The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined.We evaluated the effects of dasatinib on bone marrow-derived mesenchymal stromal cells (MSC) differentiation into osteoblasts, in the presence or absence of a mixture of dexamethasone, ascorbic acid and β-glycerophosphate (DAG) for up to 21 days. The differentiation kinetics was assessed by evaluating mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers .Dasatinib significantly increased the activity of ALP and the level of calcium deposition in MSC cultured with DAG after, respectively, 7 and 14 days; it upregulated the expression of BSP and OPN genes independently of DAG; and it markedly downregulated the expression of RANKL gene and protein (decrease in RANKL/OPG ratio), the key factor that stimulates osteoclast differentiation and activity.Our results suggest a dual role for dasatinib in both (i) stimulating osteoblast differentiation leading to a direct increase in bone formation, and (ii) downregulating RANKL synthesis by osteoblasts leading to an indirect inhibition of osteoclastogenesis. Thus, dasatinib is a potentially interesting candidate drug for the treatment of osteolysis through its dual effect on bone metabolism. Osteoblasts originate from mesenchymal osteoprogenitor cells and play a key role in physiological bone turnover and pathological disorders including osteoporosis , Paget'sIn recent years, much interest emerged for the bone marrow-derived mesenchymal stromal cells (MSC) due to their ability to self-renew, proliferate and differentiate into a variety of cell types of mesodermal, endodermal and ectodermal origins . There a527Tyr. The dephosphorylation of this residue by phosphatases leads to intramolecular autophosphorylation at 416Tyr, promoting the kinase activity  and osteoprotegerin [OPG]). β-actin was used as housekeeping gene. The tested genes could be classified into three groups Fig. . The secOf note, ALP activity Fig.  did not Thus, this part of the study allowed us to identify mineralization process, ALP activity and expression of RANKL, BSP and OPN as strong markers of osteoblastic differentiation of MSC since they were clearly changing between each stage of the differentiation process Fig. . This mo-9 to 10-4 M) for 3, 7 or 10 days. Changes in cell proliferation were measured by WST-1 assay. Dasatinib induced a concentration-dependent decrease in cell proliferation: it had no effect at the lowest tested concentrations (10-9 and 10-8 M), had low inhibitory effects between 10-7 and 10-5 M and was clearly toxic at 10-4 M  strongly decreased the phosphorylation of the tyrosine kinase  similarly potentiated the effect of DAG, increasing ALP activity after 7 days  whose levels of expression are informative of the osteoblastic differentiation status of MSC (see above). The expression of these genes reflecting MSC differentiation status was assessed by real-time quantitative PCR in MSC treated or not with DAG and exposed or not to 10nib Fig. . Dasatin-8 and 10-7 M dasatinib, in absence of DAG, for 3 and 7 days. OPG protein was also measured by ELISA in the same samples and results are presented as RANKL/OPG ratio  and a dramatic decrease in RANKL expression . This result strongly confirms our above observation at mRNA level.To further investigate the effects of dasatinib on the expression of RANKL at protein level in short-term experiments, RANKL protein was evaluated by ELISA in 24-hours conditioned medium of MSC exposed to 10We assessed the effects of the Src inhibitor dasatinib on the osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (MSC) treated or not with a combination of dexamethasone, ascorbic acid and β-glycerophosphate (DAG).Firstly, we have searched for the most informative markers of osteogenic differentiation of MSC induced by DAG and found a set of 5 markers  that best characterize the 4 stages of osteoblast differentiation: native stage (undifferentiated MSC), early stage (osteoprogenitors), intermediate stage (mature osteoblasts), and late stage (late osteoblasts). Of note, many genes related to the osteoblastic phenotype or activity have been previously used to define the osteoblastic status of different cellular types ,31. Howe416Tyr) of Src and an increase in phosphorylation of its inhibitor site (527Tyr) [-/- mice exhibit accelerated differentiation and elevated levels of osteoblast-related markers [Based on our selection of markers, we have evaluated the effects of the Src inhibitor dasatinib on the differentiation of MSC into osteoblasts. Our interest in Src inhibitors stems from a recent study showing that the differentiation of the MC3T3-E1 pre-osteoblasts was accompanied by a decrease in phosphorylation of the activator site ((527Tyr) . This in(527Tyr) , and ost markers . Taken t markers  and cKIT markers . The rol markers ,45, and  markers . On the  markers . Altoget-8 M) able to inhibit Src kinase activity (inhibition of Src phosphorylation) in accordance with a previous report showing inhibition of the kinase activity of purified Src protein with an IC50 of 3 × 10-9 M [419Tyr-Src phosphorylation was inhibited by dasatinib. Moreover, knockdown of Src by lenti-shRNA in osteoblasts enhanced their differentiation, suggesting that dasatinib stimulated osteoblast differentiation through the inhibition of Src. Of note, the authors also reported that osteoblast differentiation by dasatinib could be mediated through the inhibition of Abl, however, they found that Abl was expressed at a low level in osteoblasts, suggesting a limited impact.In this study, we found that dasatinib promoted time-dependent changes in osteoblast-related markers in MSC. These changes matched the ones induced by DAG and corresponded to the progressive differentiation of MSC into osteoblasts see Fig. . Indeed,× 10-9 M . Altoget× 10-9 M ,22,23,27× 10-9 M . The autIn the other hand, we observed that dasatinib alone was able to decrease RANKL mRNA expression as well as RANKL protein released in culture medium (decrease in RANKL/OPG ratio) in undifferentiated MSC (no DAG induction) cultured up to 7 days, suggesting a possible indirect inhibitory effect on osteoclastogenesis and, consequently, on bone resorption. In this context, recent studies reported that tyrosine kinase inhibitors are effective in inhibiting the differentiation and activity of human osteoclasts , and in Furthermore, as far as the bone loss associated with metastases is concerned, recent preclinical and clinical studies indicated that dasatinib could potentially have inhibitory effects both on osteoclasts and tumor cells. Indeed, dasatinib decreased the osteolysis induced by prostate cancer cells injected into tibiae of SCID mice and inhibited the growth of cancer cells , while iOur data also suggest that patients with bone loss could benefit from dasatinib therapy in accordance with critical findings using imatinib, another tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML). Indeed, short-term imatinib treatment increased OPG/RANKL ratio and osteocalcin levels in serum of CML patients , while lOur study reports an original dual effect of dasatinib on MSC at non toxic concentrations: (i) it is able to significantly speed up the osteogenic differentiation of MSC, and (ii) it can inhibit RANKL expression in undifferentiated MSC (no DAG induction) thus preventing osteoclast activation. This dual effect on osteoblasts, on top of its direct activity on osteoclasts as well as on tumor cells, may provide a therapeutic benefit in patients with diseases leading to bone loss, such as osteoporosis, tumor-induced osteolysis, and therapy-induced bone loss.MP and JJB are recipients of a research grant from Bristol Myers Squibb .HIB participated in experimental design, performed experiments and analyses. LL participated in experimental design, interpreted the data, and drafted the manuscript. MN helped to draft the manuscript. MP, GG and JJB discussed the results and critically revised the manuscript. FJ designed the experiments, assembled tables and figures, and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/298/prepub


Antibodies targeting blood stage antigens are important in protection against malaria, but the key targets and mechanisms of immunity are not well understood. Merozoite surface protein 1 (MSP1) is an abundant and essential protein. The C-terminal 19 kDa region (MSP1-19) is regarded as a promising vaccine candidate and may also be an important target of immunity.P. falciparum lines that expressed either the P. falciparum or P. chabaudi form of MSP1-19 were used to quantify MSP1-19 specific growth-inhibitory antibodies. The great majority of children had detectable IgG to MSP1-19, and high levels of IgG were significantly associated with a reduced risk of symptomatic P. falciparum malaria during the 6-month follow-up period. However, there was little evidence of PfMSP1-19 specific growth inhibition by plasma samples from children. Similar results were found when testing non-dialysed or dialysed plasma, or purified antibodies, or when measuring growth inhibition in flow cytometry or microscopy-based assays. Rabbit antisera generated by immunization with recombinant MSP1-19 demonstrated strong MSP1-19 specific growth-inhibitory activity, which appeared to be due to much higher antibody levels than human samples; antibody avidity was similar between rabbit antisera and human plasma.Growth inhibitory antibodies against asexual-stage parasites and IgG to recombinant MSP1-19 were measured in plasma samples from a longitudinal cohort of 206 children in Papua New Guinea. Differential inhibition by samples of mutant These data suggest that MSP1-19 is not a major target of growth inhibitory antibodies and that the protective effects of antibodies to MSP1-19 are not due to growth inhibitory activity, but may instead be mediated by other mechanisms. Alternatively, antibodies to MSP1-19 may act as a marker of protective immunity. Plasmodium falciparum. Although the disease burden of P. falciparum is considered to fall mainly in Africa, around 25% is thought to occur in Asia Between 300–500 million cases of clinical malaria occur each year resulting in approximately 1 million deaths, mostly in children under 5 years of age in sub Saharan Africa et al. 2004 Malaria disease occurs during blood-stage infection when merozoites invade human red blood cells (RBCs) and replicate inside them. Invasion of RBCs is a complex process involving the interaction of proteins present on the surface and within apical organelles of the merozoite with receptors on the RBC surface (reviewed in Gaur Merozoite surface protein 1 (MSP1), is a ∼200 kDa GPI anchored merozoite surface protein consisting of 17 blocks of conserved and variable regions P. falciparum malaria in vitro. Vaccination using recombinant MSP1-19 antigens and passive transfer of MSP1-19 antibodies has been reported to confer protection from malaria disease in both mouse and monkey models of malaria A number of studies have shown that high levels of MSP1-19 IgG antibodies measured by ELISA are associated with protection from Several mechanisms by which MSP1-19 antibodies may mediate protection from malaria have been suggested by vaccine studies. These include antibody binding to the 19 kDa fragment and inhibiting protein function and merozoite invasion of erythrocytes, disruption of secondary processing of the MSP1 precursor, agglutination of merozoites and opsonising parasites for destruction by phagocytic cells P. falciparum disease were facilitated by the development of MSP1-19 transgenic parasites. The 19 kDa fragment of P. falciparum isolate D10  was replaced with the 19 kDa region of the rodent malaria P. chabaudi (PcMEGF) in vitro that are specific to MSP1-19 Efforts to study the role and function of naturally acquired PfMSP1-19 antibodies in protection from in vitro growth inhibitory activity of untreated serum and plasma from malaria-exposed individuals P. falciparum after drug treatment P. yoelii MSP1-19 (PyMEGF), showed no association between PfMSP1-19 specific growth inhibitory activity and infection status Several studies using these transgenic parasites have reported that PfMSP1-19 specific antibodies are a large component of the total In this study, functional antibodies to MSP1-19 and the importance of MSP1-19 as a target of acquired immunity were evaluated. The contribution of MSP1-19 specific antibodies to the total invasion-inhibitory activity of acquired antibodies was quantified, and associations between antibodies to MSP1-19 and protection from parasitization, high grade parasitemia, and malarial illness were examined in a pediatric treatment-reinfection study in Papua New Guinea (PNG). Furthermore, differences in functional activity were evaluated among acquired human antibodies and antibodies generated in experimental animals by vaccination with recombinant protein.Samples were collected during a pediatric treatment-reinfection study undertaken from June to December 2004 at the Mugil Elementary School (n = 152), Megiar Elementary School (n = 44) and Megiar Primary School (n = 10), 50 km North of Madang, Madang Province, Papua New Guinea P. falciparum and P. vivax according to national guidelines.Enrolment with peripheral blood and data collection was conducted over a two-week period in June 2004 by staff from PNGIMR Data relating to time to reinfection after treatment, infecting malaria parasite species as indicated by PCR, parasite density as indicated by microscopy, red blood cell polymorphisms by PCR, history of illness as indicated by symptoms and physical examination were available P. falciparum MSP1-19 and P. chabaudi MSP1-19 were ligated into a modified pMal-C2x plasmid (New England Biolabs) to produce MSP1-19 fusions proteins with a N-terminal Maltose Binding Protein (MBP) tag. The fusion proteins were expressed in Escherichia coli BL21(DE3) Origami  and purified via nickel-nitrilotriacetic acid chromatography under native conditions  and were refolded as per Dutta et al.DNA sequences for Antigens were coated onto ELISA plates  at 1 µg/ml in 100 µl PBS and incubated overnight at 4°C PfMSP1-19 specific IgG concentration was estimated by coating two-fold serial dilutions (1 µg/ml-0.00781 µg/ml) of purified human IgG  and purified rabbit IgG  onto plates; other wells were coated with recombinant PfMSP1-19. After blocking wells with 10% skim milk, two-fold serial dilutions of Mugil children's plasma (1/1000–1/32000) or rabbit sera against MSP1-19 (1/4000–1/256000) were added to separate wells coated with 1 µg/ml PfMSP1-19. PBS was added to wells coated with human and rabbit IgG. After incubation for 2 hours, wells were washed, then incubated with either anti-human or anti-rabbit IgG secondary antibody, then washed and processed as described above. The MSP1-19 specific IgG concentration among children's plasma, or rabbit antisera, was estimated by reference to the standard curve of human or rabbit IgG, respectively. Estimated MSP1-19 specific IgG levels among human samples were expressed relative to the rabbit MSP1-19 anti-sera.After incubating antigen-coated wells with human plasma or rabbit anti-MSP1-19 antibodies (as described above), wells were washed and then incubated with ammonium thiocyanate  for 20 minutes at concentrations of 8.64 M, 6.91 M, 5.18 M, 4.32 M, 3.46 M, 2.16 M, 1.728 M, 1.08 M, 0.27 M, 0.135 M, 0.067 M. Wells were then washed and the assay was completed as described earlier. Primary antibodies were diluted for human  and rabbit  samples to yield OD values in the descending linear section of the absorbance curves. The ammonium thiocyanate concentration that resulted in a loss of 50% of maximum absorbance was estimated by fitting a line of best fit.Heat inactivated plasma samples (45 minutes at 56°C) were dialysed as described Immunoglobulin from twenty pooled Mugil samples with high levels of PfMSP1-19 specific antibody measured by ELISA (OD>1.7), 5 plasma samples from PNG adults and serum from non-exposed Melbourne donors were purified using ammonium sulfate precipitation et al. 2000 The PcMEGF (containing PcMSP1-19) and PfM3′ (containing PfMSP1-19) lines et al.2, 4% CO2 and 95% N2.The protocol for measurement of antibody inhibition of parasite growth used in this study is modified from Persson One cycle assays with measurement of parasitemia at ring-stage by microscopy and flow cytometry were cultured for approximately 24 hours until young to mid rings were present and no schizont stage parasites remained. For assessment of parasitemia by microscopy, thin smears of the cell pellets were methanol fixed and stained with 5% Giemsa . Slides were blinded and one thousand RBCs were counted for each slide. Only early to late rings (0 to 20 hours old) were counted as parasitised RBCs, with each infected RBC counted as a single parasitized RBC regardless of how many parasites were in the cell. Due to the variability inherent in microscopy based counts of parasitemia One cycle assays with measurement of parasitemia at the pigmented trophozoite stage were cultured for 48 hours post set-up until parasites were between 30–40 hours post invasion when they were prepared for flow cytometric analysis as appropriate. Two cycle trophozoite stage assays were supplemented at 48 hours post set-up (parasites 30–40 hours post invasion) with the addition of 5 µl to 10 µl (25 µl or 50 µl assay respectively) of fresh culture medium warmed to 37°C. Forty-eight (1 cycle assay) or 96 (2 cycle assay) hours post assay set-up, trophozoite stage assays were prepared for flow cytometry analysis as appropriate. For non-fluorescent parasite lines, RBCs were resuspended in 100 µl of PBS with 10 µg/ml ethidium bromide , then stained for 1 hour before centrifugation, removal of the supernatant and resuspension in 200 µl of PBS. GFP fluorescent parasite lines were resuspended in PBS to make a final volume of 200 µl. Parasitemia was measured on a Becton Dickinson FACSCalibur  flow cytometer with a 96 well plate sampler attached using Fl-2 (excitation wavelength 488 nm) for EtBr stained parasites and Fl-1 (excitation wavelength 488 nm) for GFP parasites. Typically, 50,000 to 80,000 RBCs were counted for each well. Samples were analysed using FlowJo software .mean) by the mean parasitemia of 2 or more wells of non-inhibitory control  and multiplying by 100 (G = {Smean/Cmea}*100). PfMSP1-19 specific inhibitory activity (InhPf) of an individual sample was then determined by subtracting growth of the PfMSP1-19 test line (Gpf) from growth of the PcMSP1-19 control line (Gpc) for each sample tested (InhPf = Gpc-Gpf). Rabbit antibodies to confirm MSP1-19 specific inhibition were raised against C-terminal fragments of P. falciparum and P. chabaudi MSP1-19 in an earlier study PfMSP1-19 growth inhibitory activity measured in this assay may include both invasion inhibition and intraerythrocytic parasite growth inhibition. Although PfMSP1-19 specific inhibitory antibodies could be acting through either or both mechanisms, the two mechanisms are not differentiated in this study. For both the PcMSP1-19 and PfMSP1-19 transfected lines, parasite growth (G) was determined by dividing the mean parasitemia of duplicate wells for a test sample (SVariation in the assay was estimated as (+/−) 2 standard deviations around the mean of the Melbourne control sera used in assays. The level of potential PfMSP1-19 specific inhibition for Melbourne control samples was determined by subtracting parasite growth of the PfMSP1-19 line from the growth of the PcMSP1-19 line for each individual sample. Any difference in growth can be attributed to assay variation since there was no MSP1-19 antibodies detected in the Melbourne control sera. Two standard deviations of MSP1-19 specific inhibition for Melbourne control sera was determined separately for each set of experimental conditions and is termed the “variation threshold”. Alternatively, the number of samples above arbitrary cutoffs of 10% and 15% are also considered for 2 cycle assays using non-dialysed and dialysed plasma as has been done in other studies P. falciparum infection and symptomatic malaria, ELISA data were stratified into 3 equal groups (tertiles) reflecting low, medium and high responders according to OD values for each antigen. GIA data was categorised into . Kaplan-Meier curves were generated for time-to-event  data and compared using a log-rank test. Cox proportional hazards models were used to calculate hazard ratios for the association of antibody variables with risk of high density P. falciparum parasitemia and symptomatic P. falciparum malaria. Assumptions of proportional hazards were assessed graphically and for antibody variables that showed non-proportional hazards, results from Cox regression including the antibody variable and antibody variable by time interaction are reported Differences between categorical variables was assessed using chi-squared or Fisher's exact test, where appropriate, and differences in medians were assessed using the Mann-Whitney U test. Spearman's correlation was used to determine correlations between two data sets. These tests were performed with Prism 5.0a . To determine the association between antibody levels and risk of E. coli) in this cohort P. falciparum as well as P. chabaudi were measured. Comparison between IgG levels to the PcMSP1-19 and PfMSP1-19 antigens allowed the measurement of antibodies specific to PfMSP1-19, accounting for cross-reactive or non-specific antibodies. PfMSP1-19 specific antibodies were calculated by deducting the reactivity to PcMSP1-19 from the reactivity to PfMSP1-19 for each sample. Quantifying the relative levels of antibodies to P. falciparum and P. chabaudi MSP1-19 was also important to interpret results from growth inhibition assays using the MSP1-19 transgenic parasites, which are described later.Previously we have described the presence of antibodies to recombinant PfMSP1-19  and the PfMSP1-19 line  for all samples showed potentially specific inhibition of the PfMSP1-19 line of 7.1%  with high levels of MSP1-19 antibodies by ELISA were pooled and IgG was purified using ammonium sulfate precipitation, allowing concentration of immunoglobulin and removal of non-specific inhibitors . Plasma in vitro growth inhibitory activity of children's plasma samples, and that there was little specific inhibition observed for the few adult samples tested. Importantly, PfMSP1-19 and PcMSP1-19 specific rabbit anti-sera tested in parallel in all assays consistently inhibited the growth of the respective lines, indicating that MSP1-19 specific inhibition could be effectively measured in these experiments ; high density parasitemia (Pf>5000/µl); moderate density parasitemia (Pf>500/µl) and reinfection (detected by both light microscopy and PCR). Specific IgG to PfMSP1-19 was defined as the reactivity to recombinant PfMSP1-19 minus the reactivity to recombinant PcMSP1-19 for each sample. There was strong evidence of a protective effect of high levels of anti-PfMSP1-19 specific antibodies measured by ELISA (IgG reactivity to PfMSP1-19 minus IgG reactivity to PcMSP1-19) with protection against high density parasitemia and symptomatic P. falciparum infection. High levels were associated with reduced risk of high density infection  and symptomatic P. falciparum infection , P. chabaudi MSP1-19 specific responses and high density parasitemia and symptomatic P. falciparum infection. In comparison to other antibody associations evaluated in this cohort, the strength of the protective association for PfMSP1-19-specific antibodies was similar to AMA1 , but not as strong as found for antibodies to some EBA and PfRh antigens .To investigate the association between PfMSP1-19 specific IgG levels by ELISA and protection against malaria or infection, children were defined as low, medium or high responders to the PfMSP1-19 antigen (based on tertiles). Levels of response were then related to four P. falciparum-specific growth inhibition data from testing both dialysed and non-dialysed samples was used. Although there was a strong association between antibodies to PfMSP1-19 measured by ELISA and protective immunity, there was no evidence that PfMSP1-19 specific inhibitory antibodies were associated with protection from malaria. Individuals with potentially significant levels of PfMSP1-19 specific inhibition (>10%) using dialysed plasma had an increased risk of symptomatic and high density parasitemia compared to those with no inhibition  was used as a measure to compare avidity between samples. The concentration required for a 50% reduction for the rabbit antisera (4.4 M) was comparable to that observed for a panel of Mugil plasma samples  observed in the study P. falciparum infection at baseline, were found to have higher levels of PfMSP1-19 antibodies measured by ELISA than younger children and children that were PCR negative. These data suggest that levels of PfMSP1-19 specific antibodies measured by ELISA are associated with recent and cumulative exposure to P. falciparum as expected.Of the 206 Mugil plasma samples collected at baseline, 95.6% were found to be PfMSP1-19 IgG positive by ELISA, suggesting the majority of children had had significant prior exposure to P. falciparum malaria episode post drug treatment indicated that high levels of PfMSP1-19 specific (PfMSP1-19 OD minus PcMSP1-19 OD) antibodies were associated with reduced risk of symptomatic malaria, which remained significant after adjusting for differences in age and location of residence. Time to first moderate or high density P. falciparum infection was significantly increased in children with high PfMSP1-19 antibody levels, which is consistent with the current understanding of acquired immunity and proposed mechanisms of action for anti-MSP1-19 antibodies. These findings are similar to a previous study of MSP1-19 antibodies in this cohort P. chabaudi in order to account for non-specific binding, which is a concern that is often expressed by researchers in the field.Analysis of the association between PfMSP1-19 antibody levels measured by ELISA and first P. falciparum malaria P. falciparum malaria or alternatively may act as a strong predictive marker of exposure, and hence immunity.The results of this study support the findings of a number of other studies which have shown an association between IgG antibody levels to PfMSP1-19 measured by ELISA and control of parasitemia or protection from symptomatic Our comprehensive testing suggests that there is little PfMSP1-19 specific growth inhibition in plasma samples from the Mugil cohort, under various experimental conditions. Furthermore, any potential MSP1-19 specific inhibitory activity was not associated with protection from malaria or re-infection. The median level of PfMSP1-19 specific inhibition for non-dialysed Mugil plasma tested in a 2 cycle assay, dialysed Mugil plasma tested in a 2 cycle assay, non-dialysed Mugil plasma in a 1 cycle assay measured by flow cytometry or microscopy, and ammonium sulfate precipitated IgG at a 1 in 10 or 1 in 5 dilution was generally minimal and lower than the levels reported in other studies Importantly, rabbit polyclonal antibodies raised against recombinant PfMSP1-19 and PcMSP1-19 were used in all assays and consistently found to specifically inhibited growth of the target parasite lines. This suggests that antibody mediated MSP1-19 specific inhibition in malaria exposed samples could be measured if the inhibitory activity of the antibodies was high enough. In this study, a variability threshold was determined from paired (PcMSP1-19 line or PfMSP1-19 line) Melbourne control samples to provide a reliable indicator of the amount of measured MSP1-19 specific inhibition that could be attributable to assay variability. In this way it was hoped to establish a more valid cut-off for defining MSP1-19 positivity or negativity than using an arbitrary value of 5 to 15% as has been adopted previously negative PfMSP1-19 specific inhibition (i.e. PcMSP1-19 specific inhibition), the exception being the two cycle non-dialysed data. Given the distribution of MSP1-19 specific inhibition it would appear likely that negative PfMSP1-19 specific inhibition reflects assay variability rather than a PcMSP1-19-like inhibitor. It is interesting to note that the maximum and minimum levels of notional MSP1-19 specific inhibition for Melbourne control samples lie at similar levels to the maximum and minimum levels of inhibition seen for the Mugil samples. Again this suggests that MSP1-19 specific inhibition detected in Mugil samples could be explained by assay variability. Importantly, the variability seen in Melbourne control samples was not an indication of poor reproducibility of flow cytometry based assays, since there was a high level of reproducibility for measurements of parasite growth between duplicate wells and between experiments across this study.It was noticeable that the number of samples displaying PfMSP1-19 specific inhibition was usually balanced by the number of samples showing P. falciparum malaria One possible explanation for the lack of PfMSP1-19 specific growth inhibition seen in this study might be related to the level of exposure for this cohort compared to others. Previous studies reported high levels of PfMSP1-19 specific inhibition in samples collected from children and adults living in highly malaria endemic areas in vitro, but may not faithfully represent avidity or affinity in vivo. Additionally, differences in the fine specificity of antibodies may also contribute to differences observed in the inhibitory activity of human and rabbit antibodies.Estimation of PfMSP-19 IgG concentration in rabbit antisera and Mugil plasma with high levels of PfMSP1-19 antibodies suggested that antibody concentration may be an important factor limiting ability to detect PfMSP1-19 specific inhibition using this assay. PfMSP1-19 specific IgG was 12.5 fold higher in rabbit antisera than in a panel of Mugil plasma. When the rabbit antisera were diluted down such that the concentration of IgG was similar to that seen in the Mugil plasma, there was loss of growth inhibitory activity. This suggests that levels of PfMSP1-19 antibodies in Mugil samples may not be high enough to allow detection of MSP1-19 specific growth inhibition in this assay. The avidity of PfMSP1-19 specific binding to the recombinant PfMSP1-19 antigen appeared comparable in rabbit antisera and Mugil plasma, suggesting that differences in antibody avidity or affinity did not contribute to the reduced PfMSP1-19 specific inhibition by Mugil plasma compared to rabbit antisera. However, these results should be interpreted with caution since using ammonium thiocyanate elution in ELISA provides an estimate of the relative avidity of antibodies et al. (2009) The limited PfMSP1-19 growth inhibitory activity detected in this study differs markedly to earlier published studies reporting greater levels of MSP1-19 specific inhibition in serum or plasma  [54], [5In conclusion, although MSP1-19 IgG levels measured by ELISA were associated with protection, there was little or no PfMSP1-19 specific growth inhibitory activity measurable in the Mugil cohort, probably because antibodies were not of a high enough concentration. This suggests that the association between MSP1-19 IgG levels measured by ELISA and protection from disease may be a marker of protective immunity rather than being directly involved in protective mechanisms, or may indicate that MSP1-19 antibodies contribute to protection by mechanisms other than growth inhibition. Studies in a humanized mouse model suggest that antibodies to MSP1-19 can protect against parasitemia through an interaction dependent on Fc Receptors and phagocytic cells, rather than inhibition of invasion Table S1aS = serum. P = plasma.bM = microscopy based assay. F = flow cytometry based assay. H = Hypoxanthine uptake assay.c Microscopy based assays for Bagabag Island and Madang samples were completed using the D10 wildtype line. Hypoxanthine uptake based assays for Bagabag Island and Madang samples were completed using the transfected D10-PfM3′ line d Interpretation from d Inhibition minimum at 6 months (16% n = 24) maximum at 24–30 months .e Inhibition range for 9 of 20 returned travelers considered to have significant PfMSP1-19 specific inhibition. Data for remaining 11 travelers not reported.f Interpretation from g Calculated median for all 137 samples listed in Summary of PfMSP1-19 specific inhibition levels reported in different studies.(DOCX)Click here for additional data file.


Atg7 or Atg5 or blocking LC3 lipidation or ATG5–ATG12 conjugation decreases ERK phosphorylation. Conversely, increasing LC3-II availability by silencing the cysteine protease ATG4B or acute trehalose exposure increases ERK phosphorylation. Decreased ERK phosphorylation in Atg5−/− cells does not occur from overactive phosphatases. Our findings thus reveal an unconventional function of ATG proteins as cellular scaffolds in the regulation of ERK phosphorylation.Autophagy is a conserved pathway that maintains cellular quality control. Extracellular signal-regulated kinase (ERK) controls various aspects of cell physiology including proliferation. Multiple signalling cascades, including ERK, have been shown to regulate autophagy, however whether autophagy proteins (ATG) regulate cell signalling is unknown. Here we show that growth factor exposure increases the interaction of ERK cascade components with ATG proteins in the cytosol and nucleus. ERK and its upstream kinase MEK localize to the extra-luminal face of autophagosomes. ERK2 interacts with ATG proteins via its substrate-binding domains. Deleting  The spatial organisation of mitogen-activated protein kinase (MAPK) signalling by scaffold proteins is an important determinant of signalling specificity. Martinez-Lopez et al. show that pre-autophagosomal structures can also act as scaffolds, recruiting the MAPK ERK1/2 and regulating its phosphorylation. Extracellular signal-regulated kinase ERK1/2)/212 prom1210Atg7−/− liver and brown adipose tissues, Atg5−/−mouse embryonic fibroblasts (MEFs), cells silenced for ATG7, ATG5 or Beclin1, and in a LC3 lipidation-defective mutant decreases cellular and nuclear ERK phosphorylation and activity. We further show that ERK2 colocalizes with ATG5–ATG12, LC3-II and WIPI1 through its substrate-binding domains, and that ATG5–ATG12 conjugation is required for ERK1/2 phosphorylation. Conversely, increasing steady-state LC3-II content by blocking ATG4B-mediated LC3-II delipidationHere we show that ERK cascade components display increased association with ATG5–ATG12-positive pre-autophagosomal structures and with lipidated ATG8 family proteins, LC3-II and GABARAPTo determine whether ATG proteins regulate ERK phosphorylation, we examined the effect of epidermal growth factor (EGF) treatment on interactions between ERK cascade components and APh/LC3-II. Immunofluorescence in NIH/3T3 cells revealed increased colocalization of ERK cascade components, P-bRAF, P-MEK and P-ERK with LC3-II  in respo17P-MEK and P-ERK colocalized with LC3-II in hypothalamic cells , in 10 min vivo we used density-gradient centrifugation to isolate APh, autophagolysosomes (APL), and lysosomes (Lys) from livers of fed miceTo determine whether MEK and ERK associate with LC3-II-positive autophagic structures As activation of ERK increases its nuclear localization, we next investigated whether exposure to EGF enhanced P-ERK/LC3-II colocalization in the nucleus. EGF-treated RALA hepatocytes  and NIH/de novo lipidation in the nucleus. To distinguish between these possibilities, we used wheat germ agglutinin (WGA) to block active protein import into the nucleusin vivo  and P-ERK2 (p42 isoform), respectively, when compared with controls  or cytoplasmic contamination (absence of IκB)  fractions from control and autophagy-deficient d P-cRAF  or P-MEKd P-cRAF , howevercontrols . Decreascontrols  that occpression . Fresh Ad intact , indicatexplants  and Atg5−/− MEFs . Moreove−/− MEFs , as the −/− MEFs  reduced  tissues , indicatr levels . Atg7−/− of IκB) . Decreasectively . In addiectively .ΔG) construct that impairs APh formationΔG-transfected cells  in CFP-LC3ΔG cells likely enabled production of ATG5–ATG12-positive pre-autophagosomal structures or GABARAP-positive vesiclesTo determine whether ERK phosphorylation requires LC3-II-positive vesicles and not autonomous functions of ATG7 or Beclin1, we transfected NIH/3T3 cells with wild-type (WT) cyan fluorescent protein (CFP)-LC3 or the lipidation-defective C-terminal glycine-deleted , a downstream ERK substrate   , left3. B levels , left, dB levels  and redu WT MEFs , right. 5−/−MEFs . In cons5−/−MEFs , and par5−/−MEFs , without/− cells .Atg5−/− cells, Atg5−/− MEFs subjected to brief serum deprivation (10 min) displayed raised P-MEK, P-ERK and P-p90RSK levels that increased further with EGF treatment  domain) that coordinate with Thr157/158 (ED domain) to modulate CD domain-substrate interaction. ERK2 also utilizes its F-site recruitment sites (FRS) that include residues Leu198, Tyr231, Leu232, Leu235 and Tyr261 ref. , mutatioConversely, ERK substrates interact with ERK2 through substrate D-domains that include basic residues followed by a hydrophobic LXL motif or via substrate F-sites that consist of the FXFP motif33Atg4B−/−heart and brainAtg5−/− MEFs Antibodies  for ATG7g for 5 min followed by centrifugation of the supernatant at 17,000 g (12 min). Pellets were resuspended in 1.9 ml 0.25 M sucrose and 2.8 ml of metrizamide (85.6%) was added. Samples were centrifuged on a 26–24–20–15% metrizamide gradient at 75,334 g for 3 h. After centrifugation, the APh fraction was collected from the 15–20% interface, APL from the 20–24% interface and Lys from the 24–26% interface. Fractions were centrifuged at 24,000 g and pellets were suspended in 0.25 M sucrose and used for assays. For the LC3-II protease protection assayAPh, APL and Lys were isolated from mouse livers and NIH/3T3 cells by differential centrifugation using discontinuous density gradients of metrizamide20462HPO4 (pH 7.4), 50 mM NaCl, 150 mM sucrose, 5 mM KCl, 2 mM dithiothreitol, 1 mM MgCl2, 0.5 mM CaCl2 and 0.2% NP40, supplemented with protease/phosphatase inhibitors), and homogenized using a dounce homogenizer (20 strokes/4 °C), and incubated on ice for 5 min. Cytosolic and nuclear fractions were isolated using ProteoExtract subcellular proteome extraction kit  as per manufacturer’s instructions. Equivalent protein loading was verified using antibodies for β-actin, glyceraldehyde 3-phosphate dehydrogenase, histone3 or Nopp140.For subcellular fractionation, 80% confluent cells or liver pieces (50 mg) were resuspended in buffer  and leupeptin (100 μM), reflects activity. Briefly, cells were cultured in the presence or absence of lysosomal proteases for 2 h following which, cells were collected and lysed and subjected to immunoblotting for LC3-II or p62. Autophagy flux was determined by subtracting the densitometric value of inhibitor-untreated LC3-II or p62 from corresponding inhibitor-treated values.Fresh livers were homogenized in 500 μl of 50 mM Tris/HCl (pH 8.5), 150 mM NaCl, 1% NP40, 5 mM EDTA supplemented with protease/phosphatase inhibitors. Nuclear fractions were obtained as detailed above. Lysates (500 μg of homogenate or 150 μg of nuclear fractions) were incubated overnight in rotation at 4 °C with 100 μl of protein-A sepharose beads (Sigma-Aldrich) crosslinked to LC3 antibody . For covalent crosslinking, beads were washed  with 0.2 M Borate/3 M NaCl buffer (pH 9.0) and crosslinked with 50 mM dimethyl pimelimidate in Borate buffer (30 min) in rotation at RT. Coupling reactions were stopped with 0.2 M ethanolamine (pH 8.0) for 2 h RT followed by incubation with 200 mM glycine (pH 2.5). Crosslinked beads were incubated with samples in rotation overnight at 4 °C. Bound proteins were eluted by boiling (95 °C for 5 min) in 2 × SDS–PAGE sample buffer. Immunoprecipitated (IP) proteins and original lysates (input) were resolved on a SDS–PAGE, and membranes were probed for bRAF, MEK, ERK, P-ERK and LC3. P-ERK1, P-ERK2, P-ERK1/2 or P-MEK1/2 bands obtained by immunoblotting were quantified using ImageJ and normalized to corresponding total ERK1, total ERK2, total ERK1/2 or total MEK1/2, respectively. Full scans of western blots are supplied in Electron microscopy was performed in NIH/3T3 cells cultured in monolayers. Cells were collected and fixed in a buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde in 100 mM sodium cacodylate (pH 7.43)2) settings from 0.1 to 10 and circularity from 0 to 1. Cellular fluorescence intensity was expressed as mean integrated density as a function of individual cell size. Nuclear fluorescence intensity was expressed as a function of nuclear size after the identification of the nuclear perimeter by the ImageJ ‘freehand selection tool’. Percentage colocalization was calculated using the JACoP plugin in single Z-stack sections of deconvolved images. Colocalization is shown in merged native images and/or as white pixels using the ‘colocalization finder’ plugin in ImageJ.Cells on coverslips were fixed with a 4% paraformaldehyde solution, blocked and incubated with primary and corresponding secondary antibodies (Alexa Fluor 488 and/or Alexa Fluor 647 conjugated) (Invitrogen). Mounting medium contained DAPI  to visualize the nucleus (Invitrogen). Images were acquired on a Leica DMI6000B microscope/ DFC360FX 1.4-megapixel monochrome digital camera  using × 100 objective/1.4 numerical aperture. Images in each experiment were acquired at same exposure times within the same imaging session. Image slices/stacks of 0.2 μm thickness were captured and deconvolved using the Leica MetaMorph acquisition/analysis software. All images were prepared using Adobe Photoshop and subjected to identical post-acquisition brightness/contrast effects. Representative native and/or inverted images are shown. Quantification was performed in individual frames after deconvolution and thresholding using the ImageJ software (NIH) in a minimum of 20 cells per slide and a minimum of 50 cells from two or more experiments. Particle number was quantified with the ‘analyse particles’ function in threshold single sections with size  and all reactions were in triplicate. The following primers were used: ERK1 (forward (f)), 5′-TCCAAGGGCTACACCAAATC-3′, (reverse (r)) 5′-GTTTTCGAGGGCAGAGACTG-3′; ERK2 (f) 5′-ACGGCATGGTTTGCTCTGCTTATG-3′, (r) 5′-TCATTTGCTCAATGGTTGGTGCCC-3′; MEK1 (f) 5′-GTGAACTCACGTGGGGAGAT-3′, (r) 5′-CAGGAGGAGGAATGGGGTAT-3′; MEK2 (f) 5′-CCCTACATCGTGGGCTTCTA-3′, (r) 5′-TGCATGATCTGGTGCTTCTC-3′; MKP3 (f) 5′-TTGAATGTCACCCCCAATTT-3′, (r) 5′-CATCGTTCATGGACAGGTTG-3′; PP2ACA (f) 5′-TACTCCGAGGGAATCACGAG-3′, (r) 5′-TCGGATGTGATCCAGTGTGT-3′; GATE-16 (f) 5′-TCTCGGGCTCTCAGATTGTT-3′, (r) 5′-GTGTTCTCTCCGCTGTAGGC-3′; ATG7 (f) 5′-TCCGTTGAAGTCCTCTGCTT-3′, (r) 5′-CCACTGAGGTTCACCATCCT; BECLIN1 (f) 5′-GGCCAATAAGATGGGTCTGA-3′, (r) 5′-GCTGCACACAGTCCAGAAAA-3′; LC3 (f) 5′-ACAAAGAGTGGAAGATGTCCGGCT-3′, (r) 5′-TGCAAGCGCCGTCTGATTATCTTG-3′; LAMP-1 (f) 5′-TAGTGCCCACATTCAGCATCTCCA-3′, (r) 5′-TTCCACAGACCCAAACCTGTCACT-3′; LAMP-2A (f) 5′-AGGTGCTTTCTGTGTCTAGAGCGT-3′, (r) 5′-AGAATAAGTACTCCTCCCAGAGCTGC-3′; TBP (f) 5′-GAAGCTGCGGTACAATTCCAG-3′, (r) 5′-CCCCTTGTACCCTTCACCAAT-3′; ZFP36 (f) 5′-TACGAGAGCCTCCAGTCGAT-3′, (r) 5′-GCGAAGTAGGTGAGGGTGAC-3′.Total RNA was isolated using the RNeasy Plus kit  and genomic DNA eliminated via gDNA Eliminator spin columns. Total RNA (1 μg) was reverse transcribed into cDNA using Superscript II (Invitrogen), and real-time PCR (qPCR) was performed using Power SYBR Green PCR Master Mix  on a StepOnePlus Real-Time PCR System . Expression of Cells were transiently transfected with 100 nM of small interfering RNA (siRNA) constructs or a non-related siRNA (siScramble) (Sigma-Aldrich) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were exposed to the silencing mix overnight following which silencing medium was replaced with fresh DMEM containing 10% NCS. The following siRNAs were used: siScramble (sense(s)) 5′-AAUUCUCCGAACGUGUCACGU-3′, (antisense (as)) 5′-ACGUGACACGUUCGGAGAAUU-3′; siATG7 (s) 5′-CUGUGAACUUCUCUGACGU-3′, (as) 5′-ACGUCAGAGAAGUUCACAG-3′; siATG5 (s) 5′-CCAUCAACCGGAAACUCAU-3′, (as) 5′-AUGAGUUUCCGGUUGAUGG-3′; siBeclin1 (s) 5′-CUGAGAAUGAAUGUCAGAA, (as) 5′-UUCUGACAUUCAUUCUCAG-3′; siATG4B (s) 5′-GAAAUCUUAUCCCUCUGAA-3′, (as) 5′-UUCAGAGGGAUAAGAUUUC-3′; siMKP3 (s) 5′-GUCUCUAGAUCCAGCAAUA-3′, (as) 5′-UAUUGCUGGAUCUAGAGAC-3′; siPP2ACA (s) 5′-CAGUAACUCCAAAGUGUCA-3′, (as) 5′-UGACACUUUGGAGUUACUG-3′. Silencing efficiency was determined by immunoblotting for the corresponding protein product or qPCR analyses for mRNA expression when the appropriate antibody was unavailable.ΔG in which the C-terminal glycine (120) is deletedCyan fluorescence protein (CFP)-LC3, mutant CFP-LC3t-test of the means. For multiple means comparisons, one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test was used to determine statistical significance.All numerical data arising from biochemical and cell fractionation analyses are represented as mean and s.e.m., and are from a minimum of three independent experiments unless otherwise stated. All imaging data are represented as mean and s.e.m, and represent data from at least 50 individual cells from two or more experiments. We determined the statistical significance of the difference between experimental groups in instances of single comparisons by the two-tailed unpaired Student’s in vivo experiments. R.S. conceived the idea, designed the experiments, analysed and interpreted the data, and wrote the manuscript. All authors discussed and commented on the manuscript.N.M.-L. performed the experiments and analysed the data. D.A. assisted with immunofluorescence microscopy. S.S. and P.M. assisted with How to cite this article: Martinez-Lopez, N. et al. Autophagy proteins regulate ERK phosphorylation. Nat. Commun. 4:2799 doi: 10.1038/ncomms3799 (2013).Supplementary Figures S1-S14 and Supplementary Table S1


Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the ‘tension checkpoint’ – the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins . Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine ( The chromosomal passenger complex (CPC) has emerged over the past decade as a critical and conserved regulator of eukaryotic cell division, with key roles in promoting chromosome bi-orientation, in the spindle assembly checkpoint, in spindle disassembly during anaphase and as a regulator of cytokinesis During anaphase, the CPC undergoes dynamic relocalization to the spindle mid-zone in vitro protein kinase assays SLI15 to encode a protein in which constitutive phosphorylation of Sli15 is mimicked, we demonstrate that phosphorylation in vivo is likely to limit the interaction of the CPC with the spindle, and show that phosphorylation of Sli15 or the acidic substitutions that mimic constitutive phosphorylation block the direct binding of Sli15 to microtubules in a novel in vitro binding assay. Furthermore, we find that mimicking constitutive phosphorylation of Sli15 on its Ipl1 phosphorylation sites interferes with the cell's ability to mount a spindle assembly checkpoint response specifically to reduced tension on sister kinetochores (the ‘tension checkpoint’). Thus in addition to affecting the spindle association of the CPC, Ipl1-dependent phosphorylation of Sli15 may act as a negative regulator of the tension checkpoint response.INCENP/Sli15 consists of three domains: the N-terminal region that mediates association with Bir1 and Nbl1, the central domain that binds microtubules and the C-terminal domain or IN-box that is involved in binding to and activating Aurora B/Ipl1 protein kinase ade2-1 his3-11, 15 leu2-3, 112 trp1-1 ura3-1 can1-100 ssd1-d2 Gal+. Plasmids used or generated in this work are summarized in 600 0.1 and then spotting 5 µl of undiluted and 10-fold serial dilutions onto YPAD agar plates or plates containing 10, 11 or 12.5 µg/ml benomyl, followed by growth for 2 days at the indicated temperatures. Chromosome loss was examined by monitoring the loss of a chromosome III fragment (CFIII) carrying URA3 and SUP11SUP11) leads to a red colony color due to the failure to suppress the ade2-1 mutation.Basic yeast methods, growth media, and routine recombinant DNA methodology were performed as previously described SLI15, a 2.55 kb BamHI-XhoI fragment carrying SLI15 together with 300 bp of upstream and downstream flanking sequence was first excised from pCJ145 and inserted into pRS303. The sli15-20A plasmid was created from pRS303-SLI15 by sequential site-directed mutagenesis using different primers on pRS303-SLI15 with the QuikChange Site-Directed Mutagenesis Kit  according to the manufacturer's instructions. The mutated plasmid was verified by DNA sequencing, linearized with BsiWI and integrated in single copy at one of the his3 loci in VMYD16 (SLI15/sli15Δ::KanMX6). Following sporulation and tetrad dissection, the required haploid sli15Δ::KanMX6 his3::sli15-20A::HIS3 strains were generated. Control strains expressing wild-type SLI15 were similarly generated using pRS303-SLI15. For creating the sli15-20D plasmid, a 1479 bp fragment of SLI15 encoding 20 substitutions to glutamate or aspartate was synthesized  and subcloned as an NcoI-MfeI restriction fragment in place of the equivalent wild-type fragment in pRS303-SLI15, generating pRS303-SLI15-20D. pRS303-SLI15-20D was integrated in VMYD16 as above. Following tetrad dissection, high levels of spore inviability were seen that were rescued following transformation with pCJ145 (pRS316-SLI15). sli15Δ::KanMX6 his3::sli15-20D::HIS3 strains were therefore generated by sporulation and tetrad dissection of the pCJ145-containing strain, followed by two rounds of selection on 5-fluoroorotic acid to evict the plasmid.For generating strains with mutant copies of pGAL-SCC1 shut-off was performed as previously described PDS1-myc18. For lysing cells sequential NaOH and trichloroacetic acid treatment was used 18 was detected by Western blotting with an anti-myc antibody . Cdc28 was detected using an anti-Cdc28 antibody from Santa Cruz Biotechnology (sc-6709). GFP was detected with anti-GFP .Whole-cell extracts were made and immunoblotted as previously described CEN5 centromeres that remained unseparated at one end of the metaphase spindle were scored as mono-oriented, whereas sister CEN5 centromeres that showed dynamic separation and reassociation were scored as bioriented. At least 100 cells were analyzed for all reported experiments. Quantification of Sli15-EGFP fluorescence was performed with Volocity software (PerkinElmer). Statistical analysis of chromosome biorientation data was carried out using Fisher's exact test. Statistical analysis of all other microscopy data made use of the Mann-Whitney U test. All P values are two tailed.The time-lapse fluorescence microscopy of live cells for monitoring chromosome biorientation was performed as previously described 6 and GST-Dam1 were expressed and purified essentially as described previously 6, GST-Sli15-20A-His6 and GST-Sli15-20D-His6, E. coli BL21(DE3) cells transformed with the appropriate construct were induced with 100 µM IPTG at OD600 = 0.6 for 12–16 h at 22°C. Cells were harvested by centrifugation at 5000 rpm, 4°C. Bacterial pellets were washed once with chilled PBS and resuspended in Lysis Buffer . Sonicated lysates were cleared by centrifugation at 40,000 rpm for 30–45 min in a Beckman Ti45 rotor at 4°C. Supernatants were filtered with 0.2 µm filters and incubated with 1 ml of Glutathione Sepharose 4 Fast Flow  per liter of bacterial culture. Beads were prewashed with PBS  and equilibrated with Lysis Buffer. After incubation for 1 h at 4°C, beads were transferred to a 10 ml Econo-Pac column (BioRad), washed with five column volumes of Lysis Buffer containing 1 M KCl prior to elution in 1 ml fractions with 40 mM reduced glutathione (Sigma-Aldrich), 100 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10%(v/v) glycerol. Elution fractions were assessed by SDS-PAGE and appropriate fractions pooled and dialyzed in 2 L Buffer I  glycerol, 5 mM β-mercaptoethanol, 30 mM imidazole) overnight. Dialyzed protein solutions were diluted to 10 ml in Buffer I, and incubated with 200 µl of equilibrated Ni-NTA slurry (Qiagen) for 2 h at 4°C with mixing by rotation. The beads were transferred to a 10 ml Econo-Pac column (BioRad), washed with three column volumes Buffer I containing 1 M KCl, followed by a two column volume wash in Buffer I. The bound proteins were eluted by the addition of 0.2 ml Buffer I containing 250 mM imidazole (Sigma-Aldrich). Eluted fractions were assessed by SDS-PAGE and appropriate fractions dialyzed in 2 L 50 mM Bis-Tris propane (pH 6.8), 150 mM KCl, 100 mM imidazole, 20%(v/v) glycerol and 5 mM β-mercaptoethanol. Aliquots of 200 µl were snap-frozen and stored at −80°C.Recombinant GST-Ipl1, GST-Sli15, Ipl1-His2 and 100 µM [γ-32P]ATP (5000 cpm/pmol) in a total reaction volume of 200 µl. The reaction was stopped by adding SDS, dithiothreitol (DTT) and Sample Buffer (Invitrogen) to give final concentrations of 1% SDS, 10 mM DTT and 1× Sample Buffer, then the samples were heated at 70°C for 5 min and separated by SDS-PAGE on 10% polyacrylamide gels, stained with Colloidal Coomassie (Invitrogen) and phosphoprotein localized by autoradiography. The 32P labeled proteins were excised and the gel pieces were digested with either endoproteinase Lys-C or with trypsin, then peptides separated by HPLC and analyzed by MALDI-TOF-TOF mass spectrometry using a Biosystems 4700 Proteomics Analyzer and Edman degradation as described previously GST-Ipl1 kinase (∼2 µg) and GST-Sli15 (∼5 µg) prepared as described previously 6 (0.06 µg) and Ipl1-His6 (0.3 µg) were carried out in 20 µl reactions with [γ-32P]ATP (>800 cpm/pmol) essentially as previously described Protein kinase assays using recombinant GST-Sli15-His2, 1 mM EGTA) containing 1 mM GTP  and 1 mM DTT was thawed and centrifuged to remove insoluble protein for 5 min at 42,000 rpm using a Beckman TLA100 rotor at 4°C. To assemble microtubules, the reaction was incubated at 37°C for 30 min while taxol  was added in a stepwise manner to a final concentration of 20 µM to stabilize microtubules. Microtubules were then centrifuged at 50,000 rpm for 5 min at 30°C and the pellet was resuspended in warm PME buffer containing 1 mM GTP, 1 mM DTT and 20 µM taxol to a final concentration of 5 µM polymerized tubulin.Tubulin (250 µg) purified from bovine brain  was mixed with 20 µg biotin labeled tubulin  in ice-cold PME buffer , then loaded with 5 µM biotinylated polymerized tubulin (MTs). Unbound MTs were removed in a 5 min wash with Buffer A, followed by a subsequent wash with Buffer A containing 10 µM biocytin (Tocris) to block free streptavidin sites. Biocytin was removed in a 5 min wash step with Buffer A and the system was equilibrated for 5 min with Buffer B  glycerol, 5 mM β-mercaptoethanol, 100 mM imidazole, 1 mg/ml BSA, 20 mM taxol). A parallel set of SA biosensors was blocked with biocytin to act as a reference surface to correct for non-specific binding of protein to the biosensors. Serial dilutions of GST-Sli15-His6, GST-Sli15-20A-His6, GST-Sli15-20D-His6 and GST control were allowed to associate in Buffer B for 5 min and dissociation from MTs was monitored for 10 min. Data were processed using ForteBio Data Analysis Software 7.0 to determine binding. In the kinase assay reaction, Ipl1-His6 was incubated with GST-Sli15 in the presence and absence of unlabeled 10 µM ATP for 30 min at 30°C as previously described Binding assays were carried out by biolayer interferometry at 25°C in solid black 96-well plates (Greiner Bio-One) using an Octet Red384 . Streptavidin-coated biosensors  were equilibrated in Buffer A  was also generated in which the serine and threonine residues at these twenty sites were replaced with aspartate or glutamate respectively. Detection of the wild-type and mutant versions of Sli15 following EGFP-tagging indicated that all three proteins were present at similar levels in asynchronously growing cells, and also in metaphase-arrested or anaphase cells, confirming that neither the sli15-20A nor sli15-20D alleles conferred a significant change in the level of Sli15 in comparison to the wild-type protein (data not shown). Despite the multiple amino acid replacements, yeast cells relying on either sli15-20A or sli15-20D as their sole source of Sli15 grew normally at 26°C and 37°C, indicating that both mutant proteins can provide sufficient Sli15 function to support normal rates of proliferation  sli15-20A strain and the decreased levels in the sli15-20D strain were both highly statistically significant  can promote full activation of Ipl1 in an in vitro protein kinase assay using Dam1 as a substrate. Thus direct activation of Aurora B/Ipl1 via IN-box phosphorylation may not be a conserved feature of the CPC, consistent with the apparent lack of conservation of the activatory phosphorylation sites in the IN-box region It has been known for some time that INCENP/Sli15 shows Aurora B/Ipl1-dependent phosphorylation SLI15 were fully viable, in contrast to cells in which conditional mutations in either the SLI15 IN-box region IPL1 causes lethality due to failed chromosome biorientation under restrictive conditions sli15-20A and sli15-20D strains should be capable of promoting efficient chromosome biorientation. Consistently, we could detect no significant change in the efficiency of chromosome biorientation in either strain, in agreement with the properties of a similar non-phosphorylatable sli15 mutant sli15-20D strain. The robust spindle association of Sli15-20A in pre-anaphase cells could potentially reduce kinetochore-localized CPC, but efficient chromosome biorientation in the sli15-20A strain indicates that Ipl1 can still gain proper access to its substrates on incorrectly attached kinetochores. Deletion of the entire N-terminal domain of Sli15, which mediates its association with the other CPC components (Bir1 and Nbl1) involved in centromere targeting, also drives Sli15 onto the pre-anaphase spindle but has little or no effect on the efficiency of chromosome biorientation or chromosome segregation Yeast cells relying on either non-phosphorylatable or phosphomimic alleles of in vivo, Sli15-20D was completely defective in binding microtubules in vitro whereas Sli15-20A and wild-type Sli15 bound microtubules well. Binding of the wild-type protein was strongly reduced following phosphorylation by Ipl1, indicating that phosphorylation of Sli15 by Ipl1 directly affects its affinity for microtubules and confirming that addition of multiple negative charges to the microtubule domain is inhibitory to binding. The opposite behavior of Sli15-20A and Sli15-20D in relation to microtubule association is therefore in contrast to alanine or aspartate substitutions at the six Cdk phosphorylation sites in Sli15, both of which conferred similar behavior in vitro microtubule binding properties of both Sli15-20D and of phosphorylated wild-type Sli15 in comparison with the non-phosphorylated protein or Sli15-20A support the contention that Sli15-20D is mimicking the constitutively phosphorylated form of the protein. Given that neither sli15-20A nor sli15-20D confer a significant chromosome biorientation defect, it is likely that the elevated chromosome loss rates observed in each mutant result from altered behavior of the spindle microtubules resulting from inappropriate levels of CPC association, although we cannot exclude the possibility of a small defect in bi-orientation that was below the limit of detection in our biorientation assay.Phosphorylation of the central domain of Sli15 on its cyclin-dependent kinase (Cdk) sites is known to regulate its interaction with spindle microtubules, and alanine substitution of either just ser-335 or all six Cdk phosphorylation sites drives Sli15 onto the spindle prematurely in metaphase-arrested cells et al.Since it is likely that Sli15-20A remains phosphorylated on its Cdk sites, Cdk phosphorylation on its own may be insufficient to block spindle association of the CPC. However, since Sli15-20D retains some ability to interact with the anaphase spindle, albeit at a reduced level, Ipl1 phosphorylation also appears insufficient to block spindle association of the CPC in anaphase when the Cdk sites have been dephosphorylated. Thus our data and those of Nakajima Association of the CPC with spindle microtubules is regulated in multiple ways: binding via the interaction between Ipl1 and Bim1 is inhibited by Cdk phosphorylation on Ser-50 and Ser-76 sli15-20A mutant, strains dependent on Sli15-20D fail to delay entry into anaphase in response to reduced tension on kinetochore-microtubule attachments – the ‘tension checkpoint’. However, both sli15 mutants showed a normal metaphase arrest in response to microtubule depolymerisation by nocodazole, indicating that the core checkpoint machinery is functional. Ipl1 in vitro between centromeric DNA and microtubules that requires the central microtubule-binding domain of Sli15 and that is inhibited by Ipl1 phosphorylation Our work has shown that in contrast to the


Conductive or sensorineural hearing loss was induced surgically in gerbils prior to hearing onset and GABA agonists were then administered for one week. IPSCs were subsequently recorded from pyramidal neurons in a thalamocortical brain slice preparation. Administration of either a GABAA receptor a1 subunit specific agonist (zolpidem), or a selective GABA reuptake inhibitor (SGRI), rescued IPSC amplitude in hearing loss animals. Furthermore, this restoration persisted in adults, long after drug treatment ended. In contrast, a GABAB receptor agonist baclofen did not restore inhibitory strength. IPSCs could also be restored when SGRI administration began 3 weeks after sensory deprivation. Together, these results demonstrate long-lasting restoration of cortical inhibitory strength in the absence of normal experience. This suggests that in vivo GABAA receptor activation is sufficient to promote maturation, and this principle may extend to other developmental disorders associated with diminished inhibitory function.Inhibitory synapse dysfunction may contribute to many developmental brain disorders, including the secondary consequences of sensory deprivation. In fact, developmental hearing loss leads to a profound reduction in the strength of inhibitory postsynaptic currents (IPSCs) in the auditory cortex, and this deficit persists into adulthood. This finding is consistent with the general theory that the emergence of mature synaptic properties requires activity during development. Therefore, we tested the prediction that inhibitory strength can be restored following developmental hearing loss by boosting GABAergic transmission  A receptor-mediated transmission An influential theory in developmental plasticity states that inhibitory synapse function regulates the excitatory synapse critical period A reduction in GABA signaling can delay or prevent the maturation of inhibitory synapses. For example, mice lacking the enzyme associated with GABA synthesis, glutamic acid decarboxylase (GAD) 65, do not exhibit ocular dominance plasticity; however, treatment with benzodiazepines restores this mechanism de novo synaptogenesis, and the augmentation of inhibitory drive Experimental approaches to repair brain function by modifying inhibition differ depending upon the goal, and the nature of the disorder. For example, research on ocular dominance plasticity seeks to reduce inhibition in adults in order to restore excitatory synaptic plasticity after the critical period has ended A receptors only , both GABAA and GABAB receptors imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride hydrochloride and GABAB receptors only . Zolpidem and SGRI restored inhibitory strength while BAC treatment was ineffective. Furthermore, inhibitory restoration persisted in adults. These findings suggest that the basic principle of activity-dependent synapse maturation can be used as a strategy to overcome the deficits that may attend early sensory deprivation.To test the role of GABA receptor activation, hearing loss was induced prior to hearing onset and GABA agonists were then administered for one week. The amplitude of inhibitory currents was assessed in thalamorecipient ACx neurons in brain slices. Three agents were chosen to activate GABASee SI Methods for details.Animal care, maintenance, surgery and pharmacological treatments were in accordance with the guidelines and rules of the Institutional Animal Care and Use Committee, New York University (NYU), approved by the Office of Laboratory Animal Welfare, Office of Extramural Research, and U.S. National Institutes of Health . Meriones unguiculatus) pups at postnatal day (P) 10 (P10) were anesthetized with the halogenated ethyl methyl ether methoxyflurane (Metofane). Anesthetic effect occurred by 10 min as tested by an absence of response to nociceptive stimuli (toe pinch). Conductive hearing loss was induced by tympanic membrane puncture and malleus removal. Cochlear ablations were performed on gerbil pups at P10, just prior to the onset of response to airborne sound See SI Methods for details on sham surgery and sham injection.Gerbil , or a selective GABA reuptake inhibitor imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid, Sigma, 10 mg/kg), or the GABAB agonist BAC . In a separate group of CHL pups, we administered SGRI (10 mg/kg) during P30–36 following CHL.A key objective in this study was an attempt to rescue inhibitory synaptic strength following conductive hearing loss. In order to be less invasive and support cortical inhibitory synapses at a slow and sustained level, we subcutaneously administered conductive hearing loss (CHL) pups once everyday for 7 days (i.e. during P11 through P17.) We administered the GABASee SI Methods for details.The doses were determined based on pilot experiments in which zolpidem, SGRI, or BAC was administered in vivo. The initial does of each drug was 1 mg/kg in 2 CHL animals, and this did not have an apparent effect on spontaneous inhibitory postsynaptic current (sIPSC) amplitudes. The dose was then increased to 10 mg/kg, and this appeared to restore sIPSC amplitude. Therefore, we chose to retain this dose for the study. For BAC, 10 mg/kg led to a decline in motor activity over several days, and we therefore reduced the BAC dosage to 2.5 mg/kg. For a comprehensive outline of the protocol, see See S1 Methods for details on whole-cell recordings and selective stimulation of putative unitary afferents for eliciting minimum evoked (me) IPSCs.Thalamocortical brain slices (500 µm) were generated as described in our previous papers Statistical tests of data distribution were followed by comparisons between groups using commercial software . A Shapiro-Wilcoxon W (Goodness-of-fit) test was performed to test for a normal distribution. For those data sets that were not normally distributed, a Wilcoxon rank sums test was performed to determine whether there was a main effect, followed by pair-wise comparisons. For the data that were normally distributed, an ANOVA test was performed to determine whether there was a main effect, followed by pair-wise comparisons using a Students' t test.A receptor agonist, zolpidem, could rescue inhibitory synaptic strength in juvenile animals reared with CHL . When zolpidem was administered for the 7 days immediately following CHL at P10, sIPSCs were significantly larger than those from untreated CHL animals . Furthermore, there was no difference between sIPSCs from control and SGRI-treated CHL animals . SGRI administration in CHL animals also restored me-IPSCs amplitudes . There was no difference between me-IPSCs from control and SGRI-treated CHLs . In contrast to these results, when animals with sham surgery were treated with SGRI, there was no significant change in either sIPSC amplitude or PPR (We next asked whether administration of a selective GABA reuptake inhibitor (SGRI) could preserve inhibitory synaptic strength in juvenile animals reared with CHL. As shown in e or PPR .B receptor agonist, could restore inhibitory synapse function in juvenile animals reared with CHL  vs. BAC-treated CHL: 15.5±1.8; n = 11 , χ2 = 12.02, p = 0.0006), or me-evoked IPSC amplitudes .In the third set of experiments, we asked whether a GABAwith CHL . Here, wCHL induced at P10 leads to a persistent reduction of sIPSC amplitude recorded in adults at P90–110 2 = 8.2, p = 0.004) 2 =  = 5.7, p = 0.01]. There was no difference between sIPSCs from adult controls and zolpidem-treated CHL adults .CHL induced at P10 resulted in a persistent reduction of sIPSC amplitude when recorded in adults at P90–110  vs. CHL adult: 16.5±2.1, n = 11 , χ2 = 3.6, p = 0.05]. There was no difference between sIPSCs from control and SGRI-treated CHL adults .To determine whether GABA receptor activation could preserve IPSC strength when treatment was delayed by about 3 weeks from CHL induction, we induced CHL at P10, administered SGRI at P30–36, and raised animals to adulthood. As shown in in vivo GABAA receptor activation is sufficient to promote the maturation of inhibitory synaptic strength in CHL animals, and this principle may apply to other disorders associated with diminished inhibition.Developmental hearing loss may originate in the periphery, yet it causes pervasive impairment of CNS synapses and membrane properties. The disruption of one such property, inhibitory synaptic strength, has been suggested to be a causative factor in many central disorders, including hearing loss A receptor a1 subunit (via zolpidem) or an increase in extracellular GABA at inhibitory synapses (via SGRI action) could each preserve sIPSC amplitude in juvenile animals reared with CHL  and paired pulse ratio of evoked IPSCs (bottom panel). n values appear within bars. The sIPSC amplitudes in sham-surgery animals were significantly different from CHL and CHL with sham injection groups (asterisk). The PPR was facilitatory in sham-surgery animals and depressing in the remainder group. See S1 (EPS)Click here for additional data file.Figure S2The scatter plot shows the distribution of all sIPSC amplitudes recorded from 4 experimental groups.(EPS)Click here for additional data file.Figure S3Inability of BAC to rescue inhibition persists in adults.A Left panel: Representative sIPSCs recorded in L2/3 pyramidal cells in ACx in slices from animals aged P90–110 (VHOLD = −60 mV). Examples are shown from control , CHL  and BAC-treated CHL  animals. B Right panel: Bar graphs (mean ± SEM) of average sIPSC amplitude from all recorded neurons showing diminished sIPSC amplitude in CHL animals is not rescued after treatment with BAC.(EPS)Click here for additional data file.Results S1(DOC)Click here for additional data file.Methods S1(DOC)Click here for additional data file.


We previously reported that some ATP competitive protein kinase C (PKC) inhibitors are either competitive or uncompetitive inhibitors with respect to substrate peptides. In this report, we demonstrate how the interactions between PKC and inhibitors change PKC activation kinetics. A substrate competitive inhibitor, bisindolylmaleimide I, targets activated PKC and stabilizes PKC in the activated conformation. This leads to transient activation and prolonged deactivation of PKC in the presence of bisindolylmaleimide I. In contrast, an uncompetitive substrate inhibitor, bisindolylmaleimide IV, targets quiescent PKC and stabilizes PKC in the quiescent conformation, which generates slower activation and suppressed translocation upon activation of PKC. ATP competitive kinase inhibitors have been widely used to identify signaling pathways. In some cases, however, pharmacological observations do not support the biochemical data. One example is the acetylcholine induced suppression of the M-type potassium channel Potential interactions between substrate peptides and ATP competitors are also suggested by crystal structure studies. To date, several crystal structures of PKC-inhibitor complexes have been solved The pseudosubstrate domain governs the activation status of many serine/threonine kinases In this paper, we investigate functional consequences of the interaction between the intramolecular pseudosubstrate domain of PKC and ATP competitive inhibitors. We show that the primary target for BIS I is activated PKC while BIS IV targets quiescent PKC. We demonstrate that these different state-dependent inhibitions change the activation kinetics of PKC and stabilize PKC in certain conformations within the cellular environment.Ckinase activity reporter, (CKAR), a fluorescence resonance energy transfer (FRET) based fluorescent probe 1 muscarinic acetylcholine receptor, CHO hm1 cells Bisindolylmaleimide I (BIS I) and bisindolylmaleimide IV (BIS IV) are structurally very similar PKC inhibitors . HoweverTo further examine whether the activation status of PKC changes BIS binding, we compared sensitivity to BIS I between preactivated PKC and quiescent PKC . PreactiIf the pseudosubstrate domain interferes with the PKC-BIS I binding, then BIS I should interfere with the binding of the pseudosubstrate domain to the substrate recognition site. To test this, we fused the pseudosubstrate domain with GFP, Psd-GFP, and examined its binding to V5 tagged PKCβII, PKCβII-V5, by immunoprecipitation. Since synthesized pseudosubstrate peptides have been used as PKC inhibitors Since the above experiments used a deletion mutant, we next intended to confirm these effects of BIS compounds using wild-type PKCβII. Since our immunoprecipitation in the non-activated condition did not show stable association between PKCβII and Psd-GFP, we tested the interaction in the presence of a PKC activator. Activation of PKC should remove the intramolecular pseudosubstrate domain from its catalytic site, which would allow Psd-GFP to bind PKC. To this end, purified PKCβII was first treated with PDBu together with BIS I or BIS IV, then Psd-GFP bound resin was added to test PKC binding. In this preactivated condition, we could detect PKCβII in Psd-GFP precipitate as expected . When BIThese results suggest that BIS compounds modify the interaction between the pseudosubstrate and the catalytic site in distinct ways: BIS I repels the pseudosubstrate domain from the catalytic site, while BIS IV stabilizes the association of the pseudosubstrate domain and the catalytic site. Our new results suggest that BIS compounds can stabilize PKC either in the activated state or in the quiescent state depending on their molecular structures. The next question is whether such stabilizations occur within the cellular environment.To address this question, we measured the activation and deactivation processes of PKC using CFP tagged PKCβII, PKC-CFP. Conventional PKC is a unique kinase in that its conformational changes can be detected as a change in subcellular localization per se is not sufficient to turn activated PKC into the quiescent state in the presence of PDBu. Taken together, these results suggest that BIS compounds can stabilize PKC in either the quiescent state or the activated state in living cells depending on how the compounds interact with the pseudosubstrate domain in the cellular environment.On the other hand, BIS I did affect the deactivation process. In the control condition, PKC-CFP restored its cytoplasmic localization after application of 100 µM pirenzepine with τ of 12.1±0.6 sec . In the To test whether these effects are specific to BIS compounds or represent common phenomena for ATP competitive inhibitors, we performed a similar series of experiments using staurosporine and K-252c. Staurosporine is a prototypical molecule for ATP competitors and is a wide spectrum kinase inhibitor that shows high affinity binding to most kinases. K-252c is a staurosporine aglycone that lacks the amino group; the structural relation between K-252c and staurosporine is analogous to that of BIS IV and BIS I . StaurosIn the present study, we have characterized the cellular pharmacology of several ATP competitive PKC inhibitors. Unlike conventional kinase assays that measure stationary activities, FRET based live-cell imaging analyses Firstly, these PKC inhibitors showed time-dependent changes in their potencies after activation of PKC. The time-dependent changes for both BIS I and BIS IV were best fitted by single exponential functions , which sSecondly, BIS I preferentially inhibited preactivated PKC. This is evidenced by higher susceptibility to inhibition of preactivated PKC and a much faster time course to reach the plateau inhibition in preactivated PKC . In contin vitro experiments that BIS I stabilizes PKC in the activated conformation Finally, BIS I bound PKC is stabilized in the activated conformation. This is suggested by a delayed recovery of cytosolic localization of PKCβII-CFP after termination of the activation signal . We prevTaken together, we speculate that the binding of BIS I and the pseudosubstrate domain to the catalytic site are mutually exclusive . If the On the other hand, BIS IV is an uncompetitive inhibitor with respect to the substrate peptide When we consider the gradual loss of BIS IV potency after activation , we beliRecently, a crystal structure of full-length PKCβII has been solved Despite the importance of state-dependent inhibition, not much attention has been paid to this aspect for kinase inhibitors. Real time monitoring of cellular kinase activity helped us to explore state-dependent inhibition. The fact that these state-dependent inhibitions were also observed for staurosporine, a wide spectrum kinase inhibitor, suggests that state-dependent inhibition is a common feature for ATP competitive inhibitors. In addition, we wish to emphasize that, as a consequence of state-dependent inhibition, kinase activity in the presence of kinase inhibitors is not a proportional miniature of the control response . This fe5′-GATCCCGCTTCGCCCGCAAAGGCGCCCTCCGGCAGAAGAACGTG-3′; reverse, 5′-AATTCACGTTCTTCTGCCGGAGGGCGCCTTTGCGGGCGAAGCGG-3′) was synthesized, annealed and ligated into pEGFP-C1 plasmid at BglII and EcoRI sites. DNA sequencing and immunoblot verified Psd-GFP constructs. V5 tagged PKCβII and (Δ2-31)PKCβII were made by PCR using wild-type PKCβII 5′- CACCATGGCTGACCCGGCTGCGGGGCCGCCGCCG-3′, (Δ2-31) forward, 5′-CACCATGCATGAGGTCAAGAACCACAAATTCACCGCCCGCTTCTTCAAGCAGCCC-3′, reverse, 5′-GCTCTTGACTTCGGGTTTTAAAAATTCAGAGTTAAC-3′). PCR fragments were subcloned using the pcDNA3.1 directional TOPO kit . All PCR derived fragments were confirmed by sequencing.CKAR expression plasmid was a generous gift from Dr. Alexandra Newton at UCSD Bisindolylmaleimide I, bisindolylmaleimide IV, staurosporine, K-252c and phorbol 12,13-dibutyrate were purchased from Sigma-Aldrich . These compounds were dissolved in DMSO as stock solutions and diluted at least 1∶10,000 for actual experiments. For the experiments involving steady state inhibitions, cells were incubated with relevant inhibitors for between 20 min and 2 hours.1 muscarinic receptor 2. HEK 293A cells  were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum. For transfections, cells were grown to 30% confluence and transfected  according to the manufacturer's instruction .CHO cells stably expressing human mFluorescence emission was acquired using an inverted microscope IX-81  and ImageEM CCD camera  controlled by MetaMorph 7.6.3 . The excitation light was generated by Lambda LS  and passed through a S436/10x or S500/20x filter. The light intensity was reduced to 3% for 436 nm and 0.07% for 500 nm to minimize photobleaching. Dual-emission images were obtained simultaneously through a dual-view module  with ET535/30m, ET480/40m emission filters and a T505lpxr dichroic mirror . Exposure time was 100 ms; images were taken every 1.5 s for experiments shown in 2, 0.5 mM MgCl2, 10 mM glucose, 10 mM HEPES (pH 7.4). All data are presented as mean ± SEM.Cells were cultured on round cover glasses (18 mm diameter) and washed twice before mounting onto the microscope with a solution consisting of 144 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM dithiothreitol, 5 mM EGTA, 10 mM HEPES (pH 7.4), 0.4% Nonidet P-40 and complete mini protease inhibitor cocktail . After centrifugation at 22,000 g for 20 min at 4°C, the lysate was precleared with protein G resin, and then immunoprecipitated with rabbit GFP antibody , and protein G resin. Relevant samples contained 200 µM ATP or 10 µM PKC inhibitors. Psd-GFP bound resin was washed three times with HSE buffer containing 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 20 mM HEPES (pH 7.4), 1% Triton X-100 and analyzed by SDS-PAGE followed by western blot.Transfected HEK cells were lysed with Hypotonic buffer containing 10 mM NaCl, 1.5 mM MgClPsd-GFP was purified from transiently transfected HEK cells by immunoprecipitation. Transfected cells were lysed with HSE buffer and complete mini protease inhibitor cocktail. After centrifugation at 22,000 g for 15 min at 4°C, the lysate was precleared with protein G resin, and then immunoprecipitated with rabbit GFP antibody  and protein G resin. Psd-GFP bound resin was washed twice with HSE buffer followed by twice with HSE buffer containing 650 mM NaCl then twice with HSE buffer. For the binding assay, 60 ng PKCβII  in 10 µl binding buffer was incubated 5 min at room temperature with or without 10 µM PKC inhibitors. Then, purified Psd-GFP attached to beads was added and further incubated 15 min at room temperature. After three washes with the binding buffer plus relevant PKC inhibitors, associated proteins were analyzed by western blots using a monoclonal antibody toward PKCβ . The binding buffer consisted of 0.1% Triton X-100, 150 mM NaCl, 100 nM PDBu, 1 mM DTT, 10 mM HEPES (pH 7.4).2, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 25 mM β-glycorol phosphate, 20 mM MOPS (pH 7.2). Relevant samples contained ATP and BIS IV. Reference samples were kept on ice during the incubation. PKC kinase activity was measured using 32P-ATP and PKC assay kit . Experiments were done as triplicates.Aliquots of PKCβII  were prepared and incubated for 30 min at 37°C in a buffer containing 1 mM CaCl