Activating mutations in the RasGTPases are the most common oncogenic lesions in human cancer. Similarly, elevated STAT3 expression and/or phosphorylation are observed in the majority of human cancers. We recently found that activated Ras requires a mitochondrial rather than a nuclear activity of STAT3 to support cellular transformation. This mitochondrial activity of STAT3 was supported by phosphorylation on serine 727 (S727) in the carboxyl-terminus of STAT3. In this study we show that the H-Ras oncoprotein engages the MEK-ERK pathway to drive phosphorylation of STAT3 on S727, while phosphoinositide 3-kinase (PI3K) and mTOR activity were superfluous. Moreover, pharmacological inhibition of MEK reduced transformation by H-, K- or N-Ras. However, cells expressing a mitochondrially restricted STAT3 with a phospho-mimetic mutation at S727 were partially resistant to inhibition of the ERK pathway, exhibiting a partial rescue of anchorage-independent cell growth in the presence of MEK inhibitor. This study shows that the MEK-ERK pathway is required for activated Ras-induced phosphorylation of STAT3 on S727, that inhibition of STAT3 S727 phosphorylation contributes to the anti-oncogenic potential of MEK inhibitors, and that mitochondrial STAT3 is one of the critical substrates of the Ras-MEK-ERK- axis during cellular transformation.  Signal Transducer and Activator of Transcription (STAT)3 was originally described as a latent cytosolic transcription factor activated by phosphorylation on tyrosine Y705) in response to stimulation by cytokines and growth factors . Phosph in respoPhosphorylation of STAT3 on serine 727 (S727) also occurs in response to cytokine stimulation and enhances its transcriptional efficacy . S727 phThe STAT3 serine phosphorylation site is embedded in a conserved PMSP sequence, which contains a mitogen activated protein kinase (MAPK) consensus target sequence (PXS/TP) . HoweverSTAT3 was recently shown to function in mitochondria, in addition to its canonical role as a nuclear transcription factor ,15,16. IIn this study, we used genetic mutants and pharmacological inhibitors to show that H-, N- and K-Ras engage the Raf-MEK-ERK pathway to cause phosphorylation of STAT3 on S727, which is necessary for the anchorage-independent growth of transformed fibroblasts as colonies in soft agar. Moreover, we show that a STAT3 mutant that mimics S727 phosphorylation (S727D) is partially refractory to the block in transformation resulting from pharmacologic inhibition of the Raf-MEK-ERK MAPK pathway.The following antibodies were obtained from commercial sources: STAT3 (4904), pY705 STAT3 (9145), pS727STAT3 (9134), pThr389 p70 S6 Kinase (9206), from Cell Signaling, Beverly, MA; Akt (SC-8312), pAkt (SC-7985), Tubulin, ERK1/2 (SC-94) and pERK1/2 (SC-7383), from Santa Cruz Biotechnology, Santa Cruz, CA, anti-Rabbit IRDye800 and anti-Mouse IRDye680 from LiCOR.gcaataccattgacctgcccatggacccccgcactttagatt. The retroviral constructs (pBabe-puro) expressing H-RasV12 mutants H-RasV12/35S, and H-RasV12/40C were kind gifts from C. Der. N-RasV12 (pBabePuro-N-RasV12) and K-RasG12V (pBabePuro-K-RasV12) were the kind gift of A. Pellicer. Retroviruses (MSCV-GFP) expressing wild type STAT3 fused to an N-terminal mitochondrial targeting sequence from cytochrome c oxidase subunit VIII were constructed as previously described and generously provided by A. Larner . The mit2. THP-1 cells were obtained from ATCC and grown in RPMI medium.3T3 immortalized STAT3-deficient mouse embryo fibroblasts were generated as described previously  and abse3VO4 and 1mM DTT. Protein preparations were resolved by 10% SDS-PAGE, transferred onto PVDF membranes (Millipore), and the membranes were incubated for 1 hour in 5% BSA in TBS-0.1% Tween-20 . Blots were probed with primary antibodies diluted in TBS-0.1% Tween-20 buffer overnight at 4°C. The blots were washed and incubated with 1:15,000 dilution of fluor-conjugated secondary antibody (Li-Cor) and scanned using an Odyssey infrared scanner (Li-Cor).Cells were lysed in a buffer containing 150mM NaCl, 50mM Tris (pH 7.4), 1mM EDTA, 0.5% Triton X-100, protease inhibitor cocktail (Sigma Aldrich), 1mM NaF, 1mM β-glycerophosphate, 1mM Na3) were seeded in 0.3% agar, prepared in standard growth media. Cells were cultured in a humidified incubator at 37°C and 5% CO2 for 14 days and colonies greater than 0.2 mm2 were quantified. Each cell line was plated in three separate wells and each experiment was performed on three separate days. Data presented represent the mean of biological triplicates.Cells , even in the absence of overt activation of the STAT3 pathway . Indeed,in vitro, and we have previously found that transformation of murine embryo fibroblasts (MEF) in vitro by each Ras-activated pathway is STAT3 dependent [Ras engages numerous signaling pathways to elicit its tumorigenic activities, primarily the MEK-ERK, phosphoinositol-3-kinase (PI3K), and Ral pathways . Activatependent . Moreoveependent . BecauseTo address this question, we employed H-Ras mutants that selectively activate individual Ras effector pathways ,22. ActiConsistent with our previous data we found that activation of Ras (H-RasV12) led to S727 phosphorylation, but not Y705 phosphorylation of STAT3 . InteresIn addition to phosphorylation at S727 of STAT3, these Ras-transformed cells expressed phosphorylated forms of ERK, S6K, and AKT . To examTo confirm that the Raf-MEK-ERK pathway is engaged by activated Ras to transduce phosphorylation of STAT3 on S727, we titrated the dose of the MEK-ERK pathway inhibitor, PD0325901 [Phosphorylation of STAT3 on S727 in Ras-transformed MEFs and its inhibition by treatment with MEK inhibitors provided strong evidence for a role for Ras-activated MAPK in STAT3 S727 phosphorylation. To extend these results from experimental murine cells to human tumor cells, we examined the STAT3 S727 phosphorylation and its inhibition by PD0325901 in the human acute myelocytic leukemia cell line, THP-1, which express mutant N-Ras . Extract-/- MEFs were engineered to express a mutant form of STAT3 exclusively localized to mitochondria (MTS-STAT3), they formed significantly (p<0.05) more colonies than RasV12/35S transduced, STAT3-/- MEFs . Introducing this mutation into STAT3The ability of mitochondrially-restricted MTS-STAT3 and MTS-STAT3-S/D to support modest anchorage independent growth of H-RasV12/35S-expressing cells prompted us to examine the role of total STAT3. STAT3-null cells were reconstituted with STAT3 and transformed with H-RasV12 and the S35 or 40C effector domain mutants. As observed previously [To probe the role of S727 phosphorylation in Ras transformation, we also assessed anchorage independent growth of cells expressing STAT3-S727A phospho-mutant and STAT3-S727D phospho-mimetic constructs. H-RasV12 was unable to transform cells lacking STAT3 or expressing STAT3-S727A . In contTaken together, the results shown in The data described above document that H-Ras activation of MEK-ERK signaling results in phosphorylation of STAT3 on S727, and that the expression of a mitochondrially-restricted phospho-mimetic STAT3 supports Ras transformation. However, these data do not address directly the requirement of mitochondrial S727 phosphorylation in Ras-transformation. If STAT3 S727 phosphorylation is a critical MEK-ERK target, then inhibition of this pathway in H-RasV12 transformed cells should impede colony formation. Moreover, if this phosphorylation event is a major ERK pathway target, then MTS-STAT3S727D mutation might be expected to support cell transformation even in the presence of MEK-ERK inhibition. To test this hypothesis, we performed colony assays on a panel of H-RasV12 transformed cells expressing wild-type STAT3, no STAT3, MTS-STAT3 or MTS-STAT3 S/D in the presence of increasing titrating concentrations of the MEK inhibitor, PD0325901.+/+, H-RasV12:MTS-STAT3 and H-RasV12:MTS-STAT3S/D cells formed equivalent colony numbers when cultured in soft agar, while H-RasV12 cells lacking STAT3 formed almost no colonies  (PD0325901 treatment was sufficient to fully block ERK phosphorylation at the higher doses tested (0.5-1µM) and to substantially impair ERK phosphorylation at the lowest dose (50nM) . Vehiclecolonies . The hig(p<0.01) . Taken tActivating mutations in H-Ras occur in bladder, testicular and thyroid cancers, but are rare in other human cancers. The overall prevalence of H-Ras mutation in cancer is small compared to K- and N-Ras mutations . Mutant -/- MEFs stably expressing either N-RasG12V or K-RasG12V oncogenes. Ras expressing STAT3-/- cells were complemented with mitochondrially-targeted STAT3 or the mitochondrially-targeted S727D substitution mutant described earlier. These cells were plated in soft agar supplemented with either vehicle or with titrating doses of PD0325901. Consistent with data observed with H-Ras, transformation by K-  increase in colony forming capacity of K- or N-Ras transformed MEFs expressing mitochondrially-targeted S727D mutant STAT3 in the presence of 50 and 100 nM PD0325901 . While aSTAT3 is over-expressed and/or post-translationally modified in the majority of human cancers. The current dogma for the contribution of STAT3 to cancer biology is as a potent transcription factor driving expression of genes controlling numerous hallmark features of tumor cells . This paRas engages multiple cell signaling pathways to elicit cellular responses, and oncogenic Ras exploits these pathways for malignant transformation. The best characterized in the context of cancer are the Raf1-MEK-ERK, PI3K, and Ral pathways. Deregulated PI3K signaling  is one of the most commonly mutated tumor suppressors seen in cancer, particularly malignancies of the central nervous system and endometrium ; constit-/- MEFs [Previous data have shown that strong constitutive activation of each of these pathways in isolation can transform MEFs, but not as potently as activated H-Ras. However, like the Ras oncogenes themselves, activation of each individual pathway could not transform STAT3-/- MEFs . As confWhilst STAT3 S727 can be phosphorylated by many kinases, ERK is the most common kinase implicated in this modification . ERK2 anThe requirement of the ERK pathway for H-Ras transformation was also observed for K- and N-Ras transformation, as indicated by the sensitivity of anchorage independent cell growth to PD0325901. Inhibition of the MEK-ERK pathway blocked colony formation driven by all three activated Ras isoforms , 3 and 4MTS-STAT3-S727D cells expressing H-, K- and N-Ras were less sensitive to inhibition of anchorage independent growth following PD0325901 treatment; however they were only partially resistant to kinase inhibition. We observed that the lowest concentration of PD0325901 tested resulted in about a 2-fold reduction in the number of colonies in MTS-STAT3-S/D cells compared to vehicle treated cells. This PD0325901 concentration was not sufficient to completely abolish ERK phosphorylation, although it did reduce phosphorylation of wild type STAT3-S727 to undetectable levels. We therefore conclude that the additional reduction of clonogenicity was due to inhibition of other targets of ERK. Indeed, ERK has more than 200 documented targets , so it iMitochondrial functions of STAT3 include STAT3 interaction with and augmentation of the activity of the electron transport chain complexes I, II and V ,15. STAT


Abnormal vascular smooth muscle (VSM) growth is central in the pathophysiology of vascular disease yet fully effective therapies to curb this growth are lacking. Recent findings from our lab and others support growth control of VSM by adenosine monophosphate (AMP)-based approaches including the metabolic sensor AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA). Molecular crosstalk between AMPK and PKA has been previously suggested, yet the extent to which this occurs and its biological significance in VSM remain unclear. Considering their common AMP backbone and similar signaling characteristics, we hypothesized that crosstalk exists between AMPK and PKA in the regulation of VSM growth. Using rat primary VSM cells (VSMC), the AMPK agonist AICAR increased AMPK activity and phosphorylation of the catalytic Thr172 site on AMPK. Interestingly, AICAR also phosphorylated a suspected PKA-inhibitory Ser485 site on AMPK, and these cumulative events were reversed by the PKA inhibitor PKI suggesting possible PKA-mediated regulation of AMPK. AICAR also increased PKA activity in a reversible fashion. The cAMP stimulator forskolin increased PKA activity and completely ameliorated Ser/Thr protein phosphatase-2C activity, suggesting a potential mechanism of AMPK modulation by PKA since inhibition of PKA by PKI reduced AMPK activity. Functionally, AMPK inhibited serum-stimulated cell cycle progression and cellular proliferation; however, PKA failed to do so. Moreover, AMPK and PKA reduced PDGF-β-stimulated VSMC migration. Collectively, these results show that AMPK is capable of reducing VSM growth in both anti-proliferative and anti-migratory fashion. Furthermore, these data suggest that AMPK may be modulated by PKA and that positive feedback may exist between these two systems. These findings reveal a discrete nexus between AMPK and PKA in VSM and provide basis for metabolically-directed targets in reducing pathologic VSM growth. Aberrant vascular smooth muscle (VSM) growth is a pivotal mechanism underlying numerous pathologies including the evolution of atherosclerotic plaque, pressure- and/or flow-mediated hypertrophy, stent-induced hyperplasia, and iatrogenic restenosis after intervention Majesky, . The “de+2/calmodulin-dependent protein kinase  is a key metabolic sensor that responds to cellular energy depletion and stress by inhibiting many ATP-reducing processes, thereby promoting ATP synthetic processes within the cell  and its canonical downstream kinase PKA are critical signaling factors that exert a variety of functional effects in cardiac and vascular tissues. It is reported that Thr 197 of the catalytic subunit of PKA is phosphorylated in proportion to PKA activity  they play in the collaborative inhibition of VSM growth. Our hypothesis was that AMPK and PKA possess biochemical crosstalk and, in turn, may act cooperatively to inhibit VSMC proliferation and migration. Novel results show that AMPK controls VSMC proliferation and migration and that these events are regulated at least in part by PKA. These findings offer insight into a potential metabolic signaling network that may provide new therapeutic targets for the reduction of vascular growth disorders.Thr172 (1:1000), pAMPKSer485 (1:5000), total ACC (1:1000), pACCSer80 (1:1000), total PKA (1:1000), pPKAThr197 (1:1000), total VASP (1:1000), or pVASPSer157 (1:1000), and α-tubulin (1:1000) or beta-actin (1:1000). Antibodies were diluted in IRDye blocking buffer . For In-Cell Western analysis IRDye anti-rabbit secondary antibodies  were used for protein detection while Draq 5  and Sapphire 7  were used for DNA staining and protein normalization. Propidium iodide (PI) and RNAse for cell cycle analysis were purchased from Invitrogen.AICAR was purchased from Toronto Research Company  and Invitrogen , Compound C was purchased from Invitrogen, forskolin, and PKI were purchased from Enzo Life Sciences , and IBMX was purchased from Calbiochem . All primary antibodies were purchased from Abcam  or Cell Signaling  and were targeted against the following: total AMPK (1:1000), pAMPK2. Cells were serially split and used only through passage 6 to avoid onset of phenotypic switching  per assay from serially split clonal cells.This investigation was approved by the Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals . Primary VSMCs were isolated from thoracic aortae of male Sprague-Dawley rats  by collagenase and elastase digestion and characterized morphologically as previously described , and added to wells of microtiter plates containing 6,8-difluoro-4methyl-umbelliferyl phosphate substrate. When activated by phosphatases, DiFMU was generated in proportion to phosphatase activity, which was read at 450 nm. For specific (PP-2A and PP-2C) phosphatase activities, additional components were added to the buffer per manufacturer's directions that allowed for activity analyses of specific phosphatases within the lysate  containing select pharmacologic agents for 24 h. As previously stated  Tulis, .Following protocols previously described with minor modifications  analysis, migration, and cell cycle analysis to detect significance among groups. For each assay, “n” is calculated from a minimum of 3 experiments, each consisting of 3 individual wells  from serially-split, clonal parent cell generations. Data are expressed as mean ± standard error of the mean (SEM), with a p < 0.05 considered statistically significant.Data were analyzed using Excel 2011 (Microsoft) and Sigma Plot 11.2 . One-Way analysis of variance (ANOVA) and Tukey's Initial experiments focused on activation of AMPK in primary VSMCs. Treatment of cells with 1 mM of the AMPK agonist AICAR (60 min), a concentration previously documented to effectively induce AMPK activity in vascular cells without observable side-effects  Ser157 phosphorylation, a reported marker of active PKA  to the reaction buffer to specifically monitor PP-2A activity  was added to the reaction buffer to selectively monitor PP-2C  toward anti-migration was observed with FSK (10 uM) across the entire 18 h experiment, Two-Way ANOVA revealed that the net migration at 18 h was statistically significant compared to controls and was reversed with PKI  significantly reduced the number of migratory cells which was fully reversed with CC  and by an American Heart Association Pre-doctoral Fellowship (Joshua D. Stone). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute, the National Institutes of Health, or the American Heart Association.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Both screening hits inhibited PKD isoforms at about 100 nM and were ATP-competitive inhibitors. Analysis of several related kinases indicated that 1-NA-PP1 was highly selective for PKD as compared to IKK-16. SAR analysis showed that 1-NA-PP1 was considerably more potent and showed distinct substituent effects at the pyrazolopyrimidine core. 1-NA-PP1 was cell-active, and potently blocked prostate cancer cell proliferation by inducing G2/M arrest. It also potently blocked the migration and invasion of prostate cancer cells, demonstrating promising anticancer activities on multiple fronts. Overexpression of PKD1 or PKD3 almost completely reversed the growth arrest and the inhibition of tumor cell invasion caused by 1-NA-PP1, indicating that its anti-proliferative and anti-invasive activities were mediated through the inhibition of PKD. Interestingly, a 12-fold increase in sensitivity to 1-NA-PP1 could be achieved by engineering a gatekeeper mutation in the active site of PKD1, suggesting that 1-NA-PP1 could be paired with the analog-sensitive PKD1M659G for dissecting PKD-specific functions and signaling pathways in various biological systems.The emergence of protein kinase D (PKD) as a potential therapeutic target for several diseases including cancer has triggered the search for potent, selective, and cell-permeable small molecule inhibitors. In this study, we describe the identification,  Following the discovery of Gleevec, it was widely demonstrated that highly potent and specific small molecule inhibitors of kinases exhibit remarkable clinical efficacy and reduced toxicity PKD is a key signaling component of the diacylglycerol (DAG) signaling network. It is a primary target of DAG and a downstream effector of protein kinase C (PKC). There are several conserved structural motifs in PKD, such as a C1 domain that binds DAG and modulates PKD localization, and a PH domain that exerts an autoinhibitory function on the kinase domain. In intact cells, PKD is directly phosphorylated by DAG-responsive PKCs on the two conserved serine residues in the activation loop of the catalytic domain, which leads to its activation. PKD often exhibits sustained activity upon activation which is mainly maintained through autophosphorylation in vitro and in intact cells. Particularly, CRT0066101 was found to potently block the growth of pancreatic tumor xenografts in vivo in mice in vivo stability and general toxicity issues with the current set of known inhibitors. Therefore, it is imperative to continue the search for novel PKD inhibitory chemotypes that demonstrate attractive target selectivity, improved pharmacokinetic profiles, and greater in vivo efficacy.In the past several years, significant progress has been made in the development of potent and specific small molecule PKD inhibitors. CID755673 and analogs in vitro and in cells, and improved metabolic profiles in vitro and in vivo applications.We have previously identified both ATP-competitive and -noncompetitive PKD inhibitors that are distinct in structure to other reported inhibitors in vitro PKD1 inhibitory activity of these compounds was evaluated based on their ability to inhibit the recombinant PKD1 protein at 1 µM concentration in a radiometric PKD kinase assay. The percent PKD1 inhibition was calculated as the percent inhibition of the total PKD1 kinase activity in the absence of inhibitors (DMSO). Sixteen compounds were identified as primary hits  in the radiometric PKD1 assay , 133.4+/−3.6 nM (n = 3), 109.4+/−6.8 nM (n = 3), respectively, while IKK-16 similarly inhibited the PKD isoforms with an IC50 of 153.9+/−7.7 nM (n = 2) for PKD1, 115.0+/−7.1 nM (n = 2) for PKD2, and 99.7+/−3.0 nM (n = 2) for PKD3. These results indicate that both 1-NA-PP1 and IKK 16 are potent pan-PKD inhibitors.The To gain a better understanding of the mode of action for 1-NA-PP1 and IKK-16, we examined the effects of increasing concentrations of ATP on PKD1 inhibition. Lineweaver-Burk plots were generated by plotting the reciprocal of reaction velocities (1/v) against the reciprocal of ATP concentrations (1/[ATP]) at different compound concentrations. The points were fitted by linear regression. As shown Next, we determined the specificity of 1-NA-PP1 and IKK-16 for PKD by examining their activity for several functionally or structurally related kinases, including PKCα, PKCδ and CAMKIIα. No significant inhibitory activities for PKC isoforms were detected at 0.1, 1 and 10 µM concentrations of 1-NA-PP1, in contrast to IKK-16 which showed a concentration-dependent inhibition for both PKCα and PKCδ and >50% inhibition at 10 µM concentration (50 of 138.7±33.2 nM (n = 3) (http://kir.fccc.edu/) as described (Table S1) Further insights on the specificity of 1-NA-PP1 could be gained by evaluating the kinome scan data on 1-naphthylmethyl PP1 (1-NM-PP1), where 1-NM-PP1, along with 178 known kinase inhibitors, was profiled against a panel of 300 recombinant human protein kinases at a single concentration  (n = 3) Fig. 3D,d]pyrimidine core structure and determine the biological response to these modifications, as well as to potentially identify more PKD subtype selective analogs. We considered 4 types of variations, as shown in The goal of our synthetic studies was to modify the substituents on the 1-NA-PP1 pyrazolopyrimidine 456N-iodosuccinimide 2B(OH)2 led to the chlorinated derivative, and the chlorine group was reduced by a catalytic hydrogen transfer process to give product 1 with R4 = H (File S1.Vilsmeier–Haack reaction of barbituric acid in vitro radiometric kinase assay was used to screen an 80 compound library for PKD1 inhibitory activity at 1 µM concentration. 1.2 µM of a HDAC5 peptide 32P] ATP (Perkin Elmer Life Sciences), 25 µM ATP, 50 ng purified recombinant PKD1 in 50 µL kinase buffer containing 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2 and 10 mM β-mercaptoethanol. The reaction was incubated at 30°C for 10 minutes and 25 µL of the reaction was spotted on Whatman P81 filter paper. The filter paper was washed 3 times in 0.5% phosphoric acid, air dried and counted using Beckman LS6500 multipurpose scintillation counter. Percent PKD1 inhibition was graphed using GraphPad Prism software 5.0.An 32P]ATP, 20 µM ATP, 50 ng of purified PKCα or PKCδ and 5 µg of myelin basic protein 4–14, 0.25 mg/mL bovine serum albumin, 0.1 mg/mL phosphatidylcholine/phosphatidylserine (80/20%) (1 µM), 1 µM phorbol dibutyrate in 50 µL of kinase buffer containing 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2 and 10 mM β-mercaptoethanol. For the CAMK assay, 50 ng of CAMKIIα and 2 µg syntide-2 substrate in 50 µL kinase buffer were incubated with 0.1 mM MgCl2, 1 µCi of [γ-32P] ATP, 70 µM ATP. 0.5 mM CaCl2 and 30 ng/µL calmodulin were preincubated for 15 min on ice and then added in the kinase reaction. The reactions were incubated at 30°C for 10 min and 25 µL of the reaction was spotted on Whatman P81 filter paper. The filter paper was washed 3 times in 0.5% phosphoric acid, air dried and counted using Beckman LS6500 multipurpose scintillation counter.The PKC kinase assay was carried out by co-incubating 1 µCi [γ-2 at 37°C. Western blot analysis was carried out as previously reported The prostate cancer cell lines  were obtained from the American Type Culture Collection (ATCC). LNCaP and DU145 prostate cancer cells were maintained in RPMI 1640, while PC3 cells were maintained in Ham's F-12 medium, supplemented with 10% fetal bovine serum (FBS) and 1000 units/L penicillin, and 1 mg/mL streptomycin in 5% COPC3 cells were seeded into 96-well plates (3000 cells/well) and allowed to attach overnight. Cells were then incubated in media containing 0.7–100 µM inhibitors for 72 h. 3--2,5-diphenyltetrazolium bromide methyl thiazolyl tetrazolium (MTT) solution was prepared at 2 mg/mL concentration in PBS, sterilized by filtering through a 0.2 µm filter, and wrapped in foil to protect from light. 50 µL MTT solution was added to each well and incubated for 4 h at 37°C. Then, media was removed and 200 µL DMSO was added to each well. The plate was mix by shaking for 5 min and the optical density was determined at 570 nm.Proliferation of PC3 cells was measured by counting the number of viable cells upon trypan blue staining as previously described PC3 or DU145 cells were grown to confluence in 6-well plates. Migration was initiated by scraping the monolayer with a pipette tip, creating a “wound.” The indicated concentration of compound was added to the media, and the wound was imaged immediately under an inverted phase-contrast microscope with 10× objective. After 24 h, a final image was taken. The wound gap was measured, and % wound healing was calculated. The average % wound healing was determined based on at least 6 measurements of the wound gap.4 cells/ml) in RPMI containing 0.1% fetal bovine serum (FBS) were seeded into the top chamber of BioCoat control inserts (pore size 8 µm) or BioCoat Matrigel invasion inserts with Matrigel-coated filters (BD Pharmingen). To stimulate invasion, media in the lower chamber of the insert contained 20% FBS. Inhibitors were added at 30 µM concentration to both the upper and lower chambers, and cells were incubated for 22 h. After incubation, noninvasive cells were removed using a cotton swab, and invasive cells were fixed in 100% methanol and stained with 0.4% hematoxylin. After staining, cells were counted under a microscope (200× magnification). The percentage invasion was determined by cell counts in 5 fields of the number of cells that invaded the Matrigel matrix relative to the number of cells that migrated through the control insert.DU145 cells  at 100 MOI. After 24 h, cells were replated in migration chambers and incubated with vehicle or 30 µM 1-NA-PP1 for 20 h. The number of cells that migrated was determined by cell counts in 6 fields. Percentage migration was calculated as the percent of the Adv-null treated with vehicle DMSO (set to 100%). The experiment was repeated three times and data from a representative experiment are shown.(TIF)Click here for additional data file.File S1The Synthesis of New Pyrazolopyrimidine Inhibitors of Protein Kinase D. Experimental details and spectroscopic data for 1-NA-PP1 analogs.(DOCX)Click here for additional data file.Table S1http://kir.fccc.edu/). The kinase activity was determined using a radiometric HotSpot assay which directly measures kinase catalytic activity toward a specific substrate.The specificity of 1-NM-PP1 in a kinome scan. The activity of 1-NM-PP1, along with 178 known kinase inhibitors, was profiled against a panel of 300 recombinant human protein kinases at a concentration of 0.5 µM in the presence of 10 µM ATP. Data were extracted at a cut-off of <50% residual kinase activity from the Kinase Inhibitor Resource (KIR) online tool ((DOCX)Click here for additional data file.


TP63 implicates ΔNp63 isoforms in preservation of replicative capacity and cellular lifespan within adult stem cells. ΔNp63α is also an oncogene and survival factor that mediates therapeutic resistance in squamous carcinomas. These diverse activities are the result of genetic and functional interactions between TP63 and an array of morphogenic and morphostatic signals that govern tissue and tumor stasis, mitotic polarity, and cell fate; however the cellular signals that account for specific functions of TP63 are incompletely understood. To address this we sought to identify signaling pathways that regulate expression, stability or activity of ΔNp63α. An siRNA-based screen of the human kinome identified the Type 1 TGFβ receptor, ALK5, as the kinase required for phosphorylation of ΔNp63α at Serine 66/68 (S66/68). This activity is TGFβ-dependent and sensitive to either ALK5-directed siRNA or the ALK5 kinase inhibitor A83-01. Mechanistic studies support a model in which ALK5 is proteolytically cleaved at the internal juxtamembrane region resulting in the translocation of the C-terminal ALK5-intracellular kinase domain (ALK5IKD). In this study, we demonstrate that ALK5-mediated phosphorylation of ΔNp63α is required for the anti-clonogenic effects of TGFΒ and ectopic expression of ALK5IKD mimics these effects. Finally, we present evidence that ultraviolet irradiation-mediated phosphorylation of ΔNp63α is sensitive to ALK5 inhibitors. These findings identify a non-canonical TGFβ-signaling pathway that mediates the anti-clonogenic effects of TGFβ and the effects of cellular stress via ΔNp63α phosphorylation.Genetic analysis of  TP63 is a member of the p53 family of transcriptional regulators 4A/ARF locus http://cbioportal.org) ΔNp63α has been shown to play important roles in cancer initiation and progression suggesting that pharmacologic strategies that disrupt the activity of ΔNp63α have the potential for therapeutic benefit. ΔNp63α is an oncogene that suppresses the activity of the InkTGFβ is a highly pleiotropic cytokine that governs diverse aspects of cell biology including cell cycle progression, senescence, differentiation and apoptosis IKD. This translocation results in the phosphorylation of ΔNp63α, which is required for the anti-clonogenic activities of TGFβ. Consistent with this model, ectopic expression of ALK5IKD is sufficient to phosphorylate ΔNp63α and recapitulates the anti-clonogenic and anti-proliferative effects of TGFβ. Finally, we show that phosphorylation of ΔNp63α at S66/68 in response to ultraviolet (UV) irradiation is mediated by ALK5 indicating that the ALK5/ΔNp63α signaling pathway may mediate aspects of the cellular response to stress. Together these studies identify ΔNp63α as a target of a novel non-canonical ALK5 signaling pathway that mediates cellular responses to TGFβ.The involvement of ΔNp63α in cancer initiation and progression suggests that signaling pathways that govern ΔNp63α activity or stability may be targeted for therapeutic benefit. Multiple phosphorylation sites have been identified within ΔNp63α and other TP63 isoforms 2 and lysates were harvested 1 hr later. For stability experiments, cells were plated at 30% confluency and treated with 20 ug/ml cycloheximide (CHX).Immortalized Mammary Epithelial Cells (IMECs) The expression construct pcDNA3.1-ΔNp63α-WT was generated as previously described 5′-CGATGCTCTCGCTCCAGCACCCGCCATCCCCTCC-3′, and antisense primer 5′-GGAGGGGATGGCGGGTGCTGGAGCGAGAGCATCG-3′. TGFβRI expression constructs- pRK5 TGFβRI wt Flag, pRK5 TGFβRI (T202D) Flag, and pCMV5B- TGFβRI (K232R) were purchased from Addgene (Cambridge MA) and were used as previously described IKD was generated by PCR amplification of the kinase domain region of the pRK5 TGFβR1-wt-Flag plasmid using the following primers designed to incorporate Nhe1 and HindIII overhangs; sense primer, 5′-GATCGCTAGCATGATTGTGCTACAAGAAAGCATC-3′ and the antisense primer 5′-GATCAAGCTTCTTGTCGTCGTCGTCCTTGTAGTC-3′. Fragments generated from PCR were then cloned into the pcDNA3.1 vector. All plasmid transfections were carried out using Lipofectamine2000  according to manufacturers protocol.sense primer The Silencer Select Human Kinase siRNA Library  from Ambion was used to perform a genome wide kinome screen. The assay was performed in a 96 well format. Three different siRNAs for a single gene were co-transfected per well at a concentration of 0.5 pmoles using siPORT NeoFX Transfection Agent (Cat#AM4511). Forty-eight hours post siRNA transfection; cells were infected with ΔNp63α-WT adenovirus resulting in 90% infection efficiency. Quantitative immunofluorescence assays were then performed using the anti-phospho-p63 (Ser160/162) antibody. Immunofluorescence assays were performed as described below. Fluorescent values were obtained using the Molecular Devices Gemini XS Fluorescent Microplate Reader . Raw fluorescence values were normalized against total cell count, three different negative controls and a positive control. To obtain total cell count, cells after IF readings were stained with crystal violet. After a rigorous wash with distilled water, stain from the cells were eluted in a 20% methanol and 10% acetic acid solution, values were read at 595 nm using the Molecular Devices Thermo Max Microplate reader at 595 nm.Silencer select pre validated small-interfering RNA against TGFβRI and TGFβRII  were purchased from Ambion. Ambion’s siPORT NeoFX Transfection Agent was used in all siRNA transfections according to the manufacturer’s protocol. Ambion Silencer®Negative Control #2 siRNA (Cat#AM4613) was used as control for all siRNA transfections. Forty eight hours post siRNA transfection, cells were transfected with GFP, ΔNp63α-WT or ΔNp63α-AA mutant plasmids, and harvested 24 hrs after plasmid addition in 1XSDS lysis buffer for further analysis by immunoblotting.Cells were lysed in 1XSDS lysis buffer with β-mercaptoethanol and were resolved by 10% SDS- PAGE. Antibodies used for western and Immunofluorescence were anti-p63 Clone 4A4  for total P63, anti-phospho-p63 (Ser160/162) , anti-phospho-Smad2 (Ser465/467) , anti-Smad2 , anti-TGFβ RII (L-21) , anti-TGFβ RI (V-22) antibody from  and anti-β-Actin antibody from . All primary antibodies were detected with their respective secondary HRP- conjugated antibodies using Millipore chemiluminescence. Primary antibodies for Immunofluorescence assays were detected with either anti-mouse-Alexa Fluor 488 (Invitrogen#A11029) or anti-rabbit-Alexa Fluor 555 (Invitrogen#A21429).IMEC cells were plated at 1000 cells per well in a 6-well dish. Cells were fed every other day with TGFβ1± A83-01 for 10–14 days. Cells transfected with p63 constructs were selected for in the presence of G418. Colonies were fixed in ice-cold 80% methanol and stained with 0.5% crystal violet.Recombinant ALK5 kinase was produced by transfecting H1299 cells with an expression vector programmed to produce C-terminally flag-tagged ALK5 Receptor. Immunoprecipitation of the Flag-tagged ALK5 kinase was performed using Peirce Classic IP kit and monoclonal ANTI-FLAG M2 antibody(Sigma Cat # F1804). Recombinant ALK5 was eluted from beads by incubation with 100 ug/ml of soluble flag peptide (Sigma Cat # F3290) in 10 mMTris, 150 mM NaCl2, pH7.4 buffer. GST-ΔNp63α was produced via IPTG induced expression in Y1090 cells. Bacteria were collected by centrifugation and lysed by sonication in the presence of a cocktail of protease inhibitors. Lysates were cleared by centrifugation and GST-ΔNp63α was enriched on glutathione-sepaharose beads. For the kinase reaction either 1 or 5 µL of soluble ALK5 was added to fixed amounts of GST-ΔNp63α bound to beads in 1X kinase buffer  with 10 uM ATP. Reactions were incubated at 30°C for 30 min. Reactions were stopped by the addition of 2 × SDS Sample Buffer and western blots were done as described previously.high and ALDHlow populations in IMEC cells were identified using the Aldefluor assay kit (Stem Cell Technologies). IMEC cells were plated at 25% confluence and treated with Vehicle, TGFβ1 or A83-01 for 24 and 48 hours. Cells were then harvested and stained with ALDEFLOUR reagent as per the manufacturers protocol.ALDHCells were collected by trypsinization and gentle centrifugation before being re-suspended in ice cold PBS. An equal volume of ice cold 80% methanol was added with gentle vortexing and cells were fixed on ice for 30 minutes. Fixed cells were collected by centrifugation and re-suspended in PBS supplemented with 0.5 µg/ml of RNAse A. After 45 minutes at 37°C, cells were stained with propidium iodide and samples were analyzed on a BD FACScan instrument. Nuclear and cytoplasmic extracts were prepared using the EpiQuikTM Nuclear Extraction Kit I according to the manufacturer’s protocol.To identify the signaling pathways governing the diverse activities of ΔNp63α a siRNA-based screen of the human kinome was carried out in H1299 lung adenocarcinoma cells . H1299 cΔNp63α phosphorylation suggested that TGFβ-signaling governs ΔNp63α phosphorylation. We therefore sought to determine if TGFβ stimulation was sufficient to enhance ΔNp63α phosphorylation. For these studies, an hTERT-immortalized mammary epithelial cell (IMEC) line was used due to its robust expression of ΔNp63α The finding that ALK5 is necessary and sufficient for Previous studies have shown that ΔNp63α is phosphorylated at S66/68 in response to ultraviolet (UV) irradiation IKD) would preferentially localize in the nucleus. Western analysis of nuclear and cytoplasmic extracts from IMECs indicated that the 34-kDa ALK5 C-terminal fragment was selectively localized to the nucleus  were derived via retroviral transduction of the catalytic subunit of human telomerase (hTERT) into primary human mammary epithelia and clones were selected for their ability to bypass replicative senescence pulation . This enfraction  The redude novo protein synthesis selectively repressed ΔNp63α protein levels indicating that TGFΒ may destabilize ΔNp63α  mediates TGFβ-stimulated phosphorylation of ΔNp63α and the anti-clonogenic effects of TGFβ. To address this, an expression vector was developed to produce ALK5IKD and transfected into IMECs. Consistent with the proposed model, ectopic ALK5IKD accumulates in the nucleus  in a manner that is independent of p53 Previous studies have shown that ΔNp63α is phosphorylated at S66/68 in response to UV irradiation IKD. This implies that inhibition of this protease may result in disruption of ΔNp63α phosphorylation. Further studies will be necessary to identify this protease, however a recent study has shown that the TNF-α Converting Enzyme (TACE) is able to mediate proteolysis of ALK5 and that TACE activity is required for accumulation of ALK5 in the nucleus IKD and phosphorylation of ΔNp63α is unknown because in that study TACE was shown to target the ALK5 ectodomain, which is predicted to produce a fragment greater than 34 kDa. This also raises questions regarding the mechanism(s) by which an ALK5 fragment that retains the transmembrane domain might translocate to the nucleus. Presently the protease(s) that account for generation of ALK5IKD remain unknown and their identification represents an important step in testing our model for ALK5-mediated ΔNp63α phosphorylation and also in identifying pharmacologically accessible pathway components.Finally, recent studies have indicated unacceptable levels of cardiac and inflammatory toxicity associated with selective ALK5 kinase inhibitors and it is likely that these adverse effects will limit their development and clinical utility Figure S1600 readings for crystal violet staining. Primary hits progressed to the secondary screen and kinases for which all three siRNAs repressed phosphorylation of DNp63a were selected.Schematic representation of the siRNA-based screen of the human kinome. The table at the bottom lists the top 14 hits in the screen showing the data produced from fluorescence plate readings of the IF and subsequent Abs(PDF)Click here for additional data file.Figure S2The molecular determinants of DNp63a phosphorylation by ALK5 are distinct from those necessary for SMAD2/3 phosphorylation. H1299 cells were co-transfected with wild-type ALK5, the T202D mutant which had previously been shown to constitutively activate TGFb signaling, the K232R mutant which had been previously shown to inhibit TGFb signaling and an ALK5-GFP fusion. At 24 hours post transfection protein was harvested and analyzed by western blot. Comparison of the P-p63 and P-SMAD2/3 signals indicated that T202D was unable to phosphorylate DNp63a but was able to phosphorylate SMAD2/3 (Lane 3). Remarkably the K232R mutant was able to phosphorylate DNp63a but not SMAD2/3 (Lane 4). These results suggest that the molecular mechanisms by which ALK5 phosphorylates DNp63a are distinct from those that phosphorylate SMAD2/3.(PDF)Click here for additional data file.Figure S3Effects of three TGFbR2-directed siRNAs on expression of TGFbR2 and SMAD2 phosphorylation. H1299 cells were transfected with the indicated siRNAs and TGFbR2 and phospho-SMAD2 were analyzed to confirm the efficacy of the siRNA. SiRNA-C was used in the experiment shown in (PDF)Click here for additional data file.Figure S4Schematic representation of signal transduction pathways known to be downstream of the TGFb receptor complex. Kinases associated with these pathways are shown in pink and the phospho-p63 vs total p63 IF score is shown as is the relationship of that score to the mean.(PDF)Click here for additional data file.Figure S5Transfection of H1299 cells with ALK5-directed siRNA ablates immunoflourescent detection of ALK5. This data confirms the specificity of ALK5 detection presented in (PDF)Click here for additional data file.Figure S6Low from ALDHHigh fractions.Repesentative Aldefluor data from which (PDF)Click here for additional data file.Figure S7A. The anticlonogenic effects of TGFb on IMECs are partially rescued by the phospho-ablative DNp63a-AA mutant. Colony forming assay shown is representative of multiple experiments and corresponds to the graphical data displayed in B. Ectopic expression of ALK5IKD is anti-clonogenic in IMEC cells. IMECs were tranfected with pcDNA3.1-GFP and pcDNA3.1-ALK5IKD and selected in 200 µg/ml G418 for 12 days. Colonies were fixed in alcohol and stained with crystal violet. Graph at right represents a quantification of the colony formation in which colonies from three random 1 cm × 1 cm squares were analyzed using ImageJ software. Bars represent the average of three counts and error bars represent the standard error of the mean.The anti-clonogenic effects of TGFb are phenocopied by ectopic ALK5IKD. (PDF)Click here for additional data file.


Humulus lupulus L.). Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH●) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.Xanthohumol is the principal prenylated flavonoid in the hop plant ( Humulus lupulus L.) have long been used in the brewing industry as a preservative and flavoring agent to add bitterness and aroma to beer i mobilization was measured using a fluorescence spectrophotometer  with excitation wavelengths of 340 and 380 nm, and an emission wavelength of 500 nm ,(see ).P < 0.05 was considered statistically significant.Experimental results are expressed as the means ± S.E.M. and are accompanied by the number of observations. Experiments were assessed by an analysis of variance (ANOVA). If this analysis indicated significant differences among group means, then each group was compared using the Newman-Keuls method. μM) exhibited very potent activity in inhibiting platelet aggregation and the ATP-release reaction stimulated by 1 μg/mL collagen (μM AA at higher concentrations (3 and 10 μM)  (50) value of xanthohumol for platelet aggregation induced by collagen was approximately 1.5 μM  . Howeverd 10 μM) . The 50%y 1.5 μM . The soly 1.5 μM . In subsμg/mL collagen evoked a marked increase in relative Ca2+ mobilization, and this increase was markedly inhibited in the presence of xanthohumol . As shown in αIIbβ3 disintegrin which inhibits platelet aggregation by directly interfering with fibrinogen binding to the αIIbβ3 integrin i and finally inhibition of platelet aggregation. Platelet aggregation plays important pathophysiological roles in a variety of thromboembolic disorders. Therefore, the novel role of xanthohumol in antiplatelet activation may represent high therapeutic potential for treating or preventing such diseases, in addition to it originally being considered as a chemopreventive agent. In conclusion, the most important findings of this study demonstrate for the first time that the very potent antiplatelet activity of xanthohumol may initially inhibit the PI3-kinase/Akt, p38 MAPK and PLC


Interference screw is a popular fixation device used to rigidly fix bone-patellar tendon-bone (B-PT-B) graft both in femoral and tibial tunnels in anterior cruciate ligament (ACL) reconstruction. Parallel placement of screw is difficult in transtibially drilled femoral tunnel but always desired as it affects pullout strength of the graft. Commonly, interference screw into the femoral tunnel is inserted through the anteromedial (AM) or accessory AM portal. These portals are not-in-line with the transtibially drilled femoral tunnel. Furthermore, these portals increase the divergence of the interference screw in the femoral tunnel. We hypothesized that interference screw placement through patellar tendon (PT) portal (through donor defect) in transtibially drilled femoral tunnel can be less divergent. We report the prospective randomized study to investigate the difference of divergence of interference screw placed through PT portal and AM portal and its clinical relevance.Forty-one patients underwent femoral tunnel B-PT-B graft fixation through AM portal (group 1) and other 41 (group 2) through PT portal. Femoral tunnel-interference screw divergence was measured on postoperative digital lateral X-rays. Ha’s method was used to grade divergence. The clinical outcome was assessed by postoperative intervention knee documentation committee grading (IKDC) and Lysholm score at 2 years followup.P<0.0001). In AM portal group, 82.9% patients had divergence in either grade 3 or 4 category, whereas in PT portal group, 82.9% patients were in grade 1 or 2 category (P<0.0001). Mean Lysholm score were 92.8 and 94.5 at two-year follow-up in both groups which were statistically not significant. The International knee documentation committee grades of patients in both groups were similar and had no statistical significance.Mean tunnel-screw divergence in sagittal plane through AM portal was 13.38° (95% CI: 12.34-14.41) and through PT portal was 7.20° (95% CI: 6.25-8.16) (Femoral interference screw placement through the PT portal leads to significantly less screw divergence as compared with screw placement through the AM portal. However, this difference in divergence is not reflected in clinical outcome. Bone-patellar tendon-bone (B-PT-B) graft is a reliable method for anterior cruciate ligament (ACL) reconstruction.2021The primary aim of this prospective randomized comparative study was to investigate whether an interference screw placed through the PT portal shows less divergence as compared with a screw placed through the AM portal. The secondary aim was to investigate the difference in the clinical outcome between the two modes of screw placement.et al.Eighty two consecutive patients who underwent ACL reconstruction by between January 2006 and January 2010 by B-PT-B graft were included in this study. The block randomization method was used with allocation ratio of 1:1. After proper informed consent, ACL reconstruction by B-PT-B graft for unstable ACL-deficient knee was performed with standard technique. Of 82, 41 patients underwent graft fixation in femoral tunnel by AM portal (group 1), while other 41 patients through PT portal (group 2) through donor defect. All ACL reconstructions were performed by a single surgeon. Postoperatively, tunnel-screw divergence was measured (degree) in sagittal plane on a digital lateral X-ray of the knee with the help of software embedded in GE-PACS . Angle was measured in degrees between the longitudinal axis of tunnel and interference screw by an experienced radiologist on two separate occasions. The radiologist was blinded for the technique used . Screw divergence was classified into four grades, as described by Kwon Ick Ha P value <0.05 was considered as significant.Data were entered in statistical package of social sciences 13.0  and analyzed. The difference in angle measured was analyzed for any statistical significance. Intraobserver variation was analyzed at two different occasions. Mean angle of divergence of both groups was calculated. Means of two groups were compared using independent T test. Association between grades of divergence between two groups was done using chi square test. After spinal or general anesthesia, diagnostic arthroscopy was performed under tourniquet through standard AM and anterolateral (AL) portal. All patients underwent single incision ACL reconstruction using B-PT-B graft. A 9 mm wide × 25 mm long bone plug was harvested from patella and tibial tuberosity by standard technique. After performing diagnostic arthroscopy, the intercondylar notch and lateral wall were prepared. The tibial tunnel was prepared by serial drilling from 6 to 9 mm cannulated reamers over a guide wire exiting 6 mm anterior to the anterior fibers of posterior cruciate ligament, just medial to medial tibial spine in the posterior footprint of torn ACL fibers. With the knee at 80 to 90° of flexion, femoral tunnel was drilled over a guide wire which was kept 7 mm anterior to posterior edge of lateral femoral condyle at 10:30 o’clock in right knee and 1:30 o’clock in left knee using a femoral offset of 6 mm. Bicortical drilling was done with 4-mm cannulated reamer. Then, 30 mm long femoral tunnel was drilled serially with 7- to 9-mm cannulated reamers. Then, femoral tunnel was notched supero-laterally through the portal, which was decided for passing the interference screw. B-PT-B graft was passed into the femoral tunnel through the tibial tunnel over a beath pin which exited from anterolateral aspect of thigh. The cancellous portion of graft was facing anterolaterally. Knee was flexed to 110° and a guide wire advanced into the notched portion of tunnel to minimize divergence through the selected portal, AM or PT portal, through the donor defect [Figures P<0.0001) using independent sample test. Grade of divergence in both groups are illustrated in P<0.0001). Two-year follow-up of patients in both groups had average postoperative Lysholm score of 92.8 and 94.5, respectively. The differences between Lysholm score were statistically not significant, indicating that there was no difference in clinical outcome of the two groups. The IKDC grades of patients in both groups are mentioned in There were 71 males and 11 females. Forty-one patients underwent femoral tunnel fixation by titanium interference screw through AM portal (group 1) and other 41 patients through PT portal (group 2). The intraobserver variation of tunnel-screw angle was statistically not significant. The mean tunnel-screw divergence in sagittal plane through AM portal was 13.38° (95% CI: 12.34-14.41) and through PT portal through donor defect was 7.20° (95% CI: 6.25-8.16), which is illustrated in et al. reported that pullout strength decreases with increasing divergence  in his study on 20 porcine knees.et al. reported that divergence more than 30° leads to loss of fixation and significantly decreased pullout strength.et al. in a bovine model reported that divergence less than 15° did not affect fixation characteristics.2021et al. assessed the fixation strength of hamstring graft in femoral tunnel and effect of screw divergence in coronal place.Interference screw fixation of B-PT-B graft in femoral and tibial tunnels still remains a popular method of fixation.et al. described modified Paulos technique to minimize the divergence.Several techniques have been described by various authors to minimize the divergence of interference screw in femoral tunnel. Brodie Recently, a newer technique has been developed by Chan and Wang where they also have used the tibial tunnel for femoral screw insertion while keeping the graft in AM portal which prevents any possibility of graft laceration while screw insertion.To our knowledge, there are no published studies in English literature comparing two methods of interference screw placement in femoral tunnel and divergence. Our results prove that acceptable levels of divergence can be achieved by using PT portal through donor defect with correct knee positioning. The mean divergence remained 7.2° which is within normal limits of divergence and does not affect pullout strength of graft. The technique does not require the making of an additional portal  or special instrument. The fear of damaging infrapatellar fat pad by traversing interference screw seems to be exaggerated. While inserting the interference screw, no undue pressure should be applied as it can force the fat pad into the joint and can obscure the vision temporarily. Gentle screwing action is sufficient to negotiate the interference screw through fat pad. Divergence within 30° is said to be acceptable as far as clinical result is concerned. However, the more in line PT portal through donor defect is much effective in reducing the divergence and keeps it to the minimum level. Also, to our knowledge, there are no published studies which compared the divergence of interference screw by different methods with respect to the clinical result in patients. In our study, the average postoperative Lysholm score were similar in both groups 97.14 and 98.02 , indicating that though there were radiological differences between both groups; there was no difference in clinical outcome. The IKDC objective grades were also similar in both groups, indicating that though there is angle difference between the two groups, it may not lead to any clinical difference as long as the divergence angle is within acceptable limits.Interference screw divergence in the femoral tunnel can also lead to bending of intrafemoral tunnel portion of guide wire. If the guide wire bends, it becomes difficult to pull out the guide wire. In order to accomplish that, sometimes it might be necessary to remove the interference screw and reinsert, which weakens the interference fit. So, using an appropriate portal (PT) can minimize the guide wire bending as it avoids excess divergence. Rarely, divergence can also lead to cortical penetration causing blow out or graft fracture in femoral tunnel.The limitation of this study is that only sagittal plane divergence has been evaluated. It is difficult to accurately outline the femoral tunnel in the coronal plane in AP view of X-ray. However, a randomized study with larger sample size is desired to study the relation between multiplanar divergence (using computed tomography scan) and clinical outcome using these two portals.Femoral interference screw placement through the PT defect provides significantly less screw divergence as compared with screw placement through the AM portal. However, this difference in divergence is not reflected in the clinical outcome.


Lepidium sativum is widely used in folk medicine for treatment of hyperactive airways disorders, such as asthma, bronchitis and cough. The crude extract of Lepidium sativum (Ls.Cr) inhibited carbachol  and K+ (80 mM-) induced contractions in a pattern similar to that of dicyclomine. Ls.Cr at 0.03 mg/mL produced a rightward parallel shift of CCh curves, followed by nonparallel shift at higher concentration (0.1 mg/mL), suppressing maximum response, similar to that caused by dicyclomine. Pretreatment of tissues with Ls.Cr (0.1–0.3 mg/mL) shifted Ca++ concentration-response curves (CRCs) to right, as produced by verapamil. Ls.Cr at low concentrations (0.03–0.1 mg/mL) caused leftward shift of isoprenaline-induced inhibitory CRCs, like that caused by rolipram, a phosphodiesterase (PDE) inhibitor. These results indicate that bronchodilatory effect of Lepidium sativum is mediated through a combination of anticholinergic, Ca++ antagonist and PDE inhibitory pathways, which provides sound mechanistic background for its medicinal use in the overactive airways disorders. Most of the disorders of respiratory system results from the hyperactivity of airways. Asthma is a major congestive respiratory disorder, characterized by episodic wheezing, cough, and chest tightness, associated with airflow obstruction. The worldwide prevalence of asthma has been increasing particularly in children. According to the World Health Organization, it affects about 5–10% of adults and 10% of children globally .The conventional therapy for these congestive airways disorders particularly asthma is not always safe, efficacious and is beyond the access and/or affordability of large proportion of world population, who look for alternative therapeutic measures. Phytotherapy is the most popular alternative remedy, and a number of traditional systems of medicine are heavily based on the use of herbs as medicine , 4. At tLepidium sativum Linn. belongs to family Cruciferae (cabbage family) and is commonly known as “Common cress,” “Garden cress,” or “Halim.” The plant is called “Hab el Rashaad” or “Thufa” in Saudi Arabia and is a popular herbal plant grown in many regions of Saudi Arabia, such as Hijaz, AL-Qaseem, and the Eastern Province [Lepidium sativum seeds are thought to be an effective medicinal remedy to cure respiratory disorders, like bronchitis and asthma [Province , 8. In Ed asthma . The plad asthma . In Soutd asthma , 12.β-carotenes, ascorbic acid, linoleic acid, oleic acid, palmitic acid, stearic acid [Lepidium sativum is reported to exhibit antihypertensive [++ channels and phosphodiesterase (PDE) enzyme; thus, this study provides sound pharmacological rational for its effectiveness in the airways hyperactive disorders.The plant is known to contain imidazole, lepidine, semilepidinoside A and B , β-carotric acid , sinapicric acid . Lepidiurtensive , diuretirtensive , anti-inrtensive , antirhertensive , hypoglyrtensive , laxativrtensive , antidiartensive  propertirtensive  and bronrtensive  potentiaLepidium sativum were purchased from herbal store  in March, 2010. The plant was authenticated by Dr. Mohammed Yusuf, King Saud University, and the specimen has been preserved at the herbarium of the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia and also at Natural Product Research Unit, Department of Biological and Biomedical Sciences, Aga Khan University, Karachi with voucher no. Ls-SE-04-10-98. The Lepidium sativum seeds were soaked in 70% methanol for three days [Lepidium sativum (Ls.Cr), yielding approximatly 15%.Seeds of ree days  and thenAtropine sulphate, carbachol (CCh), dicyclomine, isoprenaline, verapamil, and rolipram were purchased from Sigma Chemicals Company, St. Louis, MO, USA. Guinea-pigs (500–550 g) of either sex and local breed were kept at the Animal House of the Aga Khan University, maintained at 23–25°C, and were given standard diet and tap water. Experiments were performed in compliance with the rulings of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council  and apprμM) and K+0020(80 mM) were used to induce sustained contractions, and relaxant activity of crude extract was assessed by adding in cumulative fashion. High K+ (>30 mM) is known to cause smooth muscle contractions through opening of voltage-dependent L-type Ca++ channels, thus allowing influx of extracellular Ca++ causing a contractile effect and the substance causing inhibition of high K+-induced contraction is considered as inhibitor of Ca++ influx [++ channel blockade (CCB) effect of test substance was confirmed via building Ca++-CRCs in the Ca++-free medium [Tracheal ring strips obtained from guinea-pigs sacrificed by cervical dislocation were mounted individually in 20 mL tissue bath containing Kreb's solution, at 37°C and aerated with carbogen. A tension of 1 g was applied to each of the tissues and equilibrated for 1 hr before the addition of any drug. Carbachol  with 95% confidence intervals (CIs) are provided. CRCs were analyzed by nonlinear regression using GraphPad program .The median effective concentrations (ECμM-) and K+ (80 mM-) induced contractions, Ls.Cr inhibited the CCh-induced contractions at lower concentration with EC50 value of 0.32 mg/mL , compared to its effect against K+ (80 mM) with EC50 value of 5.4 mg/mL (4.3–7.2) as shown in 50 values of 0.28 μM (0.20–0.40) and 4.6 μM (3.0–7.2), whereas verapamil was more potent against K+-induced contractions with EC50 value of 0.14 μM (0.10–0.20), when compared with CCh-induced contractions (2.7 μM (1.7–3.5)) as shown in μM-) induced contraction, with EC50 value of 0.006 μM (0.002–0.01), without any effect on K+ (80 mM-) induced contractions  also showed a similar pattern of shift (μM) produced a nonparallel rightward shift at both tested concentration with suppression of the maximum response (μM) caused rightward parallel shift without the suppression of the maximum contractile effect  produced rightward shift in the Ca++ curves  also caused similar leftward shift of the isoprenaline curves, as shown in Pretreatment of tissues with Ls.Cr at low concentrations (0.03–0.1 mg/mL) shifted the isoprenaline-induced inhibitory CRCs to the left , showingLepidium sativum in the hyperactive airways disorders, its aqueous-methanol extract was tested for the possible bronchodilatory effect. In guinea-pig tracheal preparations, plant extract inhibited the CCh- and high K+-induced contractions, being more potent against CCh. Dicylomine, a dual blocker of muscarinic receptors and Ca++ influx [++ antagonist [+-induced contractions than those induced by CCh, while atropine, a muscarinic receptor antagonist [++ channels. The presence of anticholinergic and CCB actions was further confirmed, respectively, through constructing the CCh and Ca++ CRCs in the presence of different concentrations of the plant extract. A parallel displacement of CCh curves without suppression of the maximum effect was observed at the lower concentration of Ls.Cr, a characteristic of a competitive or specific antagonist, like atropine [++ antagonists [++ curves to the right accompanied by the suppression of maximum response, similar to that caused by verapamil, confirming the Ca++ antagonistic effect.In view of the medicinal use of + influx , showed tagonist , was mortagonist , relaxedatropine , followeagonists . Dicyclo++ antagonist activity [Lepidium sativum also exhibits PDE enzyme inhibition component(s), isoprenaline inhibitory CRCs were constructed against CCh-induced contractions by pretreatment of tissues with Ls.Cr, as PDE inhibitors are known to potentiate the isoprenaline effect [Lepidium sativum was confirmed when the plant extract potentiated isoprenaline relaxant effect, by causing leftward shift of isoprenaline-induced inhibitory curves, similar to that caused by rolipram, a PDE4 inhibitor, predominant enzyme in airways [Lepidium sativum is mediated through combined anticholinergic, CCB, and PDE inhibitory pathways; thus, this study provides sound mechanistic basis for its application in airways hyperactivity disorders. The usefulness of anticholinergics and PDE inhibitors in asthma is well established [++ antagonists are also useful in bronchoconstriction [Lepidium sativum is perhaps meant by nature to offset the tachycardia, usually associated with anticholinergics or PDE inhibitors when used alone. This scenario strengthens the concept that natural remedies possess “effect enhancing and/or side-effects neutralizing” potential in addition to cost-effectiveness and offers merit in evidence-based studies [ We have experienced that plants with medicinal use in the overactive airways disorders usually possess PDE inhibitory effect, which usually coexists with Caactivity , 36, 37.e effect . The pre airways . These fablished , though ablished , 42. Inttriction  and are triction . The coe studies . Lepidium sativum offers a unique combination of bronchodilator activities , which may explain its medicinal use in the hyperactive airways disorders, such as cough and asthma. However, further in-depth studies are required to probe nature of chemicals constituents and molecular base of biological activities.In conclusion, these results show that


The GPCR-activated PI3Kγ is also a key enzyme downstream of the IgE high affinity receptor FcεRI. PKCβ-dependent phosphorylation of PI3Kγ on Ser582 is the ‘missing link’ that functions as a molecular switch to divert PI3Kγ from GPCR inputs. 2+- influx in mast cells. Phosphorylation of p110γ correlates with the release of the p84 PI3Kγ adapter subunit from the p84-p110γ complex. Ser582 phospho-mimicking mutants show increased p110γ activity and a reduced binding to the p84 adapter subunit. As functional p84-p110γ is key to GPCR-mediated p110γ signaling, this suggests that PKCβ-mediated p110γ phosphorylation disconnects PI3Kγ from its canonical inputs from trimeric G proteins, and enables p110γ to operate downstream of Ca2+ and PKCβ. Hydrogen deuterium exchange mass spectrometry shows that the p84 adaptor subunit interacts with the p110γ helical domain, and reveals an unexpected mechanism of PI3Kγ regulation. Our data show that the interaction of p110γ with its adapter subunit is vulnerable to phosphorylation, and outline a novel level of PI3K control.All class I phosphoinositide 3-kinases (PI3Ks) associate tightly with regulatory subunits through interactions that have been thought to be constitutive. PI3Kγ is key to the regulation of immune cell responses activated by G protein-coupled receptors (GPCRs). Remarkably we find that PKCβ phosphorylates Ser582 in the helical domain of the PI3Kγ catalytic subunit p110γ in response to clustering of the high-affinity IgE receptor (FcεRI) and/or store-operated Ca Phosphoinositide 3-kinases (PI3Ks) are involved in most essential cellular processes. Class I PI3Ks are heterodimers: class IA PI3Ks are made up of one of a group of regulatory p85-like subunits and one p110α, p110β, or p110δ catalytic p110 subunit, and are activated via binding of their p85 subunit to phosphorylated tyrosine receptors or their substrates. The only, class IB PI3K member, PI3Kγ, operates downstream of G protein-coupled receptors (GPCRs). Recent work suggested that PI3Kγ also operates downstream of IgE-antigen complexes in mast cell activation, but no mechanism was provided. We show that clustering of the high-affinity IgE receptor FcεRI triggers a massive calcium ion influx, which leads to PKCβ activation. In turn, PKCβ phosphorylates Ser582 of the PI3Kγ catalytic p110γ subunit's helical domain. Downstream of GPCRs, p110γ requires a p84 adapter to be functional. Phospho-mimicking mutations at Ser582 disrupt the p84-p110γ interaction, and cellular Ser582 phosphorylation correlates with the loss of p84 from p110γ. Thus our data suggest that PKCβ phosphorylates and activates p110γ downstream of calcium ion influx, while simultaneously disconnecting the phosphorylated p110γ from GPCR signaling. Exploration of the p84-p110γ interaction surface by hydrogen- deuterium exchange mass spectrometry confirmed that the p110γ helical domain forms the main p84-p110γ contact surface. Taken together, the results suggest an unprecedented mechanism of PI3Kγ regulation. P3] and consist of a p110 catalytic and a regulatory subunit. The class IA catalytic subunits, p110α, β, and δ, are constitutively bound to p85-related regulatory proteins that link them to the activation by protein tyrosine kinase receptors. The only class IB PI3K member, p110γ, is activated downstream of G protein-coupled receptors (GPCRs), and interacts with p101 or p84  adaptor subunits Class I phosphoinositide 3-kinases (PI3Ks) produce the lipid second messenger phosphatidylinositol-trisphosphate ) Bordetella pertussis toxin (PTx) pretreatment 3AR; [ADORA3]), A3AR null mice are still sensitive to passive systemic anaphylaxis, and degranulation in A3AR−/− bone marrow-derived mast cells (BMMCs) upon antigen stimulation remains functional −/− BMMCs suggest that GPCR signaling does not generate the full input to PI3Kγ-dependent degranulation, but a GPCR-independent activation mechanism for PI3Kγ has yet to be defined.Interestingly, genetic ablation of p110γ blocks high-affinity IgE receptor (FcεRI)-dependent mast cell degranulation in vitro and in vivo 2+ influx activate PI3Kγ, (ii) PI3Kγ is operationally linked to the FcεRI specifically by PKCβ (PRKCB), (iii) and that the phosphorylation of Ser582 located in the helical domain of p110γ by PKCβ leads to the dissociation of the p84 adapter to decouple phosphorylated p110γ from GPCR inputs. Further we characterize the p110γ-p84 interface, and delineate an activation process that seems to be conserved among class I PI3Ks.Here we identify a mechanism that activates PI3Kγ independently of GPCRs: we demonstrate that (i) IgE/antigen complexes and extracellular Ca2+ by store-operated Ca2+ entry (SOCE) 2+ reuptake ATPase (SERCA), causes depletion of Ca2+ stores, triggering SOCE. The latter achieves full-scale degranulation of BMMCs PTx treatment of BMMCs , and a subsequent Ras activation by the Ras guanine nucleotide exchange factor RasGRP4 as described earlier in neutrophils PTx-insensitive Ca2+ release from mast cells To exclude that autocrine/paracrine signaling to p110γ occurred through Altogether, these results clearly illustrate that thapsigargin stimulates BMMCs via a PI3Kγ-dependent activation pathway, which operates separately from adenosine-induced activation of Gαi/o trimeric G proteins.2+ concentration ([Ca2+]i). We therefore chelated extracellular Ca2+ using EDTA, and buffered intracellular Ca2+ with the cell permeable Ca2+ chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM). Extracellular and intracellular Ca2+ chelation prevented phosphorylation of PKB/Akt induced by thapsigargin or the Ca2+ ionophore ionomycin, while IL-3 and adenosine signaling to PKB/Akt remained unperturbed , while thapsigargin -adenosine-5′-N-methylcarbox-amide [IB-MECA]) is sensitive to PTx, thapsigargin-induced SOCE is not. Ca2+-induced activation of PI3Kγ only occurs after SOCE, and is therefore clearly separated from the GPCR/trimeric G protein/PI3Kγ axis.Interestingly, the concentration of Casigargin  and IgE/−/− BMMCs (−/− and PKCγ−/− BMMCs remained intact (Protein kinase C (PKC) inhibitors  targeting classical and atypical PKCs, and the inhibitor PKC412, which mainly inhibits classical PKCs, all substantially blocked PKB/Akt phosphorylation in response to thapsigargin and phorbol 12-myristate 13-acetate (PMA)  and S2A./− BMMCs , signalid intact . Deletiod intact , demonstP3 triggered by thapsigargin and PMA, but not adenosine denosine . Interes3 peaks . ImpaireCo-expression of p110γ with tagged full length or truncated PKCβ2  revealedP[582]LKHPK; spectra in Analysis of phosphorylated, catalytically inactive p110γ by liquid chromatography tandem mass spectrometry (LC-MS/MS) identified Ser582 as a target residue of PKCβ  and intracellular (BAPTA/AM) Ca2+ chelation  incorporated equal amounts of phosphate into free and p84-bound p110γ. In the same experiment, PKCβ preferentially phosphorylated free p110γ, illustrating a preference of PKCβ for p110γ surfaces obscured in the p84-p110γ complex. CAMK also substantially phosphorylated p84, which was borderline in in vitro assays with PKC (12 versus 3 mol %) . As mast cell activation is accompanied by a massive influx of extracellular Ca3 mol %) . Probing3 mol %) .2+-triggered responses. The above, and the fact that PKCβ−/− BMMCs showed a major reduction in phospho-Ser582 after PMA or IgE/antigen stimulation   was produced. The activity of the p110γ Ser582Glu mutant was enhanced approximately 2-fold independently of the substrate used ylation) . Ser582 As mutations in the helical domain of p110α attenuate contacts with the p85 regulatory subunit Most importantly, the correlation of phosphorylation of Ser582 on p110γ and the release of p84 could also be established in wild type BMMCs: when stimulated with PMA, the amount of p84 that could be co-precipitated with p110γ was reduced significantly, and was linked to Ser582 phosphorylation of p110γ. In the inverse co-immunoprecipitation, anti-p84-associated p110γ was reduced, and phosphorylation was below detection levels in the remaining p84-associated p110γ . The colIn order to understand how p84 could mask Ser582 phosphorylation, and to map the p110γ-p84 contact interface, hydrogen deuterium exchange mass spectrometry (HDX-MS) was used. HDX-MS elucidated contacts of class IA p110δ with its p85 regulatory subunit, and the mechanism of action of cancer-linked mutations in p110α 2H) incorporation into free and p84-complexed p110γ was analyzed at seven time points . Differences in 2H-exchange of free and complexed p110γ were mapped onto the crystal structure of p110γ lacking the N-terminal domain , to visualize conformational changes induced by p84 (2H incorporation (>1.0 Da) clustered to the RBD-C2 linker, the C2-helical domain linker, and the helical domain. The 2H-incorporation in the presence of p84 is visualized as integrated average difference in exchange at all seven time points in Deuterium  in the presence of p84, this interaction seems to be dispensable. The N-terminus of p110γ instead has a role in stabilizing the intact catalytic subunit. The helical domain is the main location of interaction with p84; however, it appears that this interaction is vulnerable and easily broken, as a single phosphorylation at Ser582 is able to disrupt the contact.The N-terminus of p110γ was shown to stabilize the p110γ-p101 heterodimer The activation of PI3Kγ has been tightly linked to GPCRs-triggered dissociation of trimeric Gαi proteins, and has been shown to require the interaction of Gβγ subunits with p110γ and the PI3Kγ adaptor subunits p101 and p84 2+ ionophores can modulate phosphoinositide levels in a variety of cells, including platelets Non-GPCR-mediated activation of PI3Kγ has not been reported so far, but it has been shown that phorbol esters and Ca3AR, and is sensitive to PTx and ADA PTx did not diminish the PI3Kγ-dependent, thapsigargin-induced phosphorylation of PKB/Akt, points to a novel mechanism of PI3Kγ activation, which is clearly distinct from GPCR action. This Ca2+-mediated PI3Kγ activation requires SOCE and [Ca2+]i >600 nM. In contrast, GPCRs yield phosphorylation of PKB/Akt in mast cells even in the absence of a change in [Ca2+]i. Furthermore, increases in [Ca2+]i triggered via GPCRs remain at levels incapable of engaging a Ca2+-dependent activation of PI3Kγ.PI3Kγ has been shown to be a key element in enhancing IgE/antigen output by the release of adenosine. This process involves signaling downstream of Gαi-coupled AP3) production, and triggers SOCE by the depletion of Ca2+ stores. That thapsigargin requires functional PI3K activity to induce mast cell degranulation was first demonstrated using the PI3K inhibitors wortmannin and LY294002 2+]i rise and PI3Kγ activity was established previously. The fact that inhibitors for classical PKCs only prevented the thapsigargin- and PMA-induced phosphorylation of PKB/Akt, while the GPCR-mediated phosphorylation of PKB/Akt remained intact, points to a link between classical PKCs and PI3Kγ. Experiments on BMMCs lacking PKCα, PKCβ, and PKCγ, showed that only the PKCβ null cells lost the ability to activate PKB/Akt in response to thapsigargin or PMA stimulation. As the PMA- and thapsigargin-induced phosphorylation of PKB/Akt on Ser473 showed a partial resistance to wortmannin, and as it has been reported that PKCβ can directly phosphorylate Ser473 in the hydrophobic motif of PKB/Akt P3 production directly. The lack of PtdInsP3 production in thapsigargin or PMA-stimulated BMMCs treated with the PKC inhibitor PKC412, and in cells devoid of PKCβ, is in agreement with a requirement of PKCβ upstream of PI3Kγ. That signaling from PKC to PI3K plays a role in mast cell degranulation is further supported by the close correlation of PKC inhibitor sensitivity of phosphorylated PKB/Akt and degranulation responses. Moreover, the loss of PKCβ or PI3Kγ results in a similar reduction of degranulation over a wide range of IgE/antigen concentrations. The results obtained here are in agreement with previous findings that mast cells and mice devoid of p85α/p55α/p50α Thapsigargin bypasses the signaling chain from IgE/antigen-clustered FcεRI to the activation of phospholipase Cγ (PLCγ) and inositol-trisphosphate P3 response to PMA-stimulation was transient , and the two full-length enzymes interacted only weakly. The observation that truncated, activated forms of PKCβ formed stable complexes with p110γ, suggested that PKCβ must attain an open conformation to interact with p110γ, and that PKCβ binds to p110γ via its catalytic domain. This contact resulted in phosphorylation of Ser582 on p110γ, which could be detected both in vitro and in PMA or IgE/antigen-stimulated BMMCs by mass spectrometry. The PKCβ-mediated phosphorylation of p110γ was confirmed using site-specific anti-phospho-Ser582 antibodies. Stimuli like PMA, thapsigargin, and IgE/antigen complexes all required PKC to signal to PKB/Akt, which correlated with the phosphorylation of Ser582 on p110γ. Moreover, the phosphorylation of Ser582 on p110γ was sensitive to removal of extracellular Ca2+, buffering of [Ca2+]i, and the genetic deletion of PKCβ. Adenosine stimulates PI3Kγ via GPCRs and PTx-sensitive trimeric G proteins 2+-independent process, and did not yield a detectable phosphorylation of Ser582.A mechanistic link between PKCβ and the catalytic subunit of PI3Kγ was initially difficult to establish, as the direct PtdInsP2, and the increased rate of auto-phosphorylation displayed by p110γ with a phosphate-mimicking mutation (Ser582Glu), suggests that a structural change in the helical domain of p110γ is sufficient to increase the catalytic activity independent of the presence of the p84 subunit. Previous work examining the activation of the class IA p110α, p110β, and p110δ catalytic subunits has shown that part of the activation mechanism occurs through a conformational change from a closed cytosolic form to an open form on membranes The increased turnover of PtdIns and PtdInsAlthough Ser582 is not in a direct contact with the kinase domain, it is structurally linked to it: the heat repeat HA1/HB1 housing Ser582, and the connecting intra-helical loop (residues 560 to 570), along with helix A3 (624–631) are in contact with helices kα9 and kα10 in the C-lobe of the kinase domain /inter SH2 domain interaction, the association of the p110γ subunit with its adaptor subunit is quite vulnerable, and the Ser582Glu mutant, but not Ser582Ala, abrogated the formation of a p110γ-p84 complex. HDX-MS revealed that the helical domain of p110γ was stabilized by p84. Ser582 is located in the center of the p110γ-p84 contact surface, which explains how a change in charge (Ser582Glu) breaks the interaction with p84, either by direct contact or by destabilization of the helical domain. Overexpression of p84 shields p110γ from a PMA-induced phosphorylation, and suggests that binding of p84 to p110γ, and Ser582 phosphorylation by PKCβ are mutually exclusive. This implies that the two activation modes of PI3Kγ—by GPCRs or PKCβ—are completely separated. At low -orthophosphate for 4 h. Cells were washed, stimulated, and lysed by the addition of chloroform/methanol . Lipids were extracted, deacylated, and separated by high-pressure liquid chromatography (HPLC).PtdIns6 was incubated with PtdInsP2-containing lipid vesicles , 10 µM ATP, and 4 µCi of [γ32P]-ATP in lipid kinase buffer  for 10 min at 30°C. Alternatively, PtdIns/PS vesicles (∼200 µM each) were used. Reactions were terminated by addition of 1 M HCl and CHCl3/MeOH. Lipids were isolated by chloroform extraction, separated by TLC and quantified on a Typhoon 9400.PI3Kγ-Hist test, . * or &, ** or &&, and *** or &&& refer to p-values p<0.05, p<0.005, and p<0.0005. * and & were used for comparison of different genotypes, stimuli an conditions as indicated. Calculations were carried out using Graph Pad Prism, Microsoft Excel, or Kaleidagraph software.Numeric results were tested for significance using a two-tailed Student's 6: 30 µM; Hsp110γ-C-His6/Mmp84-C-His6: 35 µM) were prepared in 20 mM Tris [pH 7.5], 100 mM NaCl, 1 mM ammonium sulfate, and 5 mM DTT. Exchange reactions were initiated by addition of 25 µl of a 98% D2O solution containing 10 mM HEPES (pH 7.2), 50 mM NaCl, and 2 mM DTT, giving a final concentration of 82% D2O. Deuterium exchange reactions were allowed to carry on for seven time periods, 3, 10, 30, 100, 300, 1,000, and 3,000 s of on-exchange at 23°C, before addition of quench buffer. On-exchange was stopped by the addition of 40 µl of a quench buffer containing 1.2% formic acid and 0.833 M guanidine-HCl, which lowered the pH to 2.6. Samples were then immediately frozen in liquid nitrogen until mass analysis. The full HDX-MS protocol can be found in Protein stock solutions . IgE-sensitized  or non-sensitized wild type BMMCs were IL-3 depleted for 3 h and stimulated with either 1 µM adenosine (Ade), 1 µM PAF, or 5 ng/ml antigen (post IgE-sensitization) ± PAF for 2 min. Subsequently, cell lysates were subjected to SDS-PAGE. Phosphorylation of Ser473 in PKB/Akt (A), (B) Ser133 in cyclic AMP-responsive element-binding protein (CREB) and Ser660 in PKCβII was monitored by immunodetection with phosphosite-specific antibodies .(EPS)Click here for additional data file.Figure S2Effect of PKC-inhibitors on PMA- or adenosine-induced PKB phosphorylation (S473) (related to). (A/B) Wild type BMMCs were starved for 3 h in IL-3 free medium supplemented with 2% FCS, and preincubated with the inhibitors for 20 min before stimulation . (C) PKB activation in response to 100 nM PMA or 1 µM Thapsigargin (2 min) was analyzed in wild type, PKCα−/− and PKCγ−/− BMMCs. Cells were deprived of IL-3 as in (A/B). PKB (S473) and MAPK (T183/Y185) phosphorylation was determined by Western blotting. (D) Wild type, PKCβ−/− and PI3Kγ−/− BMMCs were stimulated with different concentrations of thapsigargin, before degranulation was quantified by the measurement of β-hexosaminidase release into cell supernatants ; & only the p110γ−/− dataset reached significance).(EPS)Click here for additional data file.Figure S3Identification of PI3Kγ phosphorylation sites by MS (related to). (A/B) Recombinant, catalytically inactive GST-PI3Kγ  was phosphorylated in vitro by recombinant PKCβ in the presence of 100 µM ATP/[γ32P]-ATP. Proteins were separated by SDS-PAGE and trypsin-digested PI3Kγ was analyzed by LC-MSMS. (A) Enhanced product ion spectra of the tryptic phospho-S582-peptide of PI3Kγ. The y- and b-fragments detected are indicated in the sequence. Fragments showing a H3PO4 loss are marked with an asterisk. The b2, y6, and y7 fragments allow assignment of the phosphorylation to serine 3 in the peptide. (B) Enhanced product ion spectra of the non-phosphorylated form of this peptide. (C) Relevant information for the MRM analysis of the peptides containing Ser582. The amino acid numbering is as in Swiss-Prot entry P48736. (D) Sequence alignment of the beginning of the helical domain of class I PI3Ks. Alignments were done by inspection of the crystal structures of PI3Kγ (1E8Y), PI3Kα (3HHM), PI3Kβ (2Y3A), and PI3Kδ (2WXR). Secondary structure elements are labeled as indicated in the legend. S582 is colored red, while cancer-associated PI3Kα mutations are marked as blue.(TIF)Click here for additional data file.Figure S4Anti-phospho-Ser582 antibody validation (related to). (A) Wild type BMMCs were transfected with empty vector, expression plasmid for GFP-PI3Kγ wild type or the GFP-PI3Kγ S582A mutant. On the next day, cells were stimulated with 200 nM PMA for 45 s, and PI3Kγ was immunoprecipitated from cell lysates. Specificity of the anti-S582 antibodies was validated by Western blotting.(EPS)Click here for additional data file.Figure S5Global and site-specific in vitro phosphorylation of monomeric p110γ and p110γ-p84 complexes by PKCβ and CamKII (related to). (A) Equal amounts of purified recombinant p110γ-His6 (2.5 pmole) or p110γ-His6/EE-p84 complexes were incubated with 20 pmole wortmannin to eliminate auto-phosphorylation signals. Free or complexed p110γ (with p84 protein) was incubated with 10 µM ATP and 5 µCi of [32P]-γ-ATP, and equal specific activity of recombinant PKCβII and CamKII (Life Technologies assays: 30 pmol phosphate incorporation/min.) for 30 min at 30°C. Subsequently, proteins were denatured and separated by SDS-PAGE followed by Coomassie staining (lower panel). 32P-incorporation was visualized by autoradiography and quantified on a phospho-imager . Band intensities were quantified with ImageQuant TL Software . Insert: quantification of p84 phosphorylation from the reactions shown in (A), . Phospho-PKCβII levels were subtracted from phospho-p84 signals . The filled bar represents phosphorylated p84-His6 after Ni2+-NTA pull-down to minimize contaminating PKCβII autophosphorylation signals (n = 3). (B) Equal amounts of purified recombinant p110γ-His6 (2.5 pmole) or p110γ-His6/EE-p84 complexes were incubated as indicated with ATP (100 µM), recombinant PKCβII and CamKII for 30 min at 30°C. Proteins were denatured, separated by SDS-PAGE followed by immunological detection of p110γ and p84 protein, as well as site-specific phosphorylation of residue Ser582 (pp110γ S582) in p110γ. PKCβII and CamKII input was adjusted to equal protein kinase activity (30 pmol of transferred phosphate/min). For quantification, Ser582 phosphorylation levels were normalized .(EPS)Click here for additional data file.Figure S6Phosphorylation of p110γ requires active PKCβ (related to). (A) Effect of PKC inhibitors on thapsigargin induced p110γ Ser582 phosphorylation. IL-3 deprived BMMCs were preincubated with PKC inhibitors (1 µM Ro318425 or 1 µM AEB071) for 20 min before stimulation with 1 µM thapsigargin for 2 min. PI3Kγ was immunoprecipitated from cell lysates with anti-p110γ antibody . (B) Cells were stimulated as in (A). PKB (T308) and CamKII (T286) phosphorylation was determined by Western blotting. Data are the average of three independent experiments ± SEM.body see . Precipi(EPS)Click here for additional data file.Figure S7Ser582 phosphorylation releases p84 from p110γ (related to). IL-3 deprived BMMCs were stimulated with 100 nM PMA for 2 min. PI3Kγ complex was co-immunoprecipitated from cell lysates using either (A) anti-p110γ or (B) anti-p84 antibodies. The amount of p84 co-immunoprecipitated with p110γ (A) or p110γ co-immunoprecipitated with p84 (B) was normalized to the total amount of p110γ or p84, respectively. Data are the average of five independent experiments ± standard error of the mean [SEM].(EPS)Click here for additional data file.Figure S8PI3Kγ domain order and peptide coverage after pepsin digestion (related to ). Identified and analyzed peptides are shown under the primary sequence of PI3Kγ, which has been colored according to the domain boundaries shown above.(EPS)Click here for additional data file.Figure S9Changes in deuteration levels of PI3Kγ in the presence of p84 (related to). Peptides spanning PI3Kγ (labeled A–Q) that showed greater than 0.5 Da changes in deuteration in the presence and absence of p84 were mapped onto the PI3Kγ structure . The greatest difference in exchange observed at any time was used for the mapping. S582 is shown as red balls. The ATP competitive inhibitor PIK-90 is shown in green as a reference point for the kinase domain. The linker regions between the RBD and the C2 domain and the C2 and helical domain are shown as dotted lines.(EPS)Click here for additional data file.Figure S10Changes in deuteration levels of PI3Kγ peptides in the presence of p84 (related to). The graphs showing the number of incorporated deuterium atoms in the presence (o) and absence (•) of p84 at seven time points in peptides that showed a greater than 0.5 Da H/D exchange difference. Data represent mean ± standard deviation (SD) of two independent experiments.(EPS)Click here for additional data file.Figure S11Deuteration levels in free and p84-bound p110γ (related to). Changes in deuteration levels were mapped onto the crystal structure of PI3Kγ (PDB ID: 2CHX) as in (TIF)Click here for additional data file.Table S1Deuterium exchange data of all analyzed peptides of PI3Kγ in the absence or presence of p84 are summarized in tabular form (related to ). Percent hydrogen deuterium exchange was calculated for each of the seven time points and colored according to the legend. Data show the mean of two independent experiments. The charge state (Z), maximal number of exchangeable amides (#D), starting residue number (S), and ending residue number (E) are displayed for every peptide.(XLSX)Click here for additional data file.Text S1Extended experimental procedures, reference to animals and plasmids. Detailed description of experimental procedures, materials, and further reference to the origin of genetically modified mice used here, and a primer to the determination of deuterium incorporation (HDX_MS).(DOCX)Click here for additional data file.


Cefaclor is the most common cephalosporin to induce anaphylaxis. We evaluated immunologic findings of cefaclor hypersensitivity.36 patients with a history of immediate reactions to cefaclor were enrolled from Ajou University Hospital and Asan Medical Center, South Korea. Those with immediate hypersensitivity reactions to cefaclor were defined by a certain clinical history with or without specific IgE to cefaclor by immunoCAP system. Serum specific IgE, IgG1 and IgG4 levels to cefaclor-HSA conjugate were measured by ELISA, and compared with those of ImmunoCAP system. The binding specificity was evaluated by ELISA inhibition test.Anaphylaxis  was the most common phenotype, followed by urticaria . There were no significant differences in clinical characteristics, such as age, sex and atopy status between group I and II. The serum specific IgE to cefaclor by ImmunoCAP was found in total 29 (80.6%) patients, 24 (82.8%) in group I and 5 (71.4%) in group II with no significant according to clinical parameters. The prevalence of serum specific IgE, and IgG1, IgG4 to cefaclor-HSA conjugate by ELISA tended to be higher in group I  than in group II , although these differences were not statistically significant. Serum specific IgG4 to cefaclor-HSA conjugate was observed only in group I. 10.3% patients in group I had high specific IgG1 to cefaclor-HSA conjugate with no specific IgE. Significant associations were found between specific IgE and IgG1 or IgG4 antibodies . ELISA inhibition tests showed significant inhibitions by both free cefalcor and cefaclor-HSA conjugate.Most common manifestation of immediate hypersensitivities to cefaclor was anaphylaxis in which IgE mediate response is the major pathogenic mechanism. Detection of serum specific E and IgG subtypes to cefaclor-HSA conjugate may be useful to diagnose cefaclor anaphylaxis. The IgG subtype- mediated response was suggested in some cases of cefaclor anaphylaxis.