0.05 was considered significant.The results were expressed as means ± S.E. of Western blot analysis of homogenates derived from rabbit gastric smooth muscle cells using antibodies to PAR2 (1∶1000 dilution)  demonstrated presence of PAR2 in smooth muscle . The spei) and -insensitive  G proteins, but the specific Gi isoforms and G12 and G13 proteins coupled to PAR2 have not been identified i1, Gai2, Gaq, and Ga13, respectively . These results suggest that PAR2 is coupled to Gi1, Gi2, Gq and G13.Studies in various tissues and cell lines suggest that PAR2 are coupled to both pertussis toxin (PTx)-sensitive  above basal levels  (i) and -insensitive (Gq) G proteins (PAR2-AP stimulated PI hydrolysis in a concentration-dependent fashion with an ECprotein) . Treatmeproteins . Stimulaproteins . TreatmeTreatment of cells with trypsin (1 µM), which activates PAR2 also stimulated PI hydrolysis  similar to that obtained with PAR2-AP. Evidence for the involvement of PAR2 receptors in the stimulation of PI hydrolysis in response to PAR2-AP was obtained using PAR2-specific siRNA. Increase in PI hydrolysis in response to PAR2-AP was significantly inhibited  in cells transfected with PAR2 siRNA .q and Gi in mediating PI hydrolysis in response to PAR2-AP was corroborated in experiments using Ga minigenes in cultured smooth muscle cells. Expression of Ga minigenes was confirmed by RT-PCR using primers corresponding to Ga insert and vector. The primers for the Ga minigenes amplified a PCR product of 434 bp when Ga insert are present (data not shown). The synthetic peptide corresponding to the COOH terminus of Ga subunits selectively antagonized G protein activation by blocking receptor-G protein interaction i and Gaq selectively blocked Gi and Gq activation, respectively q and Gai levels   (i minigene was closely similar to inhibition of PI hydrolysis by PTx in freshly dispersed muscle cells. Expression of control minigene (random minigene) had no effect on PI hydrolysis in response to PAR2-AP . Transfection of cultured muscle cells with Gaq siRNA also partially inhibited PAR2-AP stimulated PI hydrolysis . Treatment of cells transfected with Gaq siRNA and PTx blocked PI hydrolysis . The results corroborate that PI hydrolysis by PAR2 was mediated by both Gq and Gi.The involvement of Gi levels . PAR2-AP P<0.01) . Co-exprctively) . It is wq proteins in the regulation of PI hydrolysis by PAR2-AP was obtained in cultured muscle cells. The strength and duration of Ga-GTP signaling are regulated by a family of GTPase-activating proteins known as regulators of G protein signaling (RGS). Previous studies have shown that Gaq-GTPase activity is regulated by RGS4 in smooth muscle cells q-dependent activation of PLC-b1 and augmented by inactivation of RGS4.Further evidence for the involvement of Gi-coupled PAR2 to inhibit forskolin-stimulated cAMP formation. Treatment of muscle cells with forskolin (10 µM) for 10 min significantly increased cAMP formation  in the presence of 100 µM isobutyl methyl xanthine. PAR2-AP (1 µM) inhibited forskolin-stimulated cAMP formation by 61±3%  (Freshly dispersed smooth muscle cells were used to examine the ability of Gby 61±3% . Preincuby 61±3% . The res levels) .In freshly dispersed smooth muscle cells PAR2-AP stimulated Rho kinase activity by 152±12% above basal levels  . The spe13 in PAR2 mediated Rho kinase activity was obtained by expression of Ga minigenes in cultured smooth muscle cells. Expression of Ga12 or Ga13 levels was not altered by transfection of Ga minigenes  Rho kinase activation in response to PAR2-AP, whereas expression of the Ga12 minigene had no effect , but not by the expression of the Ga13 minigene (8±4% inhibition). These results suggest that PAR2-stimulated Rho kinase activity is mediated via G13 and consistent with selective activation of G13 but not G12 by PAR2-AP.PAR2-AP also stimulated Rho kinase activity in cultured smooth muscle cells (164±13% increase) that was not significantly different from the response in freshly dispersed muscle cells. Overexpression of dominant negative RhoA (RhoA[T19N]) in cultured muscle cells significantly inhibited Rho kinase activity in response to PAR2-AP , suggestinigenes . Expresso effect , Treatme13 in the stimulation of Rho kinase activity in response to PAR2-AP was obtained using PAR2-specific and Ga13-specific siRNA. Increase in Rho kinase activity in response to PAR2-AP was significantly inhibited in cells transfected with PAR2 siRNA  or Gα13 siRNA  (Further evidence for the involvement of PAR2 and Gap<0.001) .50 of 1 nM and 6 nM, respectively , by PAR2-AP was rapid (within 30 s) and sustained (10 min) closely paralleling the biphasic nature of contraction  or the Rho kinase inhibitor Y27632 (1 µM)  contraction by PAR2-AP was abolished by the PLC-b inhibitor U73122 (10 µM) and the MLC kinase inhibitor ML-9 (10 µM) . Pretrea2 (1 µM) .696  , but not696 .176/180), IkBa degradation was measured by western blot and NF-kB activation was measured by phosphorylation of p65 subunit using phospho-specific antibody (Ser536). PAR2-AP (1 µM) induced activation of IKK2, degradation of IkBa and activation of NF-kB in cultured muscle cells. The effect of PAR2-AP was abolished by treatment of cells with the RhoA inhibitor (C3 exoenzyme (2 µg/ml)) suggesting that activation of NF-kB was downstream of RhoA  and G protein-coupled receptor agonists (e.g. lipopolysacchride). The canonical pathway for activation of NF-kB involves phosphorylation of Ik-Ba by IkBa kinase (IKK2), degradation of IkBa via the proteasomal pathway, and translocation of NF-kB dimer to the nucleus. We have examined whether PAR2s are coupled to activation of NF-kB in smooth muscle cells. Activation of IKK2 was measured by phosphorylation of IKK2 using phospho-specific substrate   .Further evidence for the release of PKA catalytic subunit from NF-kB complex in response to PAR2 activation was obtained in co-immunoprecipitation experiments. In the basal state PKA catalytic subunit was co-immunoprecipated with NF-kB. Treatment of cells with PAR2-AP attenuated the amount of PKA catalytic subunit co-immunoprecipitated with NF-kB suggesting dissociation of the catalytic subunit from NF-kB complex . The effThe gastrointestinal tract, of all the body systems, is exposed to significant amounts of serine proteases in both normal situations and during inflammatory bowel diseases. However, expression of PAR2 receptors and the mechanism underlying their effects on smooth muscle of the gastrointestinal tract are as yet not completely understood. Insights into the mechanism of the PAR2 effects on smooth muscle are important to understand the underlying mechanism involved in altered muscle contractions related to inflammatory bowel diseases.20 phosphorylation. The initial contraction reflected activation of PLC-β via PTx-sensitive, Gi1 and Gi2, and PTx-insensitive Gq. The sustained contraction reflected activation of RhoA via G13 and inhibition of MLC phosphatase via Rho kinase-mediated phosphorylation of the MLC phosphatase regulatory subunit MYPT1 at Thr696188 and causes feedback inhibition of RhoA activity  that promote exchange of GDP for GTP leukemia-associated RhoGEF). RhoGEF shares common motifs responsible for binding to activated Ga subunits and for the exchange of GDP for GTP on RhoA 20 phosphorylation and muscle contraction. Consistent with this scheme, blockade of Rho kinase activity by Y27632 inhibited sustained contraction in response to PAR2-AP.Rho kinase activity stimulated by PAR2 was inhibited by Ga188 and inhibition of Rho kinase activity was corroborated using several complimentary approaches. We first measured the activation of canonical NF-kB pathway by PAR2. In the second approach we used IKK2 inhibitor that blocks IkB phosphorylation and subsequent degradation. In the third approach we used a proteasomal inhibitor, MG-132 that prevents degradation of IkB and thus prevents release of PKA catalytic subunits. In the fourth approach we used the membrane-permeable specific PKA inhibitory peptide, myristoylated PKI 14-22 amide. PKI contains PKA pseudosubstrate sequence and specifically inhibits PKA catalytic subunit by binding to the substrate-binding site. In the fifth approach we used a dominant negative mutant IKK2 that lacks the ability to phosphorylate IkB, a prerequisite for degradation. In the sixth approach we used a mutant RhoA (S188A) that lacks the PKA phosphorylation site. In the final approach we measured PAR2-AP-induced dissociation of NF-kB and PKA catalytic subunit by co-immunoprecipitation studies.Although PAR2 are coupled to inhibition of cAMP formation, activation of PAR2 results in the stimulation of cAMP-independent protein kinase A (PKA) activity via RhoA-dependent mechanism involving activation of canonical NF-kB pathways. The notion that PKA derived from degradation of IkB results in phosphorylation of RhoA at SerWe concluded that PAR2-stimulated RhoA was inhibited in a feedback mechanism via cAMP-independent PKA derived from RhoA-dependent activation of canonical NF-kB pathway based on the following evidence: i) PAR2-AP induced phosphorylation (i.e activation) of IKK2, degradation of IkB, and phosphorylation of p65 subunit of NF-kB (activation of NFkB); ii) PAR2-AP-induced IKK2 activation, IkB degradation and NF-kB activation was abolished by a RhoA inhibitor, C3 exoenzyme; iii) PAR2-AP-stimulated Rho kinase activity was significantly augmented by PKI, IKK IV or MG132 in freshly dispersed muscle cells and in cells expressing IKK(K44A), IkBa (S32/S36A) or RhoA(S188A), and iv) PAR2-AP stimulated dissociation of the PKA catalytic subunit from NF-kB complex.i and inhibition of cAMP, they stimulated PKA. The PKA holoenzyme is a heterotetramer consisting of two catalytic subunits bound to two regulatory subunits. Classically, the mechanism of PKA activation in response to cAMP elevating agents (Gs-coupled receptor agonist or forskolin) involves release of the catalytic subunits upon binding of two cAMP molecules to each of the regulatory subunit. Apparently, a certain pool of PKA catalytic subunit is found to be associated with the NF-kB inhibitor protein, IkB. Recent studies have identified novel cAMP-independent mechanism for activation of PKA by various G protein-coupled receptor agonists Although PAR2s receptors are coupled to G2+]i and MLC20 phosphorylation. These effects of PKA are a consequence of the inhibitory action of PKA on multiple components of the Gaq/Ga13 signaling culminating in muscle relaxation.The finding that activation of PKA by a contractile agonist raises an interesting possibility for regulation of signaling molecules involved in the contractile pathway by PKA. It is well established that activation of PKA in gastric smooth muscle cells inhibits muscle contraction, resulting from decrease in [Caq, G13, Gi1, and Gi2, but not Gi3, G12, Gs, and stimulation of PI hydrolysis via both Gaq and Gbgi. PAR2 are coupled to activation of RhoA/Rho kinase via Ga13. PAR2 induced initial contraction was mediated by stimulation of PLC-b activity, generation of IP3, IP3-dependent Ca2+ release, Ca2+/calmodulin-dependent activation of MLC kinase and phosphorylation of MLC20 at Ser19. Whereas sustained contraction was mediated by sequential activation of G13, RhoA and Rho kinase, and Rho kinase-dependent phosphorylation of MYPT1 at Thr696 and inhibition of MLC phosphatase. Although PAR2s are coupled to inhibition of cAMP, they stimulated cAMP-independent PKA activity via RhoA-dependent canonical NF-kB pathway and this resulted in feedback inhibition of Rho kinase activity via inhibitory phosphorylation of RhoA at Ser188.In conclusion, our studies have demonstrated the expression of PAR2 in smooth muscle and identified the receptor-specific signal transduction pathways in mediating smooth muscle contraction. In gastric smooth muscle cells PAR2 are coupled to activation of G