Data were obtained mainly in AT1 R-3327 prostate carcinoma cells, but the principle importance was confirmed in 5 other cell types. Extracellular acidosis leads to a rapid and sustained decrease of pHi in parallel to p38 phosphorylation in all cell types and to ERK1/2 phosphorylation in 3 of 6 cell types. Furthermore, p38 phosphorylation was elicited by sole intracellular lactacidosis at normal pHe. Inhibition of ERK1/2 phosphorylation during acidosis led to necrotic cell death. No evidence for the involvement of the kinases c-SRC, PKC, PKA, PI3K or EGFR nor changes in cell volume in acidosis-induced MAPK activation was obtained. However, our data reveal that acidosis enhances the formation of reactive oxygen species (ROS), probably originating from mitochondria, which subsequently trigger MAPK phosphorylation. Scavenging of ROS prevented acidosis-induced MAPK phosphorylation whereas addition of H2O2 enhanced it. Finally, acidosis increased phosphorylation of the transcription factor CREB via p38, leading to increased transcriptional activity of a CRE-reporter even 24 h after switching the cells back to a normal environmental milieu. Thus, an acidic tumor microenvironment can induce a longer lasting p38-CREB-medited change in the transcriptional program, which may maintain the altered phenotype even when the cells leave the tumor environment.Tumor micromilieu often shows pronounced acidosis forcing cells to adapt their phenotype towards enhanced tumorigenesis induced by altered cellular signalling and transcriptional regulation. In the presents study mechanisms and potential consequences of the crosstalk between extra- and intracellular pH (pH Two microenvironments can be distinguished with respect to solid tumors: (i) the tissue environment in which the tumor cells reside  and (ii) the local environment created by the tumor cells (tumor microenvironment), that can generate a pathological tissue environment for neighboring cells. The pathological tissue environment supports tumor promotion and the tumor microenvironment supports tumor progression It is well known that the metabolic microenvironment impacts tumor cell behavior. For example, the efficacy of radiation therapy, photodynamic therapy and chemotherapeutics is impaired by the tumor environment For a detailed mechanistic understanding it is important to deconstruct this microenvironment and determine the effects of the different parameters individually in order to evaluate their contribution. Whereas there is ample literature on hypoxia, the importance of metabolic acidosis is less well investigated. Recently we showed that metabolic acidosis per se enhances chemoresistance in prostate tumor cells under normoxic and normoglycemic conditions 2 atmosphere and sub cultivated twice per week. LS513 cells  were grown under the same conditions as AT1 cells. OK cells  i) and transferred to medium without additional FCS supplementation for 24 hours. Control cells were exposed to bicarbonate-HEPES buffered Ringer solution adjusted to pH 7.4. Intracellular acidosis was induced replacing 40 mM NaCl by 40 mM lactic acid and reduction of chloride by completely replacing NaCl by sodium gluconate . Extracellular acidosis (pH 6.6) was applied using bicarbonate-MES (morpholinoethanesulfonic acid) buffered Ringer solution pH-adjustment to 6.6. The buffer capacity (β) is 5.9 mmol/lxΔpH for the bicarbonate-HEPES buffered Ringer solution at pH 7.4 and 3.9 mmol/lxΔpH for the bicarbonate-MES buffered Ringer solution.The subline AT1 of the rat R-3327 Dunning prostate carcinoma was used as described before After serum depletion for 24 h, cells were incubated with one of the above mentioned buffers (1 ml) for up to 3 h. All inhibitors or activators used were added during this incubation period.2, 1 mM MgCl2, 10 mM HEPES and 10 µM nigericin.Cytosolic pH of single cells was determined using the pH-sensitive dye BCECF -5-(and-6)-carboxyfluorescein, acetoxymethyl ester, Invitrogen, Paisley, UK) as described before The effect of acidosis on cell volume was determined electronically with a Casy cell counter . Therefore cells were incubated as mentioned above, detached by trypsinisation and cell volume and viability was measured after 10 min or 3 h in the respective Ringer solutions.Western blotting was performed according to standard protocols. Cells were lysed , cell protein determined by the BCA-methode (BC Assay Reagents from Uptima), separated by SDS-PAGE and transferred to a nitrocellulose membrane. Subsequently, membranes were incubated with antibodies specific for ERK1/2, p38, MKK3/6; phospho-ERK1/2, phospho-p38, phospho-MKK3/6, CREB and phospho-CREB . The bound primary antibody was visualized using horseradish peroxidase-conjugated secondary antibodies and the ECL system (Pierce/Thermo Fisher Scientific) with the Molecular Imager ChemiDoc XRS System . Quantitative analysis was performed with Quantity One software (Biorad).Transactivation was assessed by the Mercury™ Pathway Profiling reporter gene assay system from Clontech Inc. using secretory alkaline phosphatase (SEAP) under the control of defined cis-regulatory response elements (CRE) as reporter, essentially as described earlier LDH activity in media and in cell lysates was measured using standard protocol Cells were washed once with PBS buffer (4°C) and incubated with 100 µl cell lysis buffer  for 10 min on ice, harvested, and centrifuged at 16000g for 10 min at 4°C. 60 µl of the supernatant was incubated with 65 µl reaction buffer  containing 42 µM DEVD-AFC  at 37°C, and fluorescence of the cleaved product, 7-amino-4-trifluoromethylcoumarin (AFC), was measured at 400 nm excitation and 505 nm emission wavelength using a multiwell counter . Cleaved AFC was quantified by a calibration curve using known AFC concentrations. Protein content was determined with bicinchoninic acid assay  using bovine serum albumin as standard.pH was measured with a blood gas analyzer . For determination of the concentration of glucose, the glucose (HK) assay kit from Sigma (G3293) was used  according to the manufacturer's instructions. Lactate was determined using lactate reagent (Trinity) according to the manufacturer's instruction (10 µl sample plus 100 µl kit). Each experiment was performed at least in triplicates.2O2 once the ester bond has been cleaved by cellular esterases. Cells were seeded in 24-well-plates and incubated for 30 minutes with dye after the indicated treatments. Subsequently, fluorescence  was measured using a multiwell counter . Additionally blank values without cells and blank values without dye were determined and subtracted. The increase of fluorescence over the blank value expressed per mg protein was used as a measure for ROS formation.Formation of reactive oxygen species (ROS) was assessed with the fluorescent dye DCFDA-AM  which reacts with an increase in fluorescence in the presence of Hm collapses. By contrast, mitochondrial uptake of rhodamine 123 is maximal in the presence of nigericin, which collapses the pH gradient across the inner mitochondrial membrane. As a consequence the entire energy from the electron transport chain is transformed into Ψm. Thus, uptake of rhodamine 123 in the presence of nigericin minus uptake of rhodamine 123 in the presence of CCCP represents a crude estimate of mitochondrial activity and eliminates any non-specific effects. Uptake of rhodamine 123 was calibrated using lysis buffer containing known concentrations of rhodamine 123.In order to estimate mitochondrial activity, we determined the accumulation of rhodamine 123 after exposure to pH 7.4 or pH 6.6. After the 3 h exposure the cells were incubated for 20 min with 0.1 µM rhodamine 123 in HEPES-Ringer solution as well as in the presence of 20 µM CCCP or 2 µM nigericin If not stated otherwise, chemicals were from Sigma-Aldrich, Munich, Germany.Data are presented as mean ± SEM. For all experiments, N equals the number of culture plates or cells used to perform the measurements if not quoted otherwise. All experiments were performed with at least two passages. Statistical significance was determined by unpaired Student's t-test or ANOVA, as appropriate. Differences were considered statistically significant when P<0.05.i, albeit the degree of this reduction differed strongly between the cell lines. Under control conditions (pH 7.4) the intracellular pH was always lower than in the extracellular compartment. However, under acidosis (pH 6.6) the intracellular pH was higher in most cell lines. This reverse pH-gradient has been described previously for solid tumors in vivoin vivo situation appropriately. Even though in CHO and H358 the intracellular pH has fallen below the extracellular pH there is indication for a pronounced net proton extrusion. From thermodynamic considerations in cells without proton extruders  the intracellular pH would be approximately 0.65 units lower as compared to the extracellular pH. In the experiments (pHe 6.6) a pHi of ∼6.0 would be expected from passive distribution. Obviously, CHO and H358 cells extrude protons actively against the passive equilibrium even though their capacity seems to be somewhat smaller than in the other cell lines.i) rapidly dropped from pH 7.22±0.08 to pH 6.75±0.09 within 10 min  were observed in AT1 cells further experiments were performed with this particular cell type.Exposing cells to moderate extracellular acidosis leads to a marked activation of ERK1/2 and p38 MAP kinases. observed . Taken tIn order to assess different mechanisms of cell death the protein content per dish , induction of apoptosis (caspase-3 activity) and necrosis (LDH-release) were analyzed. Acidosis per se elicited no significant change in cell survival . Yet, whi recovers very rapidly in the presence of extracellular lactate  either isolated inhibition of bicarbonate transport (200 µM DIDS) or incubation of the cells with lactate (40 mM) resulted in a rather weak acidification by 0.1 pH-units . However lactate . Simulta lactate . These d lactate .Since increased phosphorylation could be either the result of increased kinase activity or of reduced phosphatase rate the impact of phosphatase inhibition on the acidosis-induced MAPK activation was studied. When tyrosine- or theronine/serine-phosphatases were inhibited using either orthovanadate (100 µM) or ocadaic acid (100 nM) the effect of extracellular acidosis on ERK1/2 and p38 phosphorylation was additive . In all To unveil the possible signaling pathways leading from extracellular acidosis to an activation of ERK1/2 and p38, several promising targets were analyzed by blocking them with specific inhibitors. The role of epidermal growth factor receptor (EGFR), protein kinase C (PKC), protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K) and the Src family of tyrosine kinases was studied using AG1478, bisindolylmaleimide I (BIM), Rp-isomer, LY294002 and PP2, respectively. All of these kinases can play an important role in tumor promotion and progression. However, none of the inhibitors showed a significant effect on acidosis-induced ERK1/2 or p38 activation as depicted in per se led to reduced ERK1/2 but not p38 phosphorylation  has been shown to activate the MAPK pathway acidosis . Thus, O+/K+-ATPase, an enzyme which is inhibited by acidosis and may induce MAPK activation either via alterations in cellular electrolyte homeostasis or by signaling via the EGFR +/K+-ATPase may contribute to ERK1/2 phosphorylation in AT1 cells. However, since cell volume changes which are part of the signaling cascade Another possible mechanism for pH-"sensing" would be the NaSince reactive oxygen species (ROS) can act as an intracellular signaling molecule + ratio as trigger In the presence of rotenone, an inhibitor of mitochondrial complex I, ROS formation was enhanced under acidic but not under control conditions , i.e. ac2O2, a relatively stable ROS molecule, led to a dose dependent activation preferentially of the p38 MAP kinase , which has been shown to be involved in tumor initiation, progression and metastasis, supporting its role as a proto-oncogene i>pHe) during extracellular acidosis as compared to control conditions, similar to the situation described for tumor cells before in vivo studies. Quantitatively, pHiin vivo seems to be higher as compared to the situation in culture at similar extracellular pH-values. This apparent quantitative discrepancy has been observed before and might be due to a combination of the much larger extracellular space present in cell culture settings and the high buffer capacity of the medium representing a larger pool of protons that the cells can not compensate for Inadequate perfusion in solid tumors leads to insufficient oxygen supply and forces the tumor cells to perform anaerobic glycolysis resulting in marked lactic acid formation and extracellular acidosis i and enhanced p38 phosphorylation in all cell lines tested, the link pHi→p38 seems to be of general validity. By contrast, ERK1/2 phosphorylation was not induced in all cell types tested, indicating that the link pHe→ERK1/2 is cell type-specific.In all cell lines studied extracellular acidosis induced p38 phosphorylation within a few minutes which was sustained for the entire observation period whereas ERK1/2 phosphorylation occurred only in 3 of 6 cell types. Because acidosis induced a drop in pHi and the degree of p38 phosphorylation in the different cell lines, indicating that the sensitivity of the pHi→p38 link is cell type-specific. Still, this does not exclude the existence of some kind of cell-specific threshold as suggested by We observed no significant correlation between changes in pHi→p38 link. However, in contrast to cardiomyocytes, ERK1/2 phosphorylation required additional extracellular acidosis in AT1 cells, although we cannot exclude the possibility that ERK1/2 is less sensitive to pH changes because extracellular acidosis induced a larger drop in pHi as compared to DIDS+lactic acid. Acidosis-induced p38 phosphorylation is most probably mediated by MKK3/6, because p38 itself is not pH-sensitive as shown by Our data concur with results from cardiomyocytes In principle, the question may arise whether serum starvation for 24 h (as used in our experiments) might be responsible for the activation of MAPK and for inducing autophagy. However, in all control experiments where the cells were kept at pH 7.4 comparable experimental conditions were used for the experimental groups and all samples were kept without serum for 24 h. For this reason an impact of serum depletion seems to be unlikely. Additional experiments show that in cells without serum starvation a comparable acidosis-induced MAPK activation can be observed . FinallyIn addition, autophagy has been linked with ROS induction, MAPK phosphorylation and cell death In order to unmask pathways involved in environmental acidosis-induced MAPK activation the impact of different signaling cascades was analyzed. We did not obtain any indication for the involvement of phosphatase activity, EGFR, PKC, PKA, PI3K or Src pathways. Furthermore, our data suggest that membrane proton receptors, namely OGR1 Another possible “signaling” mechanism that could be involved is cell volume, which may change under acidic conditions and modify the phosphorylation of a variety of proteins +/K+-ATPase activity could affect ERK1/2 phosphorylation via changes of intracellular sodium and subsequently calcium. Changes in the concentration of intracellular calcium in AT1 cells following acidosis have been described before +/K+-ATPase's activity, the pump can also serve as a signaling module in combination with EGFR, stimulating the ERK1/2 pathway Acidosis-induced changes in Na2O2) enhanced MAPK phosphorylation. These results are in good agreement with previous studies clearly demonstrating that the p38 pathway can be activated by ROS generated e.g. in complex I of the respiratory chain The results of the present study indicate that reactive oxygen species play a role in acidosis-induced p38 and ERK1/2 phosphorylation . Scaveng+ ratio is high + ratio, when respiration is low. An increase of mitochondrial activity under acidic conditions was not observed. Although the mechanism by which acidosis affects mitochondria still awaits final elucidation, mitochondria are the most probable source of oxygen radicals. The precise mechanisms have to be addressed in future studies.The question arises how the acidotic environment enhances ROS formation in tumor cells. ROS production was markedly stimulated by acidification (either extra- or intracellular) whereas DPI was not able to counteract this effect. These data argue against the involvement of NADPH-oxidase, NO-synthase or other flavo-proteins. Even though it is difficult to identify the source of ROS the most probable source in our case definitively are mitochondria. Here ROS will be generated at complex I when (i) mitochondrial ATP production is reduced  resulting in a high proton gradient and a reduced CoQ (coenzyme Q) pool and when (ii) the NADH/NADAcidosis-induced changes in MAPK activity are of potential importance for the tumor cell phenotype. Recently, we demonstrated that metabolic acidosis enhances chemoresistance in AT1 cells via p38 kinase e 7.4. Thus, an acidic tumor microenvironment can induce a longer lasting change in the transcriptional program, representing a memory effect which maintains the altered phenotype even when the cells leave the tumor environment. Of course, these interpretations need future evaluation in vivo. Furthermore, we will have to investigate the identity of genes up-regulated by CREB under these conditions and whether there are other transcription factors affected.Finally, the present study shows that acidosis affects transcriptional activity through CREB phosphorylation. The transcription factor CREB has been shown to be involved in tumor initiation, progression and metastasis, supporting its role as a proto-oncogene Figure S1i) in AT1 cells.Regulation of the intracellular pH (pH (A) Representative time course of pHi after rapid changes of pHe (n = 14). (B–D) Intracellular acidification and pH-recovery following the treatment with lactic acid. The accumulation of metabolically generated acids, as well as the movement of protons into the cell or bases out of the cell, constitutes a potentially deleterious acid challenge to the cell. This chronic acid load in tumor cells (to a great extend lactic acid formation) is overcome mainly by Na+/H+ antiport, Na+-dependent HCO3−/Cl− exchange or possibly Na+-HCO3− cotransport (i.e. by acid extruders). To characterize these processes in AT1 cells, which also show extensive lactic acid production (supplementary e 7.4). In the absence of any inhibitor, addition of lactic acid led to a rapid decrease (within a minute) in pHi to pH 7.02 followed by a recovery within less than 7 min, as shown in  and . (D) Inhibition of Na+/H+ exchanger by 10 µM EIPA did not show a significant effect, whereas blocking bicarbonate transporters with 200 µM DIDS abrogated the pH-recovery after intracellular acidification , n = 28–38. (*) p<0.05. This leads to the conclusion that bicarbonate transporters are crucial for pH-regulation in AT1 cells. This assumption is also supported by the following observations: (i) intracellular pH did not stay stable but decreased constantly when cells were superfused with solutions lacking bicarbonate, (ii) exposure to DIDS also led to a constant decrease in pHi  and (iii) acute reduction of [Cl−]e to 20 mM induced a prompt and reversible alkalinization , suggesting that a Cl−-dependent transporter is involved. Because the driving forces for the Cl−/HCO3−-exchanger allow Cl− entry and bicarbonate export at the prevailing pH-gradient ("acid loader"), this transporter cannot explain the data obtained (i.e. acidification during inhibition by DIDS). The present data suggest the existence of a Na+-dependent HCO3−/Cl− exchange :1697-706). In summary, exposure to lactate+DIDS generates isolated intracellular acidosis at normal pHe.(PDF)Click here for additional data file.Figure S2Glycolysis-induced alteration of media parameters. "H+-production" (A and B) as well as changes in the concentration of glucose (C) and lactate (D) within 3 h under control (pH 7.4) and acidic (pH 6.6) conditions (in 1 ml Ringer solution) were determined. AT1 cells were incubated with Ringer solutions for 3h and parameters were determined as mentioned. N = 10. The cell number (1.9±0.1 million cells per petri dish) did not differ significantly between the groups and was not affected during the three hour incubation period. Therefore the concentrations can be compared directly. (*) p<0.05.(PDF)Click here for additional data file.Figure S3Acidosis-induced MAPK activation in different cell types. There is no significant correlation between changes in pHi or absolute pHi and ERK1/2 or p38 phosphorylation in different cell types.(PDF)Click here for additional data file.Figure S4Phosphatases do not mediate the effect of acidosis. Inhibition of phosphatases by ocadaic acid (100 nM) or orthovanadate (100 µM) enhanced ERK1/2 and p38 phosphorylation per se. Acidosis exerted an additive effect on phosphorylation. (*) p<0.05. N = 4–6.(PDF)Click here for additional data file.Figure S5Role of MEK1/2 in ERK1/2 phosphorylation. Typical western blots and semi-quantitative analysis of the western blot data (N = 3) are shown. Values represent the ratio of phosphorylated protein (pp38 or pERK) divided by total protein . These values have then been normalized to the ratio of control experiments at pH 7.4. OV, 100 µM orthovanadate; OA, 100 nM ocadaic acid; 10 µM U0126. (*) p<0.05.(PDF)Click here for additional data file.Figure S6Effect of kinase inhibitors on acidosis-induced MAPK phosphorylation. 1 µM AG1478 (EGFR kinase), 1 µM PP2 (Src-kinas), 1 µM BIM (protein kinase C), 10 µM Ly294002 (PI3-kinase). Values represent phosphorylated protein divided by total protein and normalized to HEPES-HCO3−-Ringer pH 7.4. (*) p<0.05 versus pH 7.4. N = 5–8.(PDF)Click here for additional data file.Figure S7The role of cAMP in MAPK activation. Addition of cAMP (300 µM db-cAMP) or stimualtion of cAMP formation (3 µM forskolin) prevented ERK1/2 but not p38 phosphorylation. The inactive cAMP analogue Rp-Isomer (300 µM) did not affect ERK1/2 or p38 phosphorylation. (*) p<0.05 versus pH 7.4. N = 5–7.(PDF)Click here for additional data file.Figure S8+-sensors, cell volume and Na+/K+-ATPase in MAPK activation.The role of H (A) Inhibtion of the H+-receptor ORG by 10 µM Cu2+ did not prevent acidosis-induced p38 or ERK1/2 phosphorylation. Representative of three blots. (B–D) Another possible mechanism for pH-"sensing" would be the Na+/K+-ATPase which is a pH-sensitive enzyme, inhibited by acidosis, that may induce MAPK activation either via alterations in cellular electrolyte homeostasis or by signaling via the EGFR. Exposing AT1 cells to an acidic extracellular environment showed a constant decrease in cell volume (B) which could be either the result of a reduced activity of the Na+/K+-ATPase or a lower Na+-transport via the above mentioned Na+-dependent HCO3−/Cl− exchanger. Inhibiting the Na+/K+-ATPase by ouabain (100 µM) led to a reduction of the cell volume from 2093±82 fl to 1732±124 fl  which was comparable to that found under acidic conditions (pH 6.6) 1839±65 fl . As shown in (C), ouabain induced the phosphorylation of ERK1/2 but not of p38. Furthermore, the effect of extracellular acidosis on ERK1/2 but not on p38 was abrogated by ouabain. These data indicate that acidosis-induced inhibition of the Na+/K+-ATPase may contribute to ERK1/2 phosphorylation in AT1 cells. From these data it seems possible that cell shrinkage is part of the signaling cascade leading to ERK1/2 activation. However, cell volume changes were rather heterogeneous for the different cell lines (D), arguing against a decisive role for acidosis-induced ERK1/2 phosphorylation; (*) p<0.05 versus pH 7.4. N = 5(PDF)Click here for additional data file.Figure S9ERK1/2 phosphorylation in AT1 cells growing without serum starvation. In cells that were not serum deprived acidosis induced a similar ERK1/2 phosphorylation as compared to serum deprived cells (compare  compare .(PDF)Click here for additional data file.


Allergen recognition by dendritic cells (DCs) is a key event in the allergic cascade leading to production of IgE antibodies. C-type lectins, such as the mannose receptor and DC-SIGN, were recently shown to play an important role in the uptake of the house dust mite glycoallergen Der p 1 by DCs. In addition to mannose receptor (MR) and DC-SIGN the high and low affinity IgE receptors, namely FcϵRI and FcϵRII (CD23), respectively, have been shown to be involved in allergen uptake and presentation by DCs.This study aims at understanding the extent to which IgE- and IgG-facilitated Der p 1 uptake by DCs influence T cell polarisation and in particular potential bias in favour of Th2. We have addressed this issue by using two chimaeric monoclonal antibodies produced in our laboratory and directed against a previously defined epitope on Der p 1, namely human IgE 2C7 and IgG1 2C7.Flow cytometry was used to establish the expression patterns of IgE (FcϵRI and FcϵRII) and IgG (FcγRI) receptors in relation to MR on DCs. The impact of FcϵRI, FcϵRII, FcγRI and mannose receptor mediated allergen uptake on Th1/Th2 cell differentiation was investigated using DC/T cell co-culture experiments. Myeloid DCs showed high levels of FcϵRI and FcγRI expression, but low levels of CD23 and MR, and this has therefore enabled us to assess the role of IgE and IgG-facilitated allergen presentation in T cell polarisation with minimal interference by CD23 and MR. Our data demonstrate that DCs that have taken up Der p 1 via surface IgE support a Th2 response. However, no such effect was demonstrable via surface IgG.IgE bound to its high affinity receptor plays an important role in Der p 1 uptake and processing by peripheral blood DCs and in Th2 polarisation of T cells. Allergic diseases represent a major health problem affecting a large sector of the population ,2. Type Dermatophagoides pteronyssinus[Allergens, such as Der p 1, that cause these allergic reactions are generally innocuous proteins. Der p 1 is considered as the most immunodominant allergen of the house dust mite onyssinus. It is aonyssinus.DCs are professional antigen-presenting cells that occupy a central position at the interface of innate immunity and adaptive immune responses, through recognising foreign antigens, processing them and presenting them to T cell receptors via MHC molecules -9. DCs uIn recent studies in our laboratory, it was shown that the C-type lectin receptors, mannose receptor (CD206 or MR) and DC-SIGN, play a significant role in Der p 1 uptake, internalisation and presentation. It has been shown that these receptors are characterised by the presence of carbohydrate recognition domains (CRD) that recognise sugar moieties on allergens -15. The It has been previously suggested that IgE might play an important role in antigen uptake by DCs through IgE receptors . It was Together, these findings helped to formulate the hypothesis of our work, which is that IgE-mediated allergen presentation primes naïve T cells towards Th2 cell differentiation. The elucidation of this mechanism could clearly have therapeutic potential.Previous work in our laboratory generated Der p 1-specific chimeric human IgE (IgE 2C7) and IgG (IgG1 2C7) antibodies consisting of mouse variable regions (Vκ and VH) joined to a human IgE or IgG1 constant regions, respectively ,24. It hThe kinetics of expression of FcϵRI, FcϵRII (CD23), FcγRI and mannose receptor were studied during the course of generating Mo-DCs, starting from day zero (monocyte) through to days 2, 4, 6 (immature) and 8 (mature).FcϵRI was modestly expressed on monocytes at day zero, while FcϵRII and MR were less well expressed Figure . On day 5) were stained with IgE-FITC, in the presence or absence of mannan, in order to exclude IgE binding via the mannose receptor (i.e. through carbohydrates on IgE). Der p 1 uptake with and without mannan was used to show that mannan was active in the experiment. As shown in Figure Immature DCs (day 6 of culture) were stained with IgE-FITC, in the presence or absence of unlabelled IgE or IgG, in order to determine IgE binding specificity. There was substantial inhibition of IgE-FITC binding when mixed with unlabelled IgE (p ≤0.05) but reduction in uptake in the presence of IgG was not significant Figure . In furtDer p 1 can be taken up efficiently by C-type lectin receptors (such as MR) on DCs via its sugar residues. To study the influence of the uptake of Der p 1 through immunoglobulins bound to DCs on T cell differentiation without MR engagement, we deglycosylated Der p 1 to exclude binding through MR. To confirm the deglycosylation of Der p 1, Western blotting of natural Der p 1 and deglycosylated Der p 1 against GNA  was performed. Unlike natural Der p 1, no band was visible with the deglycosylated Der p 1 preparation Figure . An ELISAn ELISA was used to ascertain the Der p 1 reactivity of the IgE and IgG 2C7 antibodies used in our experiments. Results clearly indicate that the 2C7 antibodies react specifically with Der p 1 and no such reactivity can be demonstrated with the control allergen (Fel d 1) or the isotypes control antibodies Figure .To investigate the role of IgE in the uptake of Der p 1 by Mo-DCs, we incubated Cy5 labelled Der p 1, with and without IgE 2C7, with immature Mo-DCs at 37°C. The experiment showed that adding IgE 2C7 significantly increases Der p 1 uptake to a level over and above that mediated by MR Figure . The aveMyeloid DCs were stained with anti-FcϵRIα PE, anti-CD206 PC5, anti-CD23 ECD and anti-CD64 (FcγRI) FITC. As shown in Figure Since myeloid  DCs showed high level of FcϵRI and FcγRI (IgG high affinity receptor) expressions, with only minimal expressions of CD23 and MR, these cells provide an ideal opportunity to assess the role of IgE and IgG-facilitated allergen presentation in T cell polarisation with minimal or no interference by CD23 and MR.Myeloid DCs that had been loaded with natural or deglycosylated Der p 1, both with and without IgE and IgG 2C7 antibodies, were co-cultured with autologous naïve T cells. IL-4, IL-5, IL-13 and IFN-γ secretions were then detected using a FlowCytomix kit after stimulation with PMA and Ionomycin.The data demonstrated a high level of Th2 cytokine  secretion when the IgE 2C7 antibody was used with either Der p 1 or deglycosylated Der p 1, in comparison with using Der p 1 or deglycosylated Der p 1 alone Figure . HoweverIt is well accepted that FcϵRI is highly expressed on mast cells and basophils. However, a noticeable feature of FcϵRI on APCs is that its expression is highly variable, depending on the blood donor and the pathological situation . We initIgE bound specifically to its receptors on immature Mo-DCs, as its binding was inhibited by IgE but not by IgG. The optimisation of IgE binding experiment revealed that the blocking effect of unlabelled IgE was only demonstrable when added at the same time as labelled IgE (i.e. in competition), and this could be due to the rapid turnover of FcϵRIα on the cell membrane.DCs express the C-type lectin receptors MR and DC-SIGN, which have the ability to recognise mannose containing-carbohydrates on different antigens and pathogens. MR has specificity for proteins with mannose, fucose and N-acetyl glucosamine, while DC-SIGN recognises high mannose oligosaccharide structures ,28. FurtThe hypothesis that DCs could perform allergen capture and presentation via surface IgE is of great interest, since this could lead to further perpetuation of allergic tissue damage in atopic individuals where IgE circulates in very high concentrations. We were uniquely placed to test this hypothesis through the availability of IgE and IgG chimaeric human 2C7 antibodies directed against Der p 1, the major allergen of the HDM and an important cause of allergic asthma. This meant that we could study the role of IgE in allergen uptake on DCs and compare it to IgG, particularly since these antibodies have identical epitope specificity (i.e. same variable domains) and differ only in their constant region domains .Having demonstrated IgE binding to its receptors (FcϵRI and FcϵRII) on DCs, we proceeded to investigate the role of IgE in Der p 1 uptake using a flow cytometry approach, whereby DCs were loaded with labelled Der p 1-IgE 2C7 complexes. Since Der p 1 can be internalised by MR and DC-SIGN ,13,32, wThe ultimate goal of this study was to explore the role of IgE in allergen uptake and its presentation by DCs via IgE receptors particularly FcϵRI, and to compare this with IgG. In line with previous studies ,34, we f+ T cells leads their differentiation towards one of few developmental pathways such as Th1, Th2, Th17 or Treg depending on the concentration of different cytokines in the microenvironment amongst other factors [It is well understood that the activation of CD4 factors ,36. Th2 The co-culture experiments showed that IgE facilitated Der p 1 uptake by myeloid DCs supported the differentiation of naïve T cells towards a Th2 cell phenotype. Given the low expression of CD23 on myeloid DCs this effect is most likely mediated via FcϵRI however we cannot rule out possible up-regulation of CD23 after sensitisation with IgE antibody hence a contribution from CD23 remains a possibility. The Th2 bias was evidenced by high levels of Th2 cytokine secretion, particularly IL-4, when the IgE 2C7 antibody was used in combination with natural Der p 1 or deglycosylated Der p 1. However, these findings did not reach statistical significance with two (i.e. IL-5 and IL-13) of the three Th2 cytokines tested most likely due to the low number of donors and wide inter donor variations obtained. Previous analysis of 55 crystal structures of known allergens (including Der p 1) has shown that dimerisation is a very common and essential feature of allergens . We can Our results are in keeping with a recent study by Sallmann and co-workers using a new transgenic mouse model, which suggested that FcϵRI expressed by DCs does indeed play a role in antigen uptake and the development of Th2 allergic tissue inflammation .In conclusion, IgE could play an important role in Der p 1 uptake and processing by peripheral blood myeloid DCs and in the Th2 polarisation of T cells. This would clearly serve to perpetuate symptoms of allergy in atopic patients where IgE is already produced in large quantities. It is known that DCs prime Th2 cell polarisation after encountering allergens, but the particular mechanism by which DCs induce Th2, instead of Th1, development is not fully understood. The present work provides a potential mechanism by which myeloid DCs may support the development of Th2 responses and suggests a novel target for therapy.Whist C-type lectins play a key role in recognition and uptake of glyco-allergens including Der p 1, this study shows that IgE bound to its high affinity receptor plays an important role in the uptake and processing of Der p 1 by peripheral blood DCs as well as in Th2 polarisation of T cells. Such IgE mediated pathway of allergens uptake could be particularly important in individuals with high serum IgE levels who have already been sensitised to an allergen.Der p 1 was purchased from Indoor Biotechnologies  and labelled with Cyanine 5 (Cy5) using a Cy5 labelling kit  according to the manufacturer’s instructions. The concentration of labelled allergens was determined by NanoDrop prior to use.In order to deglycosylate natural Der p 1, sodium metaperiodate  was used as previously described . BrieflyDeglycosylation was then confirmed using Western blot analysis. Briefly, using 12% Tris-Glycine precast gels , gel electrophoresis was performed for 5 μg of natural and deglycosylated Der p 1 proteins, and these proteins were then transmitted to nitrocellulose membranes . Next, a DIG Glycan Differentiation kit  was used to determine the glycan content following the manufacturer’s instructions.The structural integrity of the deglycosylated Der p 1 preparation was also tested against 2C7 and biotinylated 5H8 anti-Der p 1 antibodies, clone 5H8 C12, (Indoors Biotechnologies), using standard ELISA procedure in which the binding was detected using Extra Avidin alkaline phosphatase conjugate .+ monocytes were then collected using a magnetic cell separation system .Peripheral blood samples from non-atopic donors  were used for separation of peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation on Histopaque-1077 . PBMCs were then incubated with mouse anti-human CD14 monoclonal antibody (mAb) conjugated to magnetic beads . CD14+ monocytes by culturing them with 50 ng/ml of Granulocyte-Macrophage colony stimulating factor (GM-CSF) and 250 IU/ml of IL-4  in RPMI-1640 medium, supplemented with L-glutamine, penicillin and streptomycin , and 10% FBS  (1 × 106 cells/ml) in a 24-well flat bottom culture plate  at 37°C in 5% CO2 for six days as we have previously described [2.Immature DCs were generated from CD14escribed ,40,41. T+C cells were purified by positive selection after depletion of PBMCs of CD19+B cells as previously described [In other experiments, where peripheral blood myeloid DCs (mDCs)  were used, these cells were separated from PBMCs by two magnetic separation steps using the CD1c (BDCA-1) + dendritic cell isolation kit from Miltenyi Biotech  and following the manufacturer’s guidelines. Briefly, CD1escribed .The expression of FcϵRI, FcϵRII (CD23), FcγRI (CD64) and the mannose receptor was determined during the course of generating DCs, starting from day zero (monocyte) through to days 2, 4, 6 (immature) and 8 (mature). Receptor expression was also demonstrated on peripheral blood myeloid DCs (mDCs).Following the isolation of monocytes from PBMCs and culturing them for DC generation, Mo-DCs (or mDCs were applicable) were harvested on each day of cell culture, washed with phosphate-buffered albumin  and stained with anti-CD206 PC5, clone 19.2 , anti-CD23 ECD, clone 9P25, anti-CD64 (FcγRI) FITC, clone 22  and anti-FcϵRIα PE clone AER-37(CRA-1)  for 25 minutes in the dark at 4°C. The cells were then washed with PBA and fixed with 0.5% formaldehyde. Isotype control antibodies were used to determine binding specificity. Samples were analysed using flow cytometry as described before . A minim5 immature Mo-DCs, which were first washed with PBA and tested as follows:Purified human IgE  was labelled with fluorescein using a Fluoro-Trap™ fluorescein labelling kit  according to the manufacturer’s instructions. The concentration of labelled IgE was determined by NanoDrop prior to use. In the assay for inhibition of labelled IgE uptake by DCs, unlabelled human IgE and IgG were used at different concentrations  to stain 105 each). The first set was stained with IgE-FITC (7 μg/ml), the second set treated with unlabelled IgE (20 μg/ml) plus IgE-FITC (7 μg/ml) and the third set treated with IgG (20 μg/ml) plus IgE-FITC (7 μg/ml). Later, the cells were incubated for 20 minutes at 4°C and then washed with PBA and fixed. The detection of labelled IgE binding was performed using flow cytometry.Three sets of DCs were prepared (105 each). The first set was stained with IgE-FITC (7 μg/ml), the second set treated with 200 μg/ml mannan (MR ligand used as inhibitor)  for 20 minutes at 37°C, washed and mixed with IgE-FITC (7 μg/ml), the third set incubated with Cy5-labeled Der p 1 for 20 minutes, while the fourth set was treated with 200 μg/ml mannan for 20 minutes at 37°C then washed and loaded with Cy5-Der p 1 for 20 minutes at 4°C followed by washing with PBA and fixing. The detection of labelled IgE and Der p 1 binding was performed using flow cytometry.Four sets of DCs were prepared (10Felis domisticus) (5 μg/ml) . The plates were incubated overnight at 4°C then washed three times using PBS-0.05% Tween-20 before blocking with PBS −1% BSA buffer for one hour at room temperature.The Der p 1 reactivity of IgE and IgG1 2C7 antibodies was investigated by ELISA. Maxisorp 96-well microtiter plates  were coated with Der p 1 (or deglycosylated Der p 1 (deg) where applicable) or the control allergen Fel d 1  and IgG  control antibodies were incubated at different concentrations at 37°C for two hours. Subsequently, the plates were washed three times and the binding was detected by incubation with a goat anti-human κ chain HRP-conjugated secondary antibody . After three washes with PBS-Tween, followed by a single wash with a proprietary HRP substrate buffer , the plates were subsequently developed with o-Phenylenediamine (OPD)  in a HRP substrate buffer containing 0.012% H+2 and Mg+2  and 10% FBS . The cells were then incubated for 20 minutes at 37°C.Mannan (200 μg/ml), MR’s natural ligand, was added to aliquots of Mo-DCs using uptake media containing RPMI , 30% PBS with CaDifferent mixtures of Cy5-Der p 1 with or without human IgE or IgE 2C7 were prepared and incubated at 37°C for 20 minutes. These mixtures were added to cells that had either been pre-incubated with mannan or not, and then incubated for 20 minutes at 37°C. The final concentration of Der p 1 was 0.26 μg/ml, while human IgE and IgE 2C7 were used at 1 μg/ml. The cells were then washed and fixed with 0.5% formaldehyde.In parallel, other cell aliquots were treated with 1 μg/ml or 10 μg/ml human IgE for 20 minutes at 4°C in order to block IgE receptors, following which they were washed and loaded with the mixture of Der p 1-IgE 2C7 as above for 20 minutes. The quantitative Cy5-Der p 1 uptake was then established using flow cytometry.Myeloid dendritic cells were incubated with Der p 1 or deg Der p 1 (0.26 μg/ml), with and without IgE 2C7, IgG 2C7, IgE or IgG (1 μg/ml), for two to three hours at 37˚C. Cells were then washed and cultured with autologous CD3 + CD45RO- (naïve) T cells at DC/T cell ratio of 1:5. The naïve T cells had been negatively separated from PBMCs, using a magnetic cell separation system  and following the manufacturer’s instructions.Cultures were carried out using 96-well U bottom plates  and RPMI 1640 supplemented with penicillin/streptomycin  and 10% human AB serum . IL-2  (R & D system) was added every three to four days until day 10 of culture. After 10 days of culture, T cells were re-stimulated overnight with PMA (15 ng/ml) and Ionomycin (1 μg/ml). The supernatant from each condition was collected, and FlowCytomix Human Basic kits were used in order to detect cytokine concentrations.Student’s T test and Mann–Whitney test were used to perform the statistical analysis. Data was considered statistically significant when P-values were less than 0.05. *: p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.Authors have no competing interests.IKS carried out the experimental work and drafted the manuscript. AA carried out deglycosylation studies. PF, KA and MK participated in generating the Der p 1-specific chimeric antibodies. FS, HS and AMG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


PGI2 and NO trigger the synthesis of cAMP and cGMP, respectively, which in turn leads to the activation of Protein Kinase A (PKA) and Protein Kinase G (PKG). These cyclic nucleotide-dependent protein kinases phosphorylate numerous substrate proteins involved in several aspects of platelet activation. Both, PKA and PKG can phosphorylate the same substrates including VASP, LASP, RAP1GAP2, Rap1B, HSP27, and some others. Only few substrates are known to be specific for PKA (PDE3) and PKG (PDE5) [Under normal conditions circulating platelets are maintained in a resting state by the paracrine signaling molecules, prostacyclin PGI and nitrUsing radioactive phosphate incorporation assay, we identified S587 as the major PKA phosphorylation site in CalDAG-GEFI, which was confirmed by mass spectrometry. In platelets, PKA stimulation leads to strong phosphorylation of CalDAG-GEFI and inhibition of Rap1b activation triggered by thrombin, as well as calcium ionophore, which indicates a potential role of CalDAG-GEFI phosphorylation in the inhibition of Rap1b activation. However, PKG activation did not significantly phosphorylate CalDAG-GEFI, and inhibits thrombin-stimulated Rap1b by inhibition of calcium mobilization but did not inhibit calcium ionophore-triggered Rap1b activation. In HEK293 cells transfected with CalDAG-GEFI, calcium ionophore triggered Rap1b activation and forskolin stimulated PKA phosphorylation of CalDAG-GEFI leads to inhibition of Rap1b activation. But in cells with S587A mutant, PKA activation did not inhibit Rap1b activation. The phospho-mimetic (S587D) mutant failed to activate Rap1b following calcium ionophore stimulation.These results confirmed that PKA phosphorylation of CalDAG-GEFI inhibits Rap1b activation. We propose that this is one of the important mechanisms of platelet inhibition mediated by cAMP/PKA signaling. Presented data show that PKA and PKG could utilize similar and/or diverse intracellular mechanisms involved in platelet inhibitory pathways.


The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl− secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca2+, which not only activates epithelial CaCCs, but also inhibits epithelial Na+ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl− secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca2+ and Cl− secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.Ca Spiperone, therefore, represents a potential therapeutic compound for the treatment of CF inh172 The basic defect of cystic fibrosis (CF) is a deficiency in chloride secretion due to the malfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. Defective chloride secretion leads to dried airways and thick viscous mucous in airway and other mucosal epithelia in CF patients Here, we studied the spiperone signaling pathway in human airway epithelial cells, in order to investigate the mechanism of spiperone in stimulating chloride secretion. Using a proteomic approach, we have identified protein tyrosine kinase 2 (PYK2) as the signaling molecule that mediates spiperone's effect on chloride secretion. Inhibition of PYK2 through a specific inhibitor revealed that PYK2 is not only responsible for the spiperone stimulated CaCC activation, but appears to also play a role in the activation of CFTR. Our data uncovers a new role of PYK2 in mediating chloride secretion in human airway epithelial cells.2+ and Cl− secretion through a protein tyrosine kinase dependent pathway in human airway epithelial cells Our previous study showed that spiperone triggered an increase in intracellular CaIn order to identify the identity of the protein that is detected by the anti-phosphotyrosine antibody in IB3-1 cells, mass spectrometry analysis was performed. With the purpose of increasing the amount of the protein for mass spectrometry analysis, 4 of 10 cm dishes were utilized to collect the protein lysates and an immunoprecipitation experiment was performed. 2 mg of cellular protein of the DMSO and spiperone treated cells was immunoprecipitated with the anti-phosphotyrosine antibody and the protein agarose A/G beads. The majority of the protein was loaded on an SDS-page gel for commassie blue gel staining, while a small amount of the protein was loaded in a separate gel for western blotting with the same antibody. Drosophila homologue of Kette/Hem-2 works either as a cell surface receptor or a membrane docking protein Spots were identified by LCMS/MS to obtain CID spectra with sequence information that were each matched to the human protein database by Mascot search engine with greater than 95% confidence. 32 proteins were detected and identified by spectral counting with Scaffold software. The proteins of sizes between 105–160 kd were studied further including proline-rich tyrosine kinase 2 (PYK2), Crk-associated substrates (p130cas), Nck-associated protein1 (NAP1) and cytoplasmic FMR1 interacting protein 1 (CYFIP1). PYK2 is a proline rich cytoplasmic tyrosine kinase that belongs to the same family with the focal adhesion kinase (FAK) In order to further narrow down which protein was activated and tyrosine phospohorylated by spiperone, we first investigated and confirmed the expression of the above four proteins identified by mass spectrometry analysis in IB3-1 cells. Specific antibodies for each protein were used to detect them. Because the amount of the protein expression in the cells was unknown, immunoprecipitation was used instead of western blotting in order to detect even trace amounts of protein. The antibodies against PYK2 detected a band at 116 kD in IB3-1 cells . CYFIP1 To test whether PYK2, CYFIP1 and p130CAS were tyrosine phosphorylated by spiperone, co-immunoprecipitation experiments were performed with the anti-phosphotyrosine antibody and each specific antibody against those proteins. The same amount of protein from the DMSO and spiperone treated groups was pulled down by the anti-phosphotyrosine antibody and immunoblotted with each specific antibody. As shown in P130CAS is a downstream signaling molecule of the PYK2 signaling cascade and is physically associated with PYK2 as reported previously To test whether PYK2 plays a role in the increase in intracellular calcium induced by spiperone, a specific PYK2 inhibitor tryphostin-A9 was used To understand the functional role of PYK2, we measured the spiperone stimulated chloride current in the presence and absence of PYK2 inhibitor in short-circuit current experiments. The Calu-3 cell line was used in this study, because these cells form a tight monolayer which is essential for the current measurements. Moreover, Calu-3 cells express both CFTR and CaCC channels and spiperone was able to activate both channels as previously described − current that was partially sensitive to the CFTR inhibitor 172, and further reduced in the presence of the CaCC channel blocker DIDS. The CFTR sensitive current was linear in nature with little rectification, consistent with the characteristics of CFTR mediated Cl− currents. These results support our hypothesis that spiperone is activating both CFTR and CaCC channels in transfected IB3-1 cells, and also supports our previous short circuit current data. To insure that CFTR activation was not dependent on CaCC activation or unique to bronchial epithelial cells we performed similar experiments on CHO cells transiently transfected with WtCFTR-GFP. CHO cells have little endogenous CaCC current or any other large endogenous Cl− conductances − current.To confirm spiperone's activation of CFTR in bronchial epithelial cells, we used whole cell voltage clamp techniques to characterize the spiperone-activated Cl- currents. IB3-1 cells, transiently transfected with WtCFTR-GFP, were recorded before and after exposure of the cells to spiperone . Spipero− channel function is regulated by focal adhesion kinase (FAK) at tyrosine 407. CFTR and FAK co-localize in the apical crypt of the teleost mitochondria rich salt secreting (MR) cells and where the CFTR mediated Cl− secretion is regulated by tyrosine phosphorylation Since PYK2 plays a role in spiperone stimulated chloride current, the relationship of PYK2 and CFTR was studied further. Previous studies have indicated that CFTR ClIn order to investigate whether CFTR is tyrosine phosphorylated by PYK2, the anti-phosphotyrosine antibody was used. IB3-1 cells were transiently transfected with wt-CFTR and treated with DMSO or spiperone for 2 mins. Cell lysate were pulled down by the anti-phosphotyrosine antibody. CFTR was detected in the protein complex which is precipitated by the anti-phosphotyrosine antibody and its expression was enhanced in the spiperone treated cells . These d2 (IC50 of 40 nM) and dopamine D2 (IC50 of 10 nM) 2+. PYK2, a cytosolic tyrosine kinase, was identified by mass spectrometry LCMS/MS as the signaling molecule that mediates spiperone's effect in stimulating intracellular Ca2+ elevation and Cl− secretion.Spiperone is a bytyrophenonoe antipsychotic agent with a known antagonist activity to receptors of both serotonin 5-HTIn addition, we also identified and confirmed the presence of two other proteins P130Cas and CYFIP1 by LCMS/MS, whose tyrosine residues may also be phosphorylated by spiperone. P130CAS is an adaptor protein and a substrate of PYK2 kinase via a proline rich region on the C-terminus of PYK2. When P130CAS is tyrosine phosphorylated, it recruits other proteins to form a protein complex and activates downstream signaling 2+. A variety of stimuli that elevate intracellular Ca2+ can cause tyrosine phosphorylation of PYK2 2+ is regulated by PYK2 was not known for many years. A recent study by Kohno and colleagues provided the first evidence for the nature of this mechanism. They found that the Ca2+ sensor calmodulin regulates PYK2 activation through direct interaction with the band FERM  domain of PYK2 2+, other studies show that PYK2 plays a role in the elevation of intracellular Ca2+. Matsui and colleagues have suggested that PYK2 plays a role in VEGF stimulated increase in cell Ca2+ and in PLCγ1 activation. Phosphorylation of PLCγ1 and mobilization of Ca2+ was significantly decreased in endothelial cells of PYK2 knock-out mice 2+ release was also compromised in macrophages derived from PYK2 knock-out mice. Thus, PYK2 appears to play a crucial role in chemokine-driven Ca2+ release 2+, which indicates that PYK2 is an upstream molecule of the increase of intracellular Ca2+ induced by spiperone. However, we cannot rule out the possibility that the release of Ca2+ augments further phosphorylation of PYK2.PYK2 is a non- receptor cytoplasmic tyrosine kinase which belongs to the same family with FAK − channel function is regulated by focal adhesion kinase (FAK) at tyrosine 407 C-SRC, can also phosphorylate and activate CFTR in in vitro studies + -K+ -2Cl− cotransporter is regulated by tyrosine phosphorylation initiated by prolactin through a JAK2 kinase pathway Our data suggests that PYK2 also plays a role in spiperone stimulated CFTR activation. CFTR activity is well known to be regulated by phosphorylation. The R domain of CFTR has about 20 sites for phosphorylation by PKA and PKC. Serine/threonine phosphorylation of the R domain is the major pathway for CFTR activation − secretion. We have also demonstrated that CFTR is activated by spiperone and its activation is dependent on tyrosine phosphorylation.In summary, through a proteomic approach, we have identified that the tyrosine kinase PYK2 plays a crucial role in spiperone induced ClIB3-1 is a CF human bronchial epithelial cell line that has two different CFTR mutations (ΔF508 and W1282X) IB3-1 cells and CHO were transiently transfected with wt-CFTR-gfp construct by using the transfection reagent lipofectamine 2000 . The procedure is followed as described in the manufacture protocol. 24 hs after transfection, cells were collected for biochemistry or for patch clamp electrophysiology.Cells were put on ice and washed with ice cold PBS three times. Cell lysates were collected and mixed with protein A/G beads and the corresponding antibody for specific experiments. The immunocomplex were incubated and rotated at 4°C overnight. The beads were precipitated after centrifugation and washed three times with the cell lysis buffer. The beads were incubated in the laemmli sample buffer at 95°C for 5 mins and run on a SDS page gel, followed by the immunoblot analysis.Cells were lysed in a RIPA buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM pH 8.0 Tris-Cl, and a Complete mini protease inhibitor cocktail tablet. Twenty micrograms of protein were loaded and run on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. PYK2 mouse monoclonal antibody (BD Transduction Laboratories) was diluted at 1∶1000. All other primary antibodies were diluted at 1∶500. The secondary antibody was horseradish peroxidase-labeled goat anti-rabbit IgG diluted at 1∶3,000. Enhanced chemiluminescence was used to visualize the secondary antibody.4HCO3. Gel pieces were digested by trypsin in NH4HCO3 and incubate overnight at 37°C. The tryptic peptides were extracted by 50% acetonitrile/5% formic acid. The supernatant was collected and dried in a speed vacuum. Samples were resuspended in 0.1% formic acid and analyzed by reserved phase liquid chromatography  and mass spectrometry LTQ Orbitrap . The C18 column  was used in 2D nanoLC with gradient (5–60% of 0.1% Formic acid/90% acetonitrile) over 30 minutes with a flow rate of 300 nl/min.) Data-dependent MS/MS mode was applied and the MS/MS spectra were submitted to human database NCBInr_GB_165 (updated at 04/18/2008) by MASCOT Daemon  with the missed cleavages of 2 peptides and MS scan and MS/MS tolerance 0.1 and 0.8 da. Then the data were transferred to Scaffold  for Mascot result validation and comparison of protein identifications between the individual samples.1 mg DMSO and spiperone treated cellular protein was loaded on SDS-page gels and visualized by the commassie blue staining. Gels with bands between 105 to 160 kd were cut and sliced into 1×1 mm pieces. Gel pieces were rinsed with 50% methanol and NH2+ was measured with a dual excitation wavelength microscope system. Fura-2 fluorescence was excited at 340 nm and 380 nm with xenon light. Fluorescence was measured at 510 nm emission wavelength. Data was processed and analyzed using IPLab 4.0 software. Fluorescence data of each experiment were normalized to the individual basal fluorescence value ΔF/F0 = (F−F0)/F0 to bring all the response curves to the same pretreatment starting point.Cells were seeded onto the collagen-coated cover slips for 48 h. Cell CaCalu-3 cells were cultured on Snapwell inserts until they formed a tight monolayer as monitored by measuring the transepithelial resistance. The bottom of the monolayer was taken out, mounted on an Ussing chamber and buffered by the testing solutions. The short-circuit current was measured by a VCC MC6 multichannel voltage/current clamp . Data were recorded and analyzed by Acquire and Analyze software.Whole cell patch clamp recordings were performed in IB3-1 cells and CHO cells transiently transfected with a plasmid containing human CFTR tagged with GFP. Whole-cell currents were acquired and amplified using an Axopatch 200B amplifier , visualized using a Nikon Eclipse Fluorescent inverted microscope , and analyzed using PClamp 9.2 software . Cells were bathed in a solution containing (mM): 140 NaCl, 2CaCl2, 1 MgCl2, 80 D-mannitol, and 10 HEPES, pH 7.4. The pipette solution consisted of (mM): 135 CsCl, 2 MgCl2, 2 ATP, 2 EGTA, 10 HEPES, with or without 0.0001 free Ca2+, pH 7.4. All the recordings were performed at room temperature.


The outflow facility for aqueous humor across the trabecular meshwork (TM) is enhanced by agents that oppose the actomyosin contraction of its resident cells. Phosphorylation of MYPT1 (myosin light chain [MLC] phosphatase complex of Type 1) at Thr853 and Thr696 inhibits dephosphorylation of MLC, leading to an increase in actomyosin contraction. In this study, we examined the effects of Rho kinase (ROCK) inhibitors on the relative dephosphorylation of the two sites of MYPT1 using human TM cells (GTM3).Dephosphorylation of MYPT1 at Thr853 and Thr696 was determined by western blot analysis following exposure to selective inhibitors of ROCK, namely Y-27632 and Y-39983. Consequent dephosphorylation of MLC and decreases in actomyosin contraction were assessed by western blot analysis and collagen gel contraction assay, respectively. Changes in the cell-matrix adhesion were measured in real time by electric cell-substrate impedance sensing and also assessed by staining for paxillin, vinculin, and focal adhesion kinase (FAK).50 values for Y-39983 were 15 nM and 177 nM for dephosphorylation at Thr853 and Thr696, respectively. Corresponding values for Y-27632 were 658 nM and 2270 nM. Analysis of the same samples showed a decrease in MLC phosphorylation with IC50 values of 14 nM and 1065 nM for Y-39983 and Y-27632, respectively. Consistent with these changes, both inhibitors opposed contraction of collagen gels induced by TM cells. Exposure of cells to the inhibitors led to a decrease in the electrical cell-substrate resistance, with the effect of Y-39983 being more pronounced than Y-27632. Treatment with these ROCK inhibitors also showed a loss of stress fibers and a concomitant decrease in tyrosine phosphorylation of paxillin and FAK.Both ROCK inhibitors produced a concentration-dependent dephosphorylation at Thr853 and Thr696 of MYPT1 in adherent GTM3 cells. ICY-39983 and Y-27632 oppose ROCK-dependent phosphorylation of MYPT1 predominantly at Thr853 with a corresponding decrease in MLC phosphorylation. A relatively low effect of both ROCK inhibitors at Thr696 suggests a role for other Ser/Thr kinases at this site. Y-39983 was several-fold more potent when compared with Y-27632 at inhibiting the phosphorylation of MYPT1 at either Thr853 or Thr696 commensurate with its greater potency at inhibiting the activity of human ROCK-I and ROCK-II enzymes. The outflow of aqueous humor across the trabecular meshwork (TM) is regulated by, among other factors, actomyosin contraction of the resident TM cells and altered extracellular matrix (ECM) -3. Ex vi2+-calmodulin)-dependent kinase [2+ ([Ca2+]i) activate MLCK and induce MLC phosphorylation. However, sustained contraction is dependent on the activity of MLC phosphatase (MLCP)  of the drug. The calculations assume that log [drug] versus response follow a sigmoidal shape and were performed with Prism 5.0. The reported values of R indicate goodness of fit.A one-way ANOVA was used to compare mean values for different treatments with Bonferroni’s post-test analysis . Results are expressed as mean±SEM. For analysis of ECIS  data, normalized numerical values from individual experiments were pooled and expressed as mean±SEM. A p value of less than 0.05 was considered statistically significant. “n” denotes the number of independent experiments performed. The ICMYH9) and myosin IIB (MYH10), are expressed in the GTM cells. We failed to detect the expression of myosin IIC (MYH14). The known isoforms of MYPT (MYPT1 and MYPT2) and ROCK (ROCK-I and ROCK-II) were also expressed, along with the two MLCK splice variants . 50 for dephosphorylation at Thr696 for Y-27632 was 2270 nM  make use of a lock-in amplifier to measure small changes in electrical impedance at a high-temporal resolution. We have previously employed ECIS to assess the integrity of the apical junctional complex of the corneal endothelial monolayers [ECIS involves the measurement of impedance to a small AC current applied across cells grown to confluence on planar gold microelectrodes ,28. The nolayers -31. For nolayers -32. Howenolayers ,34. In tAfter the observed ECSR reached a steady-state, cells were challenged with the ROCK inhibitors, and the time-dependence of the ECSR was followed at several frequencies . These fActomyosin contraction affects cell shape and cell-matrix adhesion through its impact on the stress fibers. The altered cell-matrix adhesion manifests in part by tyrosine phosphorylation of focal adhesion proteins such as FAK, vinculin, and paxillin. Tyrosine phosphorylation is necessary for the functional assembly of the focal adhesion complexes and their association with the actin cytoskeleton . The chaThe disposition of the stress fibers with and without the ROCK inhibitors is shown in Consistent with the immunofluorescence data, tyrosine phosphorylation of FAK and paxillin were altered in the presence of ROCK inhibitors (data for Y-27632 not shown). The addition of 10% serum to cells led to an increase in tyrosine phosphorylation of FAK Tyr397;  and paxiIn humans, more than 80% of aqueous humor exits the anterior chamber via the TM route -38. TherMLCK, MYPT, and ROCK genes. The expression of the short isoform of MLCK was first reported in smooth muscle cells , while the longer splice variant (EC-MLCK) was found in vascular endothelial cells and other non-smooth muscle cell types  [ROCK isoforms (ROCK-I and ROCK-II isoforms) is not unique to GTM cells, as their expression has been reported in the TM cells derived from bovine, monkey, and human sources [MYPT1 and MYPT2 isoforms in monkey and human TM, and hence the expression in GTM cells, was also as expected. Expression of myosin II isoforms MYH9 and MYH10, but not of MYH14, has been previously reported in normal TM cells [In the context of our specific objectives outlined above, we performed our experiments with a TM cell line  developed previously from a human donor with glaucoma . As notethelium) . This exthelium) . The exp sources ,42,45. ATM cells . The expMYPT1, MYPT2 (in striated muscle and brain), MBS85, MYPT3, and TIMAP [MYPT1 is expressed in multiple isoforms generated by alternative splicing showing differential sensitivity to cGMP/PKG [MYPT1, as seen in 2+ sensitization [Members of the MYPT family include separate gene products such as nd TIMAP . MYPT1 icGMP/PKG . The doutization . Severaltization . As notetization   and specific at inhibiting ROCK compared to Y-27632 [Employing site-specific phospho antibodies, we examined the relative phosphorylation at Thr696 and Thr853 without and with ROCK inhibitors. As expected, both Y-27632 and Y-39983 reduced the phosphorylation at Thr696 and Thr853 and led to decreased MLC phosphorylation as well as reduced actomyosin contraction. It is evident from the IC Y-27632 . Further Y-27632 . Finally Y-27632 .2+ sensitization [50 values of MLC phosphorylation closely follow the IC50 values obtained for phosphorylation at Thr853. If there were direct phosphorylation of MLC by ROCK, we would not expect to see this agreement in the IC50 values.In addition to MYPT1 phosphorylation and inactivation, ROCK has been shown to directly phosphorylate recombinant MLC at Ser19 similar to MLCK . Howevertization . Our datA recent study on TM demonstrated that expression of constitutively active RhoA led to an increase in stress fibers, actomyosin contraction, focal adhesions, and the gene expression of various ECM proteins . In the In the past, cell-ECM interactions have been studied using qualitative immunocytochemical techniques. Here, we have employed a sensitive technique, ECIS, to measure cell-ECM interactions. This technique has been previously used to study cell signaling mechanisms by monitoring the changes in cellular characteristics such as migration , cell-ceThe resistance to outflow facility through the TM is due to increased actomyosin contraction and/or alterations in the ECM. These factors are not mutually exclusive, but modulate each other. As discussed above, increased actomyosin contraction reduces the outflow facility. This is thought to lead to the compaction of the TM tissue, which leads to reduced intertrabecular pores and therefore an increase in the resistance to the flow of fluid. On the other hand, the increase in the deposition of ECM in the anterior chamber can in itself reduce TM porosity and obstruct the fluid flow. In addition, the change in the shape of the cells (retraction of cells as seen with agents that increase cAMP and disrupt actin polymerization and ROCK inhibitors) might increase the porosity of the TM and thus aid the egress of aqueous humor from the anterior chamber, leading to a reduction in IOP. Therefore, it is hoped that, along with the existing pharmaceutical strategies to treat ocular hypertension, suitable ROCK inhibitors with adequate efficacy may soon become available to treat ocular hypertension and glaucoma. In a recent study , we haveIn light of suitable drug development, this study highlights an important signaling mechanism through which ROCK inhibits the phosphatase activity, leading to increased actomyosin contraction. Specifically, the results highlight the difference in the sensitivities of the two phosphorylation sites on MYPT1 to ROCK inhibition with the phosphorylation at Thr853 regulating the phosphorylation of MLC (summarized in


Candida albicans cell surface proteins are decorated post-translationally by glycosylation. Indeed N-glycosylation is critical for cell wall biogenesis in this major fungal pathogen and for its interactions with host cells. A detailed understanding of N-glycosylation will yield deeper insights into host-pathogen interactions. However, the analysis of N-glycosylation is extremely challenging because of the complexity and heterogeneity of these structures. Therefore, in an attempt to reduce this complexity and facilitate the analysis of N-glycosylation, we have developed new synthetic C. albicans reporters that carry a single N-linked glycosylation site derived from Saccharomyces cerevisiae Suc2. These glycosylation reporters, which carry C. albicans Hex1 or Sap2 signal sequences plus carboxy-terminal FLAG3 and His6 tags, were expressed in C. albicans from the ACT1 promoter. The reporter proteins were successfully secreted and hyperglycosylated by C. albicans cells, and their outer chain glycosylation was dependent on Och1 and Pmr1, which are required for N-mannan synthesis, but not on Mnt1 and Mnt2 which are only required for O-mannosylation. These reporters are useful tools for the experimental dissection of N-glycosylation and other related processes in C. albicans, such as secretion.A large proportion of  This fungus is a frequent cause of mucosal infections , and in severely immunocompromised patients it can cause life-threatening systemic infections of the bloodstream and internal organs  that are recognised by specific host pattern recognition receptors (PRRs) during host innate immune responses  is transferred onto Asn-X-Ser/Thr acceptor sites   were designed, synthesised by DNA2.0  and subcloned between the HindIII and NheI sites of pACT-GFP , which is RPS10) . The GR  program .C. albicans ), and sheared with glass beads. Lysates were centrifuged at 15,000g for 10 min at 4 °C, and these extracts stored at −20 °C. Extracts were reduced with 3 mM dithiothreitol , S-alkylated with 13 mM iodoacetamide , digested 8 h at 37 °C with trypsin , dried by rotary evaporation , and dissolved in 0.1% formic acid before analysis by western blotting.Intracellular protein extracts were prepared using conventional protocols . BrieflyC. albicans strains were grown in 1 l SD at 30 °C for 24 h. The culture medium was separated from cells by centrifugation, the extracellular proteins concentrated using an Amicon membrane ultrafiltration system  as described . Preparations were then dialysed against phosphate-buffered saline and freeze-dried before analysis or concentrated by Vispaspin ultrafiltration .To isolate extracellular proteins, escribed , and the2.4N-mannan. Standard protocols were used for western blotting . The secondary antibody was horse radish peroxidise-conjugated anti-rabbit IgG . Signals were detected with an HRP western blotting kit .Protein concentrations were assayed using standard protocols . In someblotting  with sli33.1N-glycosylation in C. albicans. We reasoned that this reporter need not encode a functional entity, except with regard to its ability to act as a glycosylation target. Therefore, we chose to create a synthetic reporter that carries multiple features to facilitate downstream characterisation of its N-glycosylation. This reporter carries a single N-glycosylation site (to prevent heterogeneous glycosylation at multiple target sites). This site, which is derived from S. cerevisiae Suc2, has been validated biochemically (S. cerevisiae Suc2 that surrounds a well-defined glycosylation site (Asn146-Ser-Thr) in the Suc2 protein  included the 23 amino-terminal amino acids of C. albicans N-acetylglucosaminidase Hex1  were then designed, synthesized and cloned to generate the plasmids pACT1-GR2 and pACT1-GR3. In GR2 the 23 residue Hex1 signal sequence was replaced with the 63 amino acid aminoprotease  Fig. 1)-terminal3.2C. albicans of about 25 kDa, 28 kDa and 28 kDa, respectively  proteins in ectively . FollowiC. albicans CAI4 cells transformed with pACT1-GR1, pACT1-GR2 or pACT1-GR3, and from control cells lacking GR sequences  were present inside C. albicans cells. Proteolysis is unlikely to account for the multiple isoforms as protease inhibitors were included during protein extraction  by C. albicans cells (C. albicans mnt1 mnt2 cells, in which O-glycosylation is disrupted (To confirm that the Asn146-Ser-Thr site represented the only site of ns cells  confirmens cells . Althougisrupted .C. albicans och1 and pmr1 cells, which have been shown to display significant defects in N-mannan outer chain elongation (N-glycosylation in C. albicans. The GR1 and GR2 reporters only differ with respect to their amino-terminal sequences. GR1 carries the Hex1 signal sequence whereas GR2 has the pre-pro- region from Sap2. Our data suggest that both the Hex1 and Sap2 signal sequences work in the context of these synthetic GR reporters (However, the elaboration of outer mannan chains on GR1 and GR2 was compromised in ongation . Therefoongation  suggestieporters .N-glycosylation reporters (3 and His6 tags that facilitate their detection (N-glycosylation site (6 tag. Thirdly, an additional purification step, possibly based on immuno-affinity chromatography of the FLAG-tag (C. albicans proteins that co-purify with GR reporters on the nickel columns. Even after purification, some heterogeneity in N-glycosylation patterns is likely to remain, because individual GR molecules probably vary in their N-glycan structures. Nevertheless, our system simplifies this heterogeneity by facilitating the analysis of a specific GR sequence carrying a single N-glycosylation site. Therefore, these GR reporters should prove to be useful tools for the dissection of N-glycosylation in C. albicans and its interactions with the host. We also note that the GR reporters may prove useful for mechanistic analyses of other virulence-related related processes in C. albicans such as secretion, cell wall biogenesis and adhesion.The GR1 and GR2 reporters carry other features that are designed to facilitate their exploitation as eporters . Convenieporters . The repetection  and partetection C. Functietection C are locion site , facilitFLAG-tag , might b


In response to CDK7 inhibition, p21-binding to CDK4 increased concomitantly with disappearance of the most abundant phosphorylation of p21, which we localized at S130 and found to be catalyzed by both CDK4 and CDK2. The S130A mutation of p21 prevented the activating CDK4 phosphorylation, and inhibition of CDK4/6 and CDK2 impaired phosphorylations of both p21 and p21-bound CDK4. Therefore, specific CDK7 inhibition revealed the following: a crucial but partly indirect CDK7 involvement in phosphorylation/activation of CDK4 and CDK6; existence of CDK4-activating kinase(s) other than CDK7; and novel CDK7-dependent positive feedbacks mediated by p21 phosphorylation by CDK4 and CDK2 to sustain CDK4 activation, pRb inactivation, and restriction point passage.Cell cycle progression, including genome duplication, is orchestrated by cyclin-dependent kinases (CDKs). CDK activation depends on phosphorylation of their T-loop by a CDK–activating kinase (CAK). In animals, the only known CAK for CDK2 and CDK1 is cyclin H-CDK7, which is constitutively active. Therefore, the critical activation step is dephosphorylation of inhibitory sites by Cdc25 phosphatases rather than unrestricted T-loop phosphorylation. Homologous CDK4 and CDK6 bound to cyclins D are master integrators of mitogenic/oncogenic signaling cascades by initiating the inactivation of the central oncosuppressor pRb and cell cycle commitment at the restriction point. Unlike the situation in CDK1 and CDK2 cyclin complexes, and in contrast to the weak but constitutive T177 phosphorylation of CDK6, we have identified the T-loop phosphorylation at T172 as the highly regulated step determining CDK4 activity. Whether both CDK4 and CDK6 phosphorylations are catalyzed by CDK7 remains unclear. To answer this question, we took a chemical-genetics approach by using analogue-sensitive CDK7(as/as) mutant HCT116 cells, in which CDK7 can be specifically inhibited by bulky adenine analogs. Intriguingly, CDK7 inhibition prevented activating phosphorylations of CDK4/6, but for CDK4 this was at least partly dependent on its binding to p21 In the cell cycle, duplication of all the cellular components and subsequent cell division are governed by a family of protein kinases associated with cyclins (CDKs). Related CDK4 and CDK6 bound to cyclins D are the first CDKs to be activated in response to cell proliferation signals. They thus play a central role in the cell multiplication decision, especially in most cancer cells in which CDK4 activity is highly deregulated. We have identified the activating T172 phosphorylation instead of cyclin D expression as the highly regulated step determining CDK4 activation. This finding contrasts with the prevalent view that the only identified metazoan CDK-activating kinase, CDK7, is constitutively active. By using human cells genetically engineered for specific chemical inhibition of CDK7, we found that CDK7 activity was indeed required for CDK4 activation. However, this dependence was conditioned by CDK4 binding to the CDK inhibitory protein p21, which increased in response to CDK7 inhibition. Further investigation revealed that CDK7 inhibition affects a major phosphorylation of p21, which we found to be required for CDK4 activation and performed by CDK4 itself and CDK2. Thus, depending on CDK7 activity, CDK4 and CDK2 facilitate CDK4 activation, generating novel positive feedbacks involved in the cell cycle decision. Kip1, has been shown to generate a bistable pRb-E2F switch to convert graded mitogen inputs into all-or-none E2F responses and thus cell cycle commitment The cell cycle commitment at the restriction (R) point in G1 phase is initiated by inactivating phosphorylations of the central cell cycle/tumor suppressor pRb by CDK4 and CDK6, which are activated by D-type cyclins induced by mitogenic/oncogenic signaling CDK4/6 activity is deregulated through various mechanisms in many human tumors Activation of CDK4/6 is a still poorly understood multistep process that includes an activating phosphorylation in the T-loop at T172 in CDK4 TPVVVT in CDK4 vs QMALTSVVVT in CDK6) in vitro substrates of CAK/CDK7 than CDK4 in vitro activation by CAK This determining regulation of CDK4 T172 phosphorylation contrasts with the prevalent concept that the activating phosphorylations of the different CDKs, including CDK4 and CDK6, are performed solely by the only CDK-activating kinase (CAK) known in metazoan cells, which is composed of the cyclin H-CDK7-Mat1 complex We thus hypothesized that unlike CDK2 and CDK1 RNAi-mediated  depletion of CDK7 is generally insufficient to affect cell cycle progression and thus CDK activity. Moreover, approaches based on inhibition of CDKs or their knockout (or knockdown) have generated divergent conclusions in vitro phosphorylation by recombinant CAK, and analysis of T172A-mutated CDK4 Cell cycle progression and kinetics of CDK4 phosphorylation upon stimulation of serum-deprived K7AS HCT116 cells with 10% serum were analyzed as detailed in To test whether CDK7 inhibition affects the activation of CDK4 through T172-phosphorylation, serum-deprived wild-type (wt) and K7AS HCT116 cells were re-stimulated by serum in the continuous presence or absence of the bulky adenine analog 1-NMPP1 (10 µM) to specifically inhibit CDK7 activity. As previously shown In the same experiments, we analyzed in K7AS cells the effect of CDK7 inhibition on the pRb-kinase activity of CDK4 and CDK6, both of which were immunoprecipitated using antibodies against cyclin D1, D3, p21 or CDK6. The protein composition of these complexes was also analyzed by western blotting . CDK7 inThe inhibition of pRb-kinase activity in response to selective CDK7 inhibition was associated with reduced phosphorylations of CDK4 and/or CDK6. Co-immunoprecipitation of these kinases and separation of their phosphorylated forms by 2D-gel electrophoresis showed that continuous treatment with 1-NMPP1 during serum stimulation abolished the phosphorylation of CDK4 bound to cyclin D1, cyclin D3 or p21 in K7AS but not in wt HCT116 cells . ConsistApplication of CDK7 inhibition after mitogenic stimulation has been reported to have only a delayed effect on CDK2 activity because T160 phosphorylation of CDK2 is stabilized by cyclin binding in vivo depends critically on CDK7 activity. Moreover, CDK4 phosphorylation is labile and thus more prone to rapid regulation, potentially making CDK4 the most influential target of CDK7 in G1/S transition.These results show that the activation of CDK4 and CDK6 In co-immunoprecipitations using antibodies against cyclin D1 and cyclin D3, we noticed a marked increase in the association of p21 in response to sustained CDK7 inhibition. This increase was not paralleled by a similar increase of CDK4 and CDK6 associations (lanes 3 and 5 in in vitro by cyclin H-CDK7-MAT1 (CAK) complexes in vitro phosphorylation of CDK2 and cyclin D3-CDK6 We first evaluated if CDK4 and CDK6 complexes from CDK7-inhibited cells can be re-activated by CAK. Indeed, if the impaired activation of CDK4 and CDK6 complexes in CDK7-inhibited K7AS cells was due only to the absence of activating phosphorylation, these complexes should remain phosphorylatable by CAK. We and others have shown that cyclin D3-CDK4 can be phosphorylated and activated Cyclin D1 and cyclin D3 immunoprecipitates from K7AS cells –which contained mostly unphosphorylated CDK4 and CDK6 after serum-deprivation or re-stimulation by serum in the presence of 1-NMPP1– were incubated with 2 mM ATP and active recombinant CAK. As shown in To better understand how p21 could intervene in the regulation of CDK4 activation even before the increase of p21 steady-state level, we evaluated p21 modifications in K7AS cells by 2D gel electrophoresis. As shown in in vitro phosphorylation by active recombinant CDK complexes. In the same experiments, p21 was also produced as a complex with cyclin D3 and CDK4 (immunoprecipitated by the DCS-28 cyclin D3 antibody) to evaluate how the accessibility of p21 phosphorylation sites might have been affected. As analyzed in in vitro experiments, the association of p21 with cyclin D1-CDK4 was also required for its complete S130 phosphorylation by cyclin D1-CDK4 and cyclin D1-CDK6  . The effWhen expressed alone, whether at a high or a low level, p21 was essentially unphosphorylated . Intriguin vitro experiments. That this p21 S130-kinase could itself be inhibited by p21 is suggested by the observation that S130 phosphorylation of p21 was not induced in cyclin D3-CDK4 complexes when p21 was expressed at concentrations that largely exceed the binding capacity of the CDK4 complex.All together, experiments on transfected CHO cells further established the correlation between T172 phosphorylation of CDK4 and S130 phosphorylation of p21. Binding of p21 to cyclin D3-CDK4 might expose S130 to another kinase, as also suggested by the previous Previous reports have indicated that S130 phosphorylation of p21 can be catalyzed by CDK2 to form a phosphodegron recognized by the SCF/Skp2 ubiquitin ligase complex and to signal the proteasomal degradation of cyclin/CDK-bound p21, which is required for S-phase entry To evaluate the effect of the S130 phosphorylation of p21 on the activation of CDK4 in HCT116 cells, we generated by lentiviral infection TetON-inducible K7AS cells that inducibly express 3×HA-tagged wt or S130A p21. 3×HA-p21 was induced by doxycycline and the cells were stimulated by serum. Upon induction by doxycycline, ectopic p21 expression from puromycine-selected cells was maintained well below the level of endogenous p21 and it was identical in wt and S130A 3×HA-p21 cells . From thin vitro phosphorylation of p21 by CDK2 on the subsequent phosphorylation of the p21-bound CDK4 by CAK. In the T57D mutation context, in vitro phosphorylation by cyclin A2-CDK2 reduced p21 interaction with CDK4, thereby allowing complete phosphorylation by CAK of the remaining p21-bound CDK4 .In vitro, p21 bound to cyclin D-CDK4 was efficiently phosphorylated at S130 by cyclin E- CDK2 and cyclin A-CDK2 complexes, and in transfected CHO cells S130 was phosphorylated by p21-inhibitable kinase(s). To ascertain the possible involvement of CDK2 in S130 phosphorylation of p21 and hence in CDK4 activation, we first stimulated K7AS HCT116 cells in the presence or absence of the specific CDK inhibitor roscovitine, which in vitro inhibits several CDKs but not CDK4 and CDK6 in vitro IC50 for inhibition of CDK2 than CDK7 Therefore, we also used S-CR8 (0.5 µM), a more potent and selective derivative of roscovitine, with a 20-fold lower We then compared the short term effect on CDK4 activation of roscovitine, CDK7 inhibition by 1-NMPP1, and CDK2 inhibition by CR8. Whereas continuous treatment with CR8 abolished CDK4 activation , CDK7 inin vitro experiments, CDK2 was indeed proposed to be responsible for T170 phosphorylation of CDK7 in vitro was affected only slightly by stimulating these cells with serum  might contribute to the activation of p21-bound CDK4 complexes by phosphorylating p21.We observed that CDK4 and CDK6, associated with cyclin D3 and cyclin D1, also phosphorylated p21 complexed with D-type cyclins and CDK4 in vitro . So we ein vitro . PD03329in vitro . Treatmeund CDK4 . This inund CDK4  and cyclIn asynchronously proliferating CHO cells transfected to overexpress cyclin D3 and CDK4 in the presence of a limiting amount of ectopic p21  in HCT116 cells. As performed in the previous in vivo requires S130 phosphorylation of p21 by cyclin-CDK complexes, which depends on their binding to the Cy2 site of p21.It has been reported that to phosphorylate p21 on S130, cyclin E-CDK2 has to interact with the C-terminus Cy2 cyclin-binding motif of p21 We next wanted to verify the effect of CDK7 inhibition under the experimental conditions that permitted us to propose the existence of non-CDK7 proline-directed CDK4-activating kinases, i.e. using ectopic cyclin D3-CDK4 overexpressed by cell transfection as previously We evaluated this possibility in a setting that did not depend on CDK overexpression. We have previously shown that the “CDK4-mimicking” S178P mutation of CDK6 dramatically increases its activity by inducing its almost complete T177 phosphorylation. Interestingly, phosphorylation of S178P CDK6 does not require cyclin binding (at variance with CDK4) To definitively establish the involvement of p21-binding in the requirement for CDK7 activity in the activation of endogenous CDK4, we compared the effect of the inhibition of both CDK2 and CDK7 by roscovitine in p21-null HCT116 cells and wt HCT116 cells. As p21-null HCT116 cells did not survive serum deprivation, both wt and p21-null cells were treated for 16 h with roscovitine when cycling asynchronously . PhosphoCollectively, our results indicate that in HCT116 cells the crucial requirement for CDK7 activity in activating CDK4 by phosphorylation is dependent on its binding to p21. On the other hand, kinases other than CDK7 should exist to phosphorylate p21-unbound CDK4 and S178P CDK6 during continuous CDK7 inhibition.In this study, specific inhibition of CDK7 in the human HCT116 cell line led to several novel observations that collectively reveal that the pRb-E2F switch at the R point is controlled by a complex CDK7-dependent module involving bidirectional functional interactions between CDK4 and CDK2. This process targets the central T172 phosphorylation of CDK4 and is mediated at least partly by S130 phosphorylation of p21. The model depicted in in vivo, but this requirement is conditioned by CDK4 binding to p21, and other CDK4-activating kinases must exist to phosphorylate p21-unbound CDK4 during CDK7 inhibition.CDK7 is required for CDK4 activation in vitro activation by CAK/CDK7. As reported by others The action of CDK7 on CDK4 activation is two-pronged. In addition to having an initial acute effect on T172 phosphorylation of CDK4, CDK7 inhibition induced an increased association of p21 to cyclin D-CDK4 complexes, which might have been caused by reduction of the main phosphorylation of p21 at S130. As shown by the impairment of CDK4 phosphorylation by the S130A mutation of p21, the inhibition of S130 phosphorylation likely explains in part the inhibition of the activation of p21-bound CDK4 complexes and the refractoriness of cyclin D3-CDK4 complexes from CDK7-inhibited cells to In vitro, S130 phosphorylation of p21 was performed not only by cyclin E/A-CDK2 complexes, but also by CDK4 and CDK6 associated with cyclin D1 and cyclin D3. In vivo, both S130 phosphorylation of p21 and T172 phosphorylation of p21-bound CDK4 were inhibited not only in response to CDK7 inhibition, but also in response to increased p21 expression and inhibition of CDK2 (by roscovitine and CR8) and CDK4/6 (by PD0332991). Inhibition of CDK4 phosphorylation by roscovitine and PD0332991 depended on p21.Together with a parallel study by Fisher lab In vitro, cyclin H-CDK7-Mat1 phosphorylates CDK4 independently of its +1 proline How could the rapid responsiveness of CDK4 activation to CDK7 inhibition be reconciled with the observations that raised doubts about CAK being the sole CDK4 activating kinase   .CDKN2A deletion or Cdk4 amplification) and/or CDK2 activity (e.g. due to deregulation of cyclin E). This would provide a rationale for treatments that combine inhibitory drugs targeting oncogenic signaling pathways and CDKs, as suggested recently The corollary of the central position of T172-phosphorylation of CDK4 in the cell cycle decision module is its responsiveness to various drugs or treatments used or envisaged in cancer therapy. CDK4 phosphorylation is a central node integrating oncogenic signaling cascades, as its abrogation requires combined inhibition of the Ras/MEK/ERK and mTOR pathways These procedures were preformed exactly as described 3 cells . Infected cells were selected by antibiotic resistance with a final concentration of 0.4 µg/ml of puromycin (Invivogene).HEK293T cells were transfected by calcium phosphate for 6 h with a mix of vectors: pSPAX2, pMD2G (Trono Lab), pDG2iV5puro encoding N-terminus 3×HA-tagged human p21 or its mutants, or pLVX-rtTA3 (TetON) or Tight-puro (Clontech) encoding Flag tagged CDK6 wt, CDK6 S178P mutant or cyclin D3. Multi-cistronic pDG2iV5puro is a modified version of the pLenti6 V5/DEST vector (Invitrogen). It contains an expression cassette under the control of a Tet-responsive element allowing doxycycline-inducible expression of the gene of interest and an SV40 promoter controlling rtTA3 transactivator expression followed by an IRES and a puromycin-resistance marker. Transfected HEK293T supernatant was collected after 2 days, clarified, filtered, and concentrated on Amicon filter (Millipore). HCT116 cells were infected for 24 h by adding to the culture medium a volume of lentiviruses corresponding to 100 µg of p24 capsid protein per 50×10(Co-)Immunoprecipitations were performed as described To assay pRb-kinase activity, immunoprecipitated protein complexes were incubated with ATP and a recombinant pRb fragment (QED or Sigma). The mixture was then separated by SDS-PAGE and western blotted to detect T826 phosphorylation of the pRb fragment and co-immunoprecipitated proteins in vitro phosphorylation. When indicated, immunoprecipitates were first dephosphorylated with λ-phosphatase as described 2, 20 mM EGTA, 5 mM DTT). The beads were resuspended in 50 µl of CAK buffer containing protease and phosphatase inhibitors and 2 mM ATP and incubated at 30°C for 1 h with or without 1 µg of recombinant kinases: CDK2-cyclin E1, CDK2-cyclin A2 and CDK4-cyclin D3 (Carna Biosciences); cyclin H-CDK7-MAT1 (Upstate); CDK4-cyclin D1 and CDK6-cyclin D1 (ProQinase) .Complexes containing CDK4 or CDK6 from HCT116 cells, p21 from CHO cells transfected with p21 vector alone, or with cyclin D3 or cyclin D1-Flag and CDK4 vectors, or HA-p21 from stably infected HCT116 K7AS by lentiviruses expressing inducible 3×HA-p21 wt or its mutants were immunoprecipitated and used as substrate for in vitro activation was then assessed by their pRb-kinase activity Cyclin D3-CDK4 complexes from transfected CHO cells were immunoprecipitated and dephosphorylated with λ-phosphatase as described SDS-PAGE separations of equal amounts of whole cell extract proteins Figure S1(Related to (TIF)Click here for additional data file.Figure S2(Related to (TIF)Click here for additional data file.Figure S3in vitro phosphorylation by CAK. HCT116 K7AS cells were stimulated (+) or not stimulated (−) with fetal bovine serum (FBS) for 5 h in the absence (−) or presence (+) of 1-NMPP1. Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1), anti-cyclin D3 (D3) or anti-p21 antibodies and incubated with ATP in the presence (+) or absence (−) of recombinant cyclin H-CDK7-MAT1 complex (CAK). The complexes were then separated by 2D gel electrophoresis and immunodetected with a mixture of anti-CDK4 and anti-CDK6 antibodies. In the inset, as a positive control of CAK activity in the same experiment, immunoprecipitated (D3 IP) cyclin D3-CDK4 complexes from CHO cells transfected with plasmids encoding cyclin D3 and CDK4-HA were pretreated or not with λ-phosphatase (λ PPase) and then incubated with ATP with or without CAK, before 2D gel electrophoresis and CDK4 immunodetection. Arrows indicate the position of T172/T177-phosphorylated form of CDK4/6. If the impaired activation of CDK4 and CDK6 complexes in CDK7-inhibited K7AS cells was due only to absence of activating phosphorylation, these complexes should remain phosphorylatable by CAK in vitro, as observed here using cyclin D3-CDK4 complexes produced in CHO cells (inset). By contrast, only cyclin D3-CDK6, but neither cyclin D3-CDK4, cyclin D1-CDK4 nor cyclin D1-CDK6, was phosphorylated by CAK from 1-NMPP1-treated cells. This refractoriness to CAK activation of cyclin D3-CDK4 from CDK7-inhibited cells might have been due to its increased association with p21 Click here for additional data file.Figure S4(Related to (TIF)Click here for additional data file.Figure S5(Related to (TIF)Click here for additional data file.Figure S6(Related to (TIF)Click here for additional data file.Figure S7Proteasomal inhibition increases p21 levels and mimics the effects of CDK7 inhibition on phosphorylations of pRb and CDK2 (A), pRb kinase activity (B), and T172 phosphorylation of CDK4 and S130 phosphorylation of p21 (C). HCT116 K7AS cells were restimulated or not restimulated with fetal bovine serum (+/− FBS) during 5 or 16 h in the continuous absence (−) or presence (+) of MG132. (A) Western blotting analysis was performed with the indicated antibodies from whole-cell lysates.  Lysates from cells treated for 5 h were immunoprecipitated (IP) with anti-p21 or anti-cyclin D1 (D1) antibodies and assayed for their pRb-kinase activity, separated by SDS-PAGE and immunoblotted with the indicated antibodies (B), or were separated by 2D gel electrophoresis followed by CDK4 and p21 immunodetection (C).(TIF)Click here for additional data file.Figure S8in vitro phosphorylation of p21-cyclin-CDK4 complexes by CDK2 might more efficiently affect them and the capacity of p21-bound CDK4 to be phosphorylated by CAK. As shown in in vitro phosphorylation by cyclin A2-CDK2 and/or CAK revealed that (i) cyclin A2-CDK2 phosphorylated p21 at S130, S98 and another unidentified site Click here for additional data file.Figure S9(Related to (TIF)Click here for additional data file.Figure S10in vitro activation reflected by the T826 phosphorylation of the pRb fragment (pRb-kinase). (B) Phosphorylation profiles of CDK7. Same cell lysates as in (A) were immunoprecipitated (IP) with anti-CDK7 or anti-cyclin H (cyc H) and separated by 2D gel electrophoresis followed by immunodetection by antibodies directed against CDK7 or T170-phosphorylated CDK7 (P-CDK7 (T170)). As deduced from computed isoelectric points (Related to (TIF)Click here for additional data file.Figure S11Phosphorylations of p21 and p21-bound CDK4 depend on the Cy2 cyclin-binding motif of p21. Stably infected HCT116 K7AS cells for Tet-On inducible 3×HA-p21 wt or ΔCy2 (aa 1 to 154) mutant were treated with doxycycline (1 µg/ml) for 16 h prior cell restimulation with fetal bovine serum (FBS) for 16 h in the continuous presence of doxycycline. Cell lysates were immunoprecipitated (IP) with anti-HA antibody and separated by 2D gel electrophoresis followed by simultaneous immunodetection of ectopic 3×HA-p21 and 3×HA-p21-bound endogenous CDK4 using a mixture of anti-CDK4 and p21 antibodies. Black arrow, T172-phosphorylated CDK4. Colored arrows indicate phosphorylated forms of p21. Black arrowheads indicate the unphosphorylated form of wt and ΔCy2 p21. Of note, deletion of the 10 C-terminal aminoacids (including the Cy2 motif) much modifies the isoelectric point of p21 . This explains why the phosphorylated forms are much less separated in the ΔCy2 mutant, which also precludes the formal identification of these forms (the form indicated by a blue arrow is putatively phosphorylated on S98).(TIF)Click here for additional data file.