Aspergillus fumigatus is a leading infectious killer in immunocompromised patients. Calcineurin, a calmodulin (CaM)-dependent protein phosphatase comprised of calcineurin A (CnaA) and calcineurin B (CnaB) subunits, localizes at the hyphal tips and septa to direct A. fumigatus invasion and virulence. Here we identified a novel serine-proline rich region (SPRR) located between two conserved CnaA domains, the CnaB-binding helix and the CaM-binding domain, that is evolutionarily conserved and unique to filamentous fungi and also completely absent in human calcineurin. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of A. fumigatus CnaA in vivo at four clustered serine residues  in the SPRR. Mutation of the SPRR serine residues to block phosphorylation led to significant hyphal growth and virulence defects, indicating the requirement of calcineurin phosphorylation at the SPRR for its activity and function. Complementation analyses of the A. fumigatus ΔcnaA strain with cnaA homologs from the pathogenic basidiomycete Cryptococcus neoformans, the pathogenic zygomycete Mucor circinelloides, the closely related filamentous fungi Neurospora crassa, and the plant pathogen Magnaporthe grisea, revealed filamentous fungal-specific phosphorylation of CnaA in the SPRR and SPRR homology-dependent restoration of hyphal growth. Surprisingly, circular dichroism studies revealed that, despite proximity to the CaM-binding domain of CnaA, phosphorylation of the SPRR does not alter protein folding following CaM binding. Furthermore, mutational analyses in the catalytic domain, CnaB-binding helix, and the CaM-binding domains revealed that while the conserved PxIxIT substrate binding motif in CnaA is indispensable for septal localization, CaM is required for its function at the hyphal septum but not for septal localization. We defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi in a region absent in humans. These findings suggest the possibility of harnessing this unique SPRR for innovative antifungal drug design to combat invasive aspergillosis.The fungus  in vitro against the major invasive fungal pathogens, they are also immunosuppressive in the host, limiting therapeutic effectiveness. Here we defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi that is responsible for virulence in the opportunistic human pathogen, Aspergillus fumigatus. This phosphorylation occurs on a cluster of four serine residues located in a unique serine-proline rich domain of calcineurin that is absent in humans. This finding of a new fungal-specific mechanism controlling hyphal growth and virulence represents a new potential target for antifungal drug therapy.Invasive fungal infections are a leading cause of death in immunocompromised patients. Translating molecular understanding into tangible clinical benefit has been difficult due to the fact that fungal pathogens and their hosts have similar physiology. The calcineurin pathway is an important signaling cascade in all eukaryotes, and calcineurin inhibitors are powerful immunosuppressants that have revolutionized medicine. Through both genetic and pharmacologic inhibition, we have established that calcineurin is vital for invasive fungal disease. Although the currently available calcineurin inhibitors are active  Aspergillus fumigatus, is the most frequent fungal cause of mortality in vitro against A. fumigatusInvasive fungal infections are a leading cause of death in immunocompromised patients 2+/calmodulin (CaM)-dependent protein phosphatase comprised of a catalytic A and regulatory B subunit heterodimeric complex 2+/CaM binds to calcineurin A at the CaM-binding domain (CaMBD), adjacent to the calcineurin B binding helix (CnBBH) in its regulatory domain and displaces the auto-inhibitory domain (AID) Calcineurin is a CaSchizosaccharomyces pombe, calcineurin participates in morphogenesis by affecting septal positioning, spindle body organization, and membrane trafficking Candida albicans and Cryptococcus neoformans, calcineurin regulates growth at alkaline pH and elevated temperature, membrane stress, and virulence Although calcineurin is conserved from yeasts to human, it exhibits diverse roles in different cell types, evidenced by modulating immune responses, impacting muscle development, neuronal plasticity and cell death in mammalian cells S. pombe was shown to be activated after phosphorylation by the check point kinase Cds1 at the similarly positioned serine residue within the CaMBD (S459), and at another site at the C-terminus (S521) As a protein phosphatase, calcineurin is known to dephosphorylate specific substrates A. fumigatusin vivoWe and others have previously determined that calcineurin is required for hyphal growth and virulence of A. fumigatus CnaA to investigate those required for hyphal growth, CnaA septal localization, phosphatase function, and virulence. We uncovered six novel findings, including (i) the linker between the CnBBH and CaMBD, contains a region unique to filamentous fungi (completely absent in humans), that is rich in serine and proline residues  and is phosphorylated in vivo at all 4 clustered serine residues , (ii) complementation of the A. fumigatus ΔcnaA mutant strain with calcineurin A homologs from other fungi defined a filamentous fungal-specific phosphorylation of the SPRR in CnaA, suggesting its evolutionarily conserved importance in fungal hyphal growth, (iii) GSK-3β, CK1, CDK1 and MAP kinase as potential kinases that phosphorylate the SPRR, implicating their role in the regulation of A. fumigatus CnaA, (iv) mutations in the SPRR did not affect septal localization of CnaA but resulted in significant hyphal growth and virulence defects, implicating the importance of calcineurin phosphorylation for its function in A. fumigatus and its possibility as a new antifungal target, (v) CaM is not required for septal localization of CnaA but is required for its function at the hyphal septum, and (vi) the PxIxIT substrate binding motif in CnaA is required for its localization at the hyphal septum.Here we performed mutational analyses in the functional domains of A. fumigatus strains expressing a series of truncated cnaA cDNAs  under the control of its native promoter in the ΔcnaA mutant strain , did not complement the hyphal growth defect of the ΔcnaA mutant strain and mislocalized CnaA in the cytoplasm (cnaA-T2 that included the CnBBH region (1–400 aa) showed partial growth recovery, indicating that this fragment may bind to CnaB in vivo and partially function by less efficiently localizing at the septum (cnaA-T3 (1–425 aa), containing the linker region spanning 23 aa between the CnBBH and CaMBD , also completely restored hyphal growth and properly localized CnaA at the septa  is calcineurin-mediated, and that paradoxical growth is abolished in the ΔA. fumigatus CnaA with homologs from other organisms. This alignment confirmed a high degree of conservation within the catalytic domain, CnBBH, and the CaMBD across different species (data not shown). However, the linker region showed marked variation in different species  that is relatively conserved in filamentous fungi and completely absent in the human calcineurin α-catalytic subunit  and S. cerevisiae (CNA1) and found no hyphal growth recovery and cytosolic CnaA localization (data not shown). We then complemented our A. fumigatus ΔcnaA mutant strain with CnaA counterparts from other phylogenetically distinct fungi belonging to the phyla basidiomycota and zygomycota  and we utilized the genes encoding MccnaA and MccnaC that showed variability in the SPRR  and also localized to the hyphal septa , which contained the SPRR, completely restored both calcineurin activity and hyphal growth . Further phosphoproteomic analyses by LC-MS/MS identified 6 serine residues phosphorylated in A. fumigatus CnaA  were detected in the M. grisea CnaA SPRR, and a single phosphorylated serine residue (position 423) was detected in the N. crassa CnaA SPRR  in the region aligning with the A. fumigatus CnaA SPRR, we verified its phosphorylation status in vivo and found that this serine residue was phosphorylated, along with another serine residue at position 499 in the C-terminus  from A. fumigatus and various combinations of the kinases. The phosphorylation reactions were processed for mass spectrometry after proteolytic digestion to identify the phosphorylated residues. As shown in In order to determine the potential kinase(s) that may phosphorylate the CnaA SPRR we scanned this region using Scansite 2.0, NetPhos 2.0, and NetPhosK 1.0 programs. These analyses suggested that the amino acids surrounding S406 and S413 of CnaA form a potential consensus sequence for phosphorylation by the proline-directed kinases, such as glycogen synthase kinase (GSK-3), cyclin dependent kinase 1 (CDK1), and mitogen activated protein kinase (MAP Kinase). Casein kinase I (CK1) was also predicted to phosphorylate the SPRR. Based on this prediction, we performed in vivo, we treated the CnaA-EGFP expression strain with GSK-3β and CK1 specific inhibitors, GSK-3β inhibitor VII and D4476, respectively. Both the GSK-3β inhibitor VII and D4476 showed a growth inhibitory effect in a concentration range of 0.5–0.75 µM (data not shown). The CnaA-EGFP fusion protein was isolated after treatment with 0.75 µM each of GSK-3β inhibitor VII and D4476 and analyzed for its phosphorylation status by mass spectrometry. Surprisingly, treatment with GSK-3β and CK1 inhibitors resulted in the dephosphorylation of only S406, but S408, S410 and S413 were phosphorylated, suggesting the possibility of S406 as a target for GSK-3β and CK1 in vivo, while other kinases may be involved in phosphorylating the S408, S410 and S413 residues in vivo. To investigate this possibility, we performed in vitro phosphorylation reactions in presence of the other potential proline-directed kinases, CDK1 and MAP kinase. As shown in in vivo and further work on the interaction of these enzymes with CnaA, specifically addressing the timing of phosphorylation of CnaA by these enzymes, will lead to a more definitive understanding of the role of these kinases in the regulation of CnaA.Next, to determine the role of GSK-3β and CK1 in the phosphorylation of S406, S408, S410 and S413 Since the immunosuppressant FK506 inhibits calcineurin activity by binding to the immunophilin FKBP12, we also examined the phosphorylation of CnaA and CnaB in the presence of FK506 to correlate phosphorylation versus activity. The FK506-treated sample showed a 2-fold decrease in the phosphorylation of S406 in the CnaA SPRR and a 1.2- and 1.8-fold increase in the phosphorylation of S537 and S542, respectively, in the C-terminus compared to the untreated control  and S6. 2+/CaM binds to the CaMBD to displace the AID, resulting in calcineurin activation S. pombe (S459) activated calcineurin 2+/CaM binding 2+/CaM binding to CnaA or imparts a change in structural content following Ca2+/CaM binding. To test this, we utilized the purified AfCaM and the CnaA regulatory domain  from A. fumigatus. We also expressed another recombinant AfRD in which we mutated the four SPRR serine residues to glutamate to mimic phosphorylated status, designating this as AfRD-4SE. The CD spectra of both complexes (AfRD+AfCaM and AfRD-4SE+AfCaM) indicated essentially identical secondary structural content revealing that conformational changes that occur upon Ca2+/CaM binding appear to be unaffected by the phosphorylation  to block phosphorylation and also the 4 serine residues to glutamate (CnaAmt-4SE) to mimic a fixed phosphorylated state in vivo. In comparison to the wild-type strain expressing CnaA-EGFP, only the CnaAmt-4SA strain exhibited a growth defect  in contact with the PxIxIT motif resulted in defective substrate interaction 2+/CaM bound to a 25-residue peptide spanning the CaMBD in the human calcineurin catalytic subunit also revealed that R408, V409, and F410 play a major role in rigidity and stabilization of the central helix of CaM bound to calcineurin A. fumigatus, we mutated the PxIxIT-binding NIR residues to alanines (NIR-AAA), as well as the critical Ca2+/CaM-binding RVF residues in the CaMBD to alanines , these results, coupled with our truncational analyses, confirmed that CnaA localization at the septum is CaM-independent, yet CaM is required to activate CnaA to completely restore hyphal growth.Binding studies with the human calcineurin-NFAT complex previously revealed the PxIxIT motif as a common binding site for calcineurin on its substrates RVF-AAA; . The NIRRVF-AAA; . On the RVF-AAA; , supportRVF-AAA; . The obsS. cerevisiae have been identified by substitution of V385 with an aspartic acid that disrupted the interaction between the catalytic and the regulatory subunit, and also by random mutagenesis of three residues  that led to loss of calcineurin activity but did not disrupt calcineurin A binding to Ca2+/CaM or to calcineurin B A. fumigatus CnaA, we mutated V371 to aspartic acid (V371D) and the T359, H361, and L365 to proline, leucine and serine (THL-PLS), respectively. Both mutations had a significant effect on hyphal growth, but neither affected CnaA septal localization , efficacy against emerging resistant strains, and synergism with existing antifungals l growth  and 2, wl growth . Inclusil growth .To evaluate the conservation of this linker region, we examined it in 22 eukaryotes selected based on divergence and to include model organisms and pathogens affecting both humans and plants . Phylogein vivo, we performed complementation tests in our A. fumigatus ΔcnaA mutant strain with cnaA homologs from human, S.cerevisiae, C. neoformans and M. circinelloides, all of which lacked similarity within the SPRR , C. neoformans CNA1, which exhibits 67% similarity to A. fumigatus CnaA, localized to the septum but did not restore hyphal growth , revealing that the prephosphorylation of S406 residue by CK1 may trigger the subsequent phosphorylation of S408 and S410. Interestingly, a previous study showed that the yeast Mck1 protein kinase belonging to the GSK-3 kinase family stimulated calcineurin activity by phosphorylating Rcn1, and in the absence of GSK-3 kinase, calcineurin activity was fully inhibited revealing a allosteric mechanism of calcineurin regulation by Rcn1 in vitro phosphorylation data by examining the phosphorylation status of CnaA in vivo after treating with inhibitors for GSK-3β and CK1, which confirmed the absence of phosphorylation at S406 only but not the other serine residues, leading to the notion that other kinases may also phosphorylate the CnaA SPRR in vivo. The observed phosphorylation of CnaA SPRR by CDK1 and MAP kinase in vitro strengthens this possibility. However, further analyses are required to specifically understand how these enzymes actually regulate CnaA.Phosphorylation was reduced in both the catalytic and the regulatory calcineurin subunits following treatment with FK506  and S7. 2+/CaM and the calcineurin complex. Circular dichroism studies revealed that conformational changes that occur after Ca2+/CaM binding remained unaffected between unphosphorylated and phosphomimetic SPRR constructs , located at the beginning of the long helix that forms the bulk of the CnaB binding interface of CnaA, would place a charged residue in an unfavorable hydrophobic environment and result in destabilization of the calcineurin heterodimer . MutatioAnimal studies at Duke University Medical Center were in full compliance with all of the guidelines of the Duke University Medical Center Institutional Animal Care and Use Committee (IACUS) and in full compliance with the United States Animal Welfare Act (Public Law 98-198). Duke University Medical Center IACUC approved all of the vertebrate studies under the protocol number A-038-11-02. The studies were conducted in the Division of Laboratory Animal Resources (DLAR) facilities that are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).cnaA mutant strain (ΔcnaA::A. parasiticus pyrG) A. fumigatus wild-type strain (AF293) or the strain expressing cnaA-egfp under its native promoter cnaA mutant complementation analyses, the respective truncated or mutated cDNAs encoding cnaA amplified by using pUCGH-cnaA as a template with primers listed in cnaApromo and transformants selected in the presence of hygromycin B Strains are listed in C. neoformans, M. grisea, N. crassa and M. circinelloides were amplified from respective cDNA libraries. All genes were amplified using the respective templates and primers listed in CNA-α subunit the plasmid, pET15b CnA CnB (obtained from Addgene), was used as a template. S. cerevisiae genomic DNA was used as a template to amplify S. cerevisiae CNA1. The plasmids were sequenced to verify for accuracy prior to their transformation into the A. fumigatus ΔcnaA mutant strain. Transformants were selected in the presence of hygromycin B (150 µg/ml).The calcineurin A encoding cDNAs from p-nitrophenyl phosphate as substrate at 405 nm. The difference of absorbance values between the amounts of p-nitrophenol released in the strains versus the ΔcnaA ΔcnaB double mutant control strain represented the phosphatase activity mediated by calcineurin. Each experiment consisted of two biologic replicates, with each assay consisting of 6 technical replicates; data are presented as mean ± SD of nanomoles of pNPP released/min/mg protein.Preparation of cell extracts and Western detection were performed as described earlier 2 phosphopeptide enrichment and mass spectrometry as previously described 18 75 µm I.D.×250 mm reversed-phase column. The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. Following a 5 µl injection, peptides were trapped for 5 min on a 5 µm Symmetry C18 180 µm I.D.×20 mm column at 20 µl/min in 99.9% A. The analytical column was held at 5% B for 5 min then switched in-line and a linear elution gradient of 5% B to 40% B was performed over 90 min at 300 nl/min. The analytical column was connected to a fused silica PicoTip emitter  with a 10 µm tip orifice and coupled to an LTQ-Orbitrap XL mass spectrometer. In some experiments the analytical column was connected to a fused silica PicoTip emitter  with a 10 µm tip orifice and was coupled to a Waters Synapt G2 QToF mass spectrometer through an electrospray interface operating in a data-dependent mode of acquisition. The instrument was set to acquire a precursor MS scan in the Orbitrap from m/z 400–2000 with r = 60,000 at m/z 400 and a target AGC setting of 1e6 ions. In a data-dependent mode of acquisition, MS/MS spectra of the three most abundant precursor ions were acquired in the Orbitrap with r = 7500 at m/z with a target AGC setting of 2e5 ions. Max fill times were set to 1000 ms for full MS scans and 500 ms for MS/MS scans with minimum MS/MS triggering thresholds of 5000 counts. For all experiments, fragmentation occurred in the LTQ linear ion trap with a CID energy setting of 35% and a dynamic exclusion of 60 s was employed for previously fragmented precursor ions. When using the Waters Synapt G2 QToF mass spectrometer through an electrospray interface operating in a data-dependent mode of acquisition the instrument was set to acquire a precursor MS scan from m/z 50–2000 with MS/MS spectra acquired for the three most abundant precursor ions. For all experiments, charge dependent CID energy settings were employed and a 120 s dynamic exclusion was employed for previously fragmented precursor ions.Total cell lysates were extracted and normalized to contain ∼10 mg protein in each sample before GFP-Trap® affinity purification (Chromotek) and processed for TiORaw LC-MS/MS data files were processed in Mascot distiller (Matrix Science) and then submitted to independent Mascot database searches (Matrix Science) against a SwissProt (fungus taxonomy) containing both forward and reverse entries of each protein. Search tolerances for LTQ-Orbitrap XL data were 10 ppm for precursor ions and 0.02 Da for product ions, and for Synapt G2 data were 10 ppm for precursor and 0.04 Da for product ions using trypsin specificity with up to two missed cleavages. Carbamidomethylation (+57.0214 Da on C) was set as a fixed modification, whereas oxidation (+15.9949 Da on M) and phosphorylation  were considered a variable modification. All searched spectra were imported into Scaffold (Proteome Software) and protein confidence thresholds were set using a Bayesian statistical algorithm based on the PeptideProphet and ProteinProphet algorithms which yielded a peptide and protein false discovery rate of 1%. Phosphopeptide intensities were obtained by generating selected ion chromatograms (20 ppm window around most abundant charge state of precursor ion with seven point Boxcar smoothing) from raw LC-MS data. MS response at peak apex was used for quantitating abundance.E. coli optimized genes for A. fumigatus calcineurin  and calmodulin (AfCaM) were synthesized and ligated into a pUC57. The genes were digested and ligated into the pET303/CT-His vector using XhoI and XbaI. The mutant AfRD4Ser-Glu (AfRD-4SE), containing mutations S14E, S16E, S18E, and S21E, was made using a QuikChange Site-Directed Mutagenesis Kit. AfRD and AfRD-4SE were transformed into E. coli BL-21 (DE3) cells for expression, and purified on a Ni2+/NTA column followed by a calmodulin-sepharose column. AfCaM was purified on a 2-trifluoromethyl-10-aminopropylphenothiazine (TAPP)-sepharose column. Protein concentrations were determined by bicinchoninic acid assay. Circular dichroism (CD) experiments were performed using a Jasco J-810 spectropolarimeter. Sample buffers were composed of 20 mM Tris, 200 mM NaCl, 4 mM EGTA, pH 7.5, and 20 mM CaCl2. Samples contained AfRD, AfCaM, or equimolar concentrations of AfRD and AfCaM. The CD experiments shown were all performed in the presence of an excess of calcium in order to determine the effects of the phospho-mimics on the AfRD conformation when bound by AfCaM. The concentrations of either protein alone per sample ranged from 10–20 µM. In the sample that contained both of AfRD and AfCaM, the total protein concentration per sample ranged from 10–25 µM. Spectra were collected in quartz 1 mm pathlength cuvettes. Samples were scanned from 200–260 nm in 0.5 nm increments at a scanning speed of 50 nm/sec and each spectrum is the average of 4 scans. The raw CD data (in millidegrees) was converted to molar ellipticity. The amount of secondary structure was determined using the CONTIN/LL deconvolution program.In vitro phosphorylation reactions with GSK-3β, CK1, CDK1/cyclinB and MAP kinase (New England Biolabs) contained 10 µg of recombinant CnaA-AfRD protein, reaction buffer supplied by the manufacturer, and 500 µM ATP in total volume of 50 µl. GSK-3β (2500 U), CK1 (5000 U), CDK1 (100 U) and MAP kinase (500 U) were used for each reaction. The reactions were performed either with single enzymes or combinations of the different enzymes. The reactions were incubated at 30°C for 4 h and processed for mass spectral analysis following digestion with Glu-C and TiO2 phosphopeptide enrichment.in vivo, the CnaA-EGFP expression strain was grown in presence of 0.75 µM each of the GSK-3β inhibitor VII  and D4476 (abcam) for a period of 24 h and the isolated CnaA-EGFP fusion protein was subjected to phospho enrichment and mass spectrometry as described earlier To determine the effect of GSK-3β and CK1 inhibitors on the phosphorylation status of CnaA A. fumigatus calcineurin A and B subunits were prepared using the Phyre server. A model of the fungal heterodimer was constructed by superimposing the individually created homology models onto the X-ray structure of the human calcineurin heterodimer bound to a substrate peptide (PDB ID 2P6B) A. fumigatus calcineurin to propose a structural basis for the disruption of calcineurin activity observed for the mutants described in the current study.Homology models for the mt-4SA strain Six-week-old CD1 male mice (mean weight 22.5 g) were immunosuppressed with both cyclophosphamide and triamcinolone acetonide as previously described Figure S1Clustal alignment of the calcineurin A proteins from 24 different species of the region including the CnBBH and the CaMBD. While there was clear homology observed in the CnBBH and the CaMBD, the linker that separates these conserved domains exhibited variation. Notably, filamentous fungal species contained several serine and proline residues in this linker. AfcnaA-Aspergillus fumigatus CnaA; AmCnaA-Allomyces macrogynus; BcCnaA-Botrytis cinerea CnaA; BchycnaA-Batrachochytrium dendrobatidis CnaA; CaCnaA-Candida albicans CnaA; CcCnaA-Coprinopsis cinerea CnaA; CeCnaA-Caenorhabditis elegans CnaA; CiCnaA-Coccidioides immitis; CnCnaA-Cryptococcus neoformans CnaA; DdCnaA-Dictyostelium discoideum CnaA; FvCnaA-Fusarium verticillioides CnaA; HcCnaA-Histoplasma capsulatum CnaA; HcnaA-Homo sapiens CnaA; MgCnaA-Magnaporthe grisea CnaA; MygCnaA-Microsporum gypseum CnaA; MvCnaA-Mortierella verticillata CnaA; NcCnaA-Neurospora crassa CnaA; PbCnaA-Paracoccidiodes brasilensis CnaA; PtCnaA- Puccinia triticina CnaA; RoCnaA-Rhizopus oryzae CnaA; ScCnaA-Saccharomyces cerevisiae CnaA; SpCnaA-Schizosaccharomyces pombe CnaA; TrCnaA-Trichophyton rubrum CnaA; UmCnaA-Ustilago maydis CnaA.(PDF)Click here for additional data file.Figure S2Phosphorylation of the A. fumigatus CnaB regulatory subunit. Annotated tandem mass spectrum of RA[pS]VGTSQLLDNIV[pS]ASNFDRDEVDR (3+ m/z 1008.7813) from CnaB revealed two unique phosphorylated serine residues (Ser21 and Ser33) localized with >99% confidence using AScore. The presence of each identified C-terminal (y) and N-terminal (b) product ions are indicated within the peptide sequence.(PDF)Click here for additional data file.Figure S3Tandem mass spectra of phosphorylated peptides identified from Magnaporthe grisea calcineurin. Phosphorylation in the SPRR (A) and the C-terminus (B) are shown. Annotated tandem mass spectra showing assigned fragment ion assignments from Mascot database searches imported into Scaffold are shown. For each qualitative identification, spectra were subjected to Ascore localization to assign percent confidences of amino acid specific assignments. An extracted ion chromatogram (EIC) of the precursor m/z (+/−20 ppm) of the chromatographic elution profile of each phosphorylated peptide is presented to the right of each annotated fragment ion spectra. An asterisk in the EIC plot indicated the peak which was chosen for subsequent qualitative identification.(PDF)Click here for additional data file.Figure S4Tandem mass spectra of phosphorylated peptides identified from Neurospora crassa calcineurin. Annotated tandem mass spectra showing assigned fragment ion assignments from Mascot database searches imported into Scaffold are shown. For each qualitative identification, spectra were subjected to Ascore localization to assign percent confidences of amino acid specific assignments. An extracted ion chromatogram (EIC) of the precursor m/z (+/−20 ppm) of the chromatographic elution profile of each phosphorylated peptide is presented to the right of each annotated fragment ion spectra. An asterisk in the EIC plot indicated the peak which was chosen for subsequent qualitative identification.(PDF)Click here for additional data file.Figure S5Tandem mass spectra of phosphorylated peptides identified from Mucor circinelloides calcineurin. Phosphorylation in the SPRR (A) and the C-terminus (B) are shown. Annotated tandem mass spectra showing assigned fragment ion assignments from Mascot database searches imported into Scaffold are shown. For each qualitative identification, spectra were subjected to Ascore localization to assign percent confidences of amino acid specific assignments. An extracted ion chromatogram (EIC) of the precursor m/z (+/−20 ppm) of the chromatographic elution profile of each phosphorylated peptide is presented to the right of each annotated fragment ion spectra. An asterisk in the EIC plot indicated the peak which was chosen for subsequent qualitative identification.(PDF)Click here for additional data file.Figure S62 enrichment and LC-MS/MS analysis.Extracted ion chromatogram (EIC) peak height measurements of CnaA following TiO Manual EIC generation was performed for each precursor ion (+/−20 ppm tolerance) which was qualitatively identified as a phosphorylated peptide from CnaA and resulting peak height was used as a measure of intensity. To ensure the same precursor ion was being compared across the +FK506, −FK506, and Control samples, a peak was required to have the same m/z (+/−10 ppm), have the same retention time (+/−30 seconds), and was qualitatively identified as the same species in Mascot database searches.(PDF)Click here for additional data file.Figure S72 enrichment and LC-MS/MS analysis.Extracted ion chromatogram (EIC) peak height measurements of CnaB following TiO Manual EIC generation was performed for each precursor ion (+/−20 ppm tolerance) which was qualitatively identified as a phosphorylated peptide from CnaB and resulting peak height was used as a measure of intensity. To ensure the same precursor ion was being compared across the +FK506, −FK506, and Control samples, a peak was required to have the same m/z (+/−10 ppm), have the same retention time (+/−30 seconds), and was qualitatively identified as the same species in Mascot database searches.(PDF)Click here for additional data file.Figure S8CnaA mutation strains cultured on GMM agar in the presence of varying concentrations of the cell wall inhibitor caspofungin for 5 days. The THL-PLS mutation  and the V371D mutation (CnBBH residue) in CnaA completely abolish calcineurin-mediated paradoxical growth. The NIR-AAA (PxIxIT Binding Motif) and the RVF-AAA (CaMBD residues) mutations do show slight paradoxical growth at 2 µg/ml and 4 µg/ml of caspofungin.(PDF)Click here for additional data file.Table S1Strains used in the present study. All the strains listed were constructed in the Af293 derived isogenic Af293.1 strain with pyrG auxotrophy. The ΔcnaA strain previously constructed in the Af293.1 background by utilizing the pyrG marker gene was transformed with the various cnaA constructs either under the control of its native promoter or the otef promoter with gfp tag and hygromycin (hph) resistance marker gene.(DOCX)Click here for additional data file.Table S2Primers and constructs. All the primers used for construction of the various cnaA.(DOCX)Click here for additional data file.


Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling. Drosophila homologue of SOCS5 (SOCS36E) has been shown to regulate both JAK/STAT and EGF receptor signaling in vivoEnhanced survival, proliferation, angiogenesis and/or migration are hallmarks of many human cancers The Janus kinases (JAKs) sit at the apex of many cytokine receptor pathways and their activation results in phosphorylation of the cytoplasmic domains of the receptor, leading to the recruitment and phosphorylation of the Signal Transducers and Activators of Transcription (STAT)s. In turn, the STATs induce transcription of a specific subset of genes, resulting in an appropriate cellular response that can include survival, proliferation and/or cell differentiation. However, this cellular response requires tight regulation, as aberrant signaling has been unequivocally linked to mutations in key signaling genes, such as the valine 617 mutation in the JAK2 pseudokinase (JH2) domain associated with myeloproliferative disease The eight mammalian SOCS proteins, SOCS1-7 and cytokine inducible SH2 domain-containing protein (CIS) consist of a C-terminal SOCS box, a central SH2 domain and an N-terminal region of variable sequence and length SOCS1 and SOCS3, which appear to have a unique ability to inhibit JAK catalytic activity, contain a Kinase Inhibitory Region (KIR) adjacent to the SH2 domain that is critical for their inhibition of JAK activity SOCS4, 5, 6 and 7 are distinguished from other SOCS proteins by an extended N-terminal region, which varies from 270 to 385 amino acid residues in length for the mouse proteins + T cells towards a Th1 phenotype and has been suggested to bind the IL-4 receptor (IL-4R) α chain via the first 100 residues of its N-terminal region, displacing JAK1 from the receptor complex to inhibit further signaling Leishmania majorWhilst the physiological functions of SOCS1-3, and to a lesser extent those of CIS, SOCS6 and SOCS7 have been described, the biological roles of SOCS4 and SOCS5 remain poorly characterised. Currently, SOCS5 is thought to negatively regulate interleukin (IL)-4 signaling, polarizing CD4Growing evidence now points towards a role for SOCS5 as a tumor suppressor. Early studies utilising exogenous expression of SOCS5 suggested a role in inhibition of EGF signaling, with SOCS5 shown to interact with the EGF-R complex in a ligand-independent manner The EGF-R and JAK are both validated targets for the treatment of human cancer, with inhibitors in use in the clinic and in phase III clinical trials Asc I and Mlu I restriction sites at the N- and C- termini respectively and sub-cloned into the mammalian expression vector pEF-FLAG-I, a derivative of the mammalian expression vector pEF-BOS The cDNA encoding SOCS5 has been described previously 175–244 sequence. The construct was ligated into the pGEX-2T vector  via EcoRI sites and transformed into E. coli BL21 (DE3) cells. SOCS5175–244 was expressed as a fusion protein with a glutathione S-transferase (GST) tag in 1 L of Luria-Bertani medium. The cells were grown to an OD600 0.8 at 28°C, cooled to 18°C and protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 20 h at 18°C. The fusion protein, expressed as a soluble protein, was purified using glutathione-SepharoseTM 4B  according to the manufacturer's instructions. One unit of TEV per 20 mg of fusion protein was used to cleave at 4°C for 20 h on a rotating mixer. The polypeptide corresponding to SOCS5175–244 was purified from the cleavage mixture by RP-HPLC  using a gradient of 20% to 60% acetonitrile and 0.1% trifluoroacetic acid over 20 min. The purity of SOCS5175–244 was confirmed by analytical RP-HPLC and the molecular mass determined by LC-MS (8103 Da).The fragment in the N-terminus of mouse SOCS5 (residues 175–244), corresponding to the region conserved in SOCS4, was amplified from SOCS5 cDNA and engineered to contain a Tobacco Etch Virus (TEV) protease cleavage site upstream of the SOCS5E. coli expression vectors encoding human SOCS5  and elongin B/elongin C  were co-transformed into BL21(DE3) cells for expression and purification of the trimeric SOCS5-SH2-SOCS box-elongin B/elongin C complex (GST-SOCS5-SH2 Elo B/C). Cells were grown to an O.D. of 0.8 at 37°C, cooled and protein expression induced with 1 mM IPTG for 12–16 h at 18°C. Cells were collected by centrifugation and lysed in phosphate buffered saline (PBS) containing 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 1 mM phenylmethylsulfonyl fluoride  and 0.005% (w/v) hen egg white lysozyme (Sigma) by sonication in a Sonoplus sonicator (BANDELIN). Affinity purification was performed using gravity filtration with glutathione-SepharoseTM 4B according to the manufacturer's instructions. Recombinant GST-SOCS5-SH2 Elo B/C was further purified by size exclusion chromatography with a HiLoadTMSuperdexTM 200 (16/60) column (Pharmacia Biotech) at 1.0 mL/min flow rate in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM TCEP.Recombinant SOCS5-SH2 domain was engineered to contain an N-terminal GST-tag and included the SOCS box sequences for increased stability and solubility when expressed as a ternary complex with elongins B and C, as previously described The construct for expression of recombinant murine SOCS3 lacks the first 21 amino acids and has the PEST motif (residues 129–163) replaced by a Gly-Ser x4 linker, as these modifications enhance its stability and solubility. SOCS3 protein was expressed and purified as described Recombinant JAK JH1 domains were expressed in insect cells and purified essentially as described was kindly provided by Dr. Nadia Kershaw  and was expressed and purified essentially as described Anti-phosphoJAK1 antibody was obtained from Biosource. Antibodies to phosphoJAK2 were obtained from Cell Signaling. The pan-anti-phosphotyrosine antibody (4G10) was obtained from Millipore. Rat anti-Flag antibody was a kind gift from Prof. D. Huang & Dr. L. O'Reilly, . The anti-SOCS5 antibody was generated in-house and is a mouse monoclonal antibody directed against the SOCS5 N-terminal region.293T cells Firefly luciferase reporter gene [p(Ie-IL4RE)4-luc] Renilla luciferase downstream of the Herpes-simplex virus thymidine kinase promoter (HSV-TK) (Promega). Cells were incubated overnight with or without 10 ng/mL recombinant human IL-4 (R&D systems) prior to lysis with 100 μL of Reporter Lysis Buffer (Promega) containing protease inhibitors . Firefly and Renilla luciferase activities were quantified using substrate reagents from the Luciferase Assay Dual-Reporter kit (Promega) and an automated LUMIstar Galaxy plate reader (BMG Technologies). SOCS expression was analyzed by Western blotting with polyclonal rat anti-Flag antibody.293T cells were transiently transfected with 500 ng of an IL-4-responsive promoter-3VO4 and 1 mM NaF. Proteins were immunoprecipitated using anti-Flag antibody conjugated to Sepharose . Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and electrophoretically transferred to Biotrace polyvinylidene fluoride  or nitrocellulose membranes (Amersham). Membranes were blocked overnight in 10% w/v skim milk and incubated with primary antibody for 2 h. Antibody binding was visualized with peroxidase-conjugated goat anti-rat immunoglobulin (Southern Biotech), sheep anti-rabbit immunoglobulin (Chemicon), sheep anti-mouse immunoglobulin , or goat anti-mouse immunoglobulin, light chain specific (Jackson ImmunoResearch) and the enhanced chemiluminescence (ECL) system (Amersham or Millipore). To re-blot, the membranes were stripped of antibodies in 0.1 M glycine, pH 2.9.Cells were lysed in KALB lysis buffer 2O2/25 μM Na3VO4) for 30 min and lysis in 1% NP-40 buffer . Cell lysates were pre-cleared with protein-A-Sepharose for 1.5 h prior to immunoprecipitation of Flag-tagged Shc-1 proteins with anti-Flag M2 affinity gel (Sigma). Co-immunoprecipitated SOCS5 protein was detected by Western blotting with in-house anti-SOCS5 antibodies.Cells were pre-treated with 10 μM MG132 (Sigma) for 3 h followed by treatment with pervanadate solution .JAK1 2, 2 mM MgCl2 and 2 mM DTT). The eluted proteins were then concentrated, mixed appropriately and incubated with 10 mM ATP and 1 mg/mL GST-Jak2 activation peptide as a substrate Constructs encoding full-length, Flag-tagged JAK1, SOCS1, SOCS3 and SOCS5 were independently transfected into 293T cells, and cells were lysed as described for immunoprecipitation and Western blotting. Proteins were then immunoprecipitated with anti-Flag antibody and eluted from the M2 Sepharose resin using 5× bead volumes of Flag peptide (Sigma Aldrich) at a concentration of 100 µg/mL in kinase assay buffer  to visualize proteins and were subsequently analyzed using a PhosphorImager.GST-JAK2 substrate peptide (1 mg/mL) was incubated with 50 nM JAK1 JH1 at 25°C for 15 min in kinase assay buffer together with 1 mM ATP and various concentrations of either recombinant SOCS3 or SOCS5All SPR experiments were conducted on a BIAcore3000 instrument.175–244 was diluted to 10 μg/mL in 10 mM sodium acetate, pH 4.5 and immobilised by amine coupling to a CM5 Biosensor chip  according to the manufacturer's specifications. A reference flow cell was prepared by the same procedure in the absence of protein. Increasing concentrations (62.5 nM to 2 µM) of recombinant JAK JH1 (kinase) domain were injected at a rate of 20 µL/min for 2 min in running buffer . The surface was regenerated between injections with 50 mM NaOH at a rate of 20 µL/min for 20 s. The reference flow cell sensorgrams were subtracted from the ligand flow cell sensorgrams for all analyses. Saturation curves were obtained by plotting the response signal at equilibrium as a function of the analyte concentration. These curves were fitted to a steady-state model to derive the apparent equilibrium dissociation constant (KD). KD values were representative of three (JAK1) and one (JAK3 and TYK2) experiments. GraphPad Prism software  was used for the analysis of steady-state SPR data.Recombinant SOCS510 competitor peptide concentration versus percentage of total binding was then used to fit a non-linear regression and an equilibrium dissociation constant determined. KD values are representative of two replicate experiments. All phosphopeptides were purchased from GL Biochem, China.The binding affinities of the SOCS5-SH2 domain for different tyrosine phosphorylated peptides were determined by a competitive binding assay. Shc-1 pY317 was immobilised by amine coupling to a CM5 Biosensor Chip  according to the manufacturer's instructions. Recombinant GST-SOCS5-SH2 Elo B/C was then mixed at 0.1 µM with serially diluted competitor peptides (41 nM to 10 µM) in running buffer  and injected onto the chip at a flow rate of 20 µL/min, for 2 min. Non-specific binding of the recombinant protein to a reference lane on the chip was subtracted within the experiment and regeneration was achieved with 10 mM glycine-HCL, pH 2.0. The binding affinities of the competitor peptides were determined by steady-state analysis as follows. The measured response of SOCS5 binding in the presence of competitor was expressed as a percentage of total binding (SOCS5 binding to immobilised Shc-1 pY317 peptide in the absence of competitor). A plot of the logGiven that SOCS1 and SOCS3 have been reported to interact directly with JAK and inhibit catalytic activity To investigate whether SOCS5 preferentially inhibited JAK1 activation in this system, 293T cells were transiently transfected with expression vectors encoding Flag epitope-tagged JAK1, JAK2, JAK3, or TYK2 with or without Flag-tagged SOCS1 or SOCS5. Proteins were immunoprecipitated using anti-Flag antibody and JAK phosphorylation assessed using phosphospecific or anti-phosphotyrosine antibodies, as indicated. Co-expression of SOCS5 dramatically inhibited JAK2 , but didTo determine which regions of SOCS5 were required for inhibition of JAK1 activation, SOCS5 mutants which lacked either the entire N-terminus  or part thereof , or contained a mutated SH2 domain  or SOCS box , were generated to express proteins with N-terminal Flag epitopes. We also assessed the functional importance of the region adjacent to the SOCS5-SH2 domain by mutating His360  in vitro kinase assays were performed examining JAK autophosphorylation. 293T cells were transiently transfected with constructs encoding Flag-tagged JAK1 and either Flag-tagged SOCS1 or SOCS5, lysed, and the proteins immunoprecipitated using anti-Flag antibodies. Immunoprecipitates were then incubated in the presence of [γ-32P]-ATP and phosphate incorporation analyzed. Expression of either SOCS1 or SOCS5 inhibited JAK1 autophosphorylation , which was predicted to contain some secondary structural features 175–244 fragment was immobilised by amine coupling to a CM5 biosensor chip and the binding affinity for recombinant JAK1 JH1 domain measured by SPR. The SOCS5175–244 fragment bound the JAK1 kinase (JH1) domain with an equilibrium dissociation constant (KD) of 0.5 μM (175–244 bound with comparable affinity to the JAK3 and TYK2 JH1 domains (1–2 μM) . This indicates that the region corresponding to SOCS5175–244 has the potential to bind all four JAK kinases, but an additional region/s of SOCS5 determines the selective inhibition within the JAK family. We therefore propose that the region of the SOCS5 N-terminus encompassing residues 175–244 be termed a JAK interaction region (JIR).The SOCS5f 0.5 μM  demonstrf 0.5 μM . SOCS517(1–2 μM) . By immoHaving established that SOCS5 bound directly to the JAK1 JH1 via its JIR, we next investigated whether this region was functionally important. SOCS5 has previously been shown to inhibit IL-4-induced Stat6 activity in vitro kinase assay. In contrast to recombinant SOCS3, the addition of the JIR to the reaction only inhibited JAK1 kinase activity at high levels  significantly impaired the inhibitory effect of SOCS5 on JAK1 activity  and, as Mutation of the SOCS5-SH2 domain had only a modest effect on JAK1 phosphorylation . In addiD 0.5 µM) The SOCS4 and SOCS5-SH2 domains share over 92% amino acid sequence homology, suggesting a potential functional overlap in substrate binding. As a first step towards identifying the relevant SOCS4 or SOCS5-SH2 domain interacting partner(s), a complex consisting of GST-SOCS4-SH2 and SOCS box coupled with elongins B and C, was used as bait to affinity-purify proteins from EL4 cell lysates treated with pervanadate and MG132, followed by on-column tryptic digest and Orbitrap LC-MS/MS analysis   (Shc-1 pY317 peptide was immobilised and a competitive SPR binding assay established to test binding to GST-SOCS5-SH2 Elo B/C. The Shc-1 pY317 phosphopeptide bound the SOCS5-SH2 domain with a  (pY239) . Bindingctively) ; the relKD of 0.87 µM   0.87 µM , compara 0.87 µM , indicatTo explore the binding interface on the SOCS5-SH2 domain, it was modelled in complex with the Shc-1 Tyr317 phosphopeptide. The highly related SOCS4-SH2 domain structure To confirm that SOCS5 interacts with full-length Shc-1 protein, 293T cells were transiently transfected with expression vectors encoding Myc epitope-tagged SOCS5 in the presence or absence of Flag-tagged Shc-1 or Flag-tagged SOCS5 alone. Cells were treated with MG132 for 3 h to inhibit the proteasome, and sodium pervanadate for 30 min to inhibit phosphatase action and ensure that Tyr317 in Shc-1 was phosphorylated. Cells were lysed and proteins immunoprecipitated using anti-Flag antibody, followed by Western blot with anti-SOCS5 antibody. SOCS5 was specifically associated with Shc-1 immunoprecipitates; whilst Shc-1 phosphorylation was confirmed by reprobe of anti-Flag immunoprecipitates with a phospho-specific antibody for Shc-1-Tyr317 .Collectively, these results reveal a potential new mechanism by which SOCS5 may play a role in regulating Ras/MAPK signaling, not only in the context of EGF and growth factor signaling, but also in the context of increased phosphorylation of Shc-1, as occurs during oncogenic signaling.Very little is known regarding the signaling cascades regulated by SOCS4 and SOCS5, and while both JAK and the EGF-R have been suggested as potential targets, our understanding of the biochemical mechanism/s of action employed by these two proteins is limited, and largely inferred from our knowledge of other SOCS family members. Here, we have shown using co-expression in 293T cells that while SOCS5 can specifically interact with all four JAKs it selectively inhibits the autophosphorylation of JAK1 and JAK2. The interaction is likely to be mediated by the identified, conserved JAK interacting region (JIR) in the SOCS5 N-terminus, whilst the inhibition appears to require an additional region within the SOCS5 N-terminus. Given that by homology, the JIR is also present in the SOCS4 N-terminus While caveats must be applied to observations obtained using overexpressed proteins, our results revealed a striking specificity in the ability of SOCS5 to regulate JAK, with selective inhibition of JAK1 and JAK2, but not JAK3 or TYK2 phosphorylation. Specificity did not appear to be determined by interaction of the SOCS5 JIR with JAK, as this region appeared to bind similarly to the JAK1, JAK2, JAK3 and TYK JH1 domains. Deletion analysis of the SOCS5 N-terminus indicated that additional residues, yet to be defined  in the SOCS5 inhibition of JAK phosphorylation, it is likely to have a more important role in a physiological setting. Prior to this study, no substrates had been identified for the SOCS5-SH2 domain. Our preliminary peptide binding analysis suggests a preferred consensus of “P X pY Φ N Φ S” where X denotes any residue, and Φ denotes any hydrophobic residue, and enables candidate binding targets to be interrogated for SOCS5 substrate sequences. We note that neither the JAK1 nor JAK2 JH1 domain contains a sequence corresponding to this consensus. Our studies have identified Shc-1 as a novel candidate for regulation by SOCS5. The measured binding affinity of the SOCS5-SH2 domain for Tyr317 in Shc-1 0.16 μM, , is compEGF activation of the Ras-mitogen activated protein kinase (MAPK) pathway occurs through the recruitment of Grb2 and Shc-1 to tyrosines within the EGF-R cytoplasmic domain Consistent with their high sequence homology, the SOCS4 and SOCS5-SH2 domains bind with comparable affinity to the Shc-1 Tyr317 phosphopeptide (data not shown), suggesting that these proteins might be functionally redundant in their ability to regulate Shc-1 pathways. The role of the SOCS5 N-terminus remains unclear in this context, although our previous work suggests that the N-terminus is required for recruitment to the EGF receptor complex prior to ligand stimulation The SOCS5 interaction with Shc-1 is likely to have wider consequences than regulation of EGF signaling. Shc-1 is involved in transducing signals from many tyrosine kinase receptors, such as the insulin receptor, c-Met and M-CSF receptor Although preliminary, our data show that via specific regions within its N-terminal region, SOCS5 has the potential to regulate JAK1 or JAK2 activity, while both SOCS4 and SOCS5 may retain the ability to regulate Shc-1-mediated signaling through binding of their SH2 domains to Tyr317. In conclusion, this study identifies two distinct mechanisms by which SOCS5 can regulate cytokine and growth factor signaling, and positions SOCS5 as a potential regulator of multiple growth and chemotactic stimuli, many of which are pivotal to cellular transformation and metastatic disease. Future work will address the significance of these observations in animal models of tumorigenesis.File S1Suppressor of cytokine signaling (SOCS) 5 utilises distinct domains for regulation of JAK1 and interaction with the adaptor protein Shc-1.(DOCX)Click here for additional data file.


Ki = 44 μM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for β2- and β3-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions.The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself ( Catechol O-methyltransferase (COMT) is an enzyme that has been implicated in the perception of mechanical, thermal, and inflammatory pain in both humans and rodents -3. Its aWhile commonly associated with antinociception ,6, serot2 – and β3–adrenergic receptor pathways -adenosyl-L-methionine  [2, and 1 μM DTT with a total reaction volume of 100 μL.Activity of COMT was measured through a series of experiments where the enzyme was incubated with the substrate catechol (dissolved in HCi/mmol) . ReactioTo determine the linear range of the activity assay, SAM concentration (1 μM) was held constant while COMT concentration was varied (10 – 80 ng/μL). Each reaction was placed in a clean PCR tube and incubated at 37°C for 30 minutes. After incubation, reactions were terminated by addition of 100 μL of 1 M HCl to each tube. Radiolabeled catechol products were extracted from the reaction by adding 10 mL of scintillation fluid  and quantified using a scintillation counter. Activities were normalized for each reaction by performing a duplicate reaction with the COMT inhibitor, OR-486 , and subtracting its radioactivity. We found that the linear range was between 10 and 40 ng/μL, and thus for all subsequent experiments we used a COMT concentration of 20 ng/μL.Kinetic experiments were performed at a variety of catechol concentrations  while maintaining constant SAM (1 μM) and COMT (20 ng/μL) concentrations. Reactions were set-up for a variety of time points: 0, 5, 10, 30, 60, and 120 minutes. Each time-point reaction is incubated at 37°C and then quenched with 1 M HCl at its specified time point. Products were quantified as discussed above. Serotonin-inhibition experiments were conducted for the same catechol concentrations, but with the addition of 100 μM of serotonin (Sigma) to each reaction.Kinetic parameters were determined by first fitting the data sets to a single-site Michaelis-Menten model (for reference), followed by a two-site Michaelis-Menten model as given by :Ki  of inhibition for both a competitive and non-competitive inhibitor, and compared which Ki,app yielded the lowest value. The apparent Ki for a competitive inhibitor is given bywhere catechol is abbreviated as “Cat.” To determine the mechanism of serotonin-inhibition, we calculated the apparent Km,app is the apparent Km,Cat for the serotonin-inhibited curve. The apparent Ki for a noncompetitive inhibitor is given bywhere max,app is the apparent Vmax for the serotonin-inhibited curve.where VSubjects were naïve, young adult (6–14 weeks old) outbred CD-1® (ICR:CrI) mice of both sexes . Animals were housed in groups of 2 to 6 with same-sex littermates, with food (Harlan Teklad 8604) and tap water available ad lib. Approximately equal numbers of male and female mice were used; no sex differences were noted so data from both sexes were pooled. All procedures followed the guidelines and regulations of the Canadian Council on Animal Care, and were approved by the McGill Downtown Animal Care and Use Committee.Mechanical sensitivity was measured using the automated von Frey test. Mice were placed in individual transparent Plexiglas cubicles set atop a perforated metal floor (with 5-mm diameter holes 7 mm apart), and separated from each other by opaque dividers. Prior to testing, the mice were habituated to their surroundings for 1 h. A von Frey fiber with automatically increasing force (Ugo Basile Dynamic Plantar Aesthesiometer) was applied to the mid-plantar hind paw. Three separate withdrawal threshold determinations (on each hind paw) were taken at baseline (pre-injection) and then averaged. One withdrawal threshold determination (on each hind paw) was taken at every post-injection time point.For the SAM pre-treatment study, after measuring baseline thresholds, mice received i.p. injections of S- (5’-Adenosyl)-L-methionine chloride (80 mg/kg) (Sigma), followed, 15 min later, by 20 μl i.pl. injections of serotonin hydrochloride (10 uM) (Sigma). Mice were tested at 15, 30, 45, 60, 90, 120, 180 and 240 min following the serotonin injection.For the β-adrenergic antagonist study, mice received 20 μL i.pl. injections of serotonin hydrochloride (5 uM), followed, 15 min later, by i.p. injection of a cocktail of ICI118,551 (5 mg/kg) and SR59230A (50 mg/kg) (Tocris). Mice were tested at 15, 30, 45, 60, 90, 120, 180 and 240 min following the antagonist injection.Pain hypersensitivity was quantified as the area over the time-threshold curve (post-injection), using the trapezoidal method.We utilize Generalized Estimating Equations (GEE) to determine whether SAM pretreatment or co-administration of SR and ICI treatment versus serotonin alone resulted in significantly different paw withdrawal thresholds  across tThe authors declare that they have no competing interests.DT performed the in silico simulations, SPR experiments, COMT activity assays, and wrote the paper. J.S.W. performed the mouse model studies and wrote the paper. N.R. performed the GEE analysis and wrote the paper. R.E.S. performed mouse model studies. R.L.R. performed SPR experiments. S.K.S. developed the COMT activity assay. J.S.M. designed the mouse model experiments and wrote the paper. W.M. conceived of the hypothesis, designed the activity assay experiments, and wrote the paper. N.V.D. designed the in silico experiments, developed the hypothesis, and wrote the paper. L.D. designed the activity assay experiments, developed the hypothesis, and wrote the paper.


Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants . In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402.Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca Neuronal communication in the mammalian brain mainly depends on the release of neurotransmitters Studies have shown that various protein tyrosine kinases (PTKs) are expressed in the mammalian central nervous system (CNS) 2+ influx, thereby contributing to neuronal cell apoptosis and death Pyk2 autophosphorylation has a key function in multiple intracellular signals, including the activation of migration in macrophages 2+ influx. At low concentrations, ionomycin, a Ca2+ ionophore, can increase intracellular Ca2+ concentrations in neurites at a higher extent than in neuronal cell bodies, resulting in early neurite degeneration 2+ overload. Results showed that ionomycin-induced neurotransmitter release was dependent on Y402 phosphorylation of Pyk2. Src, as a downstream signal molecule of Pyk2, exhibits an antagonistic action during neurotransmitter release. Src inhibition may prolong the duration of neurotransmitter release by extending the phosphorylation time of Pyk2 on Y402. These findings suggested that Pyk2 was involved in neurotransmitter release through Y402 phosphorylation.The increase in intracellular calcium is believed to be a major cause of neurotransmitter release and activation of Pyk2. Thus, Pyk2 may be involved in neurotransmitter release in response to increased Ca2+ triggers neurotransmitter release in at least two principal modes, synchronous and asynchronous release 2+ in cells. p<0.001 iono vs. control at 0 min to 2 min, Student’s t test). DA release reached its peak in 0 min to 2 min, and declined immediately in 2 min to 4 min . Ionomycin-induced Glu release from cerebellar granule cells (CGNs) showed similar changes .Ca changes  with DA To identify the intracellular tyrosine-phosphorylated proteins involved in ionomycin-induced neurotransmitter release, PC12 cells incubated with 1 μΜ ionomycin were collected and tested by western blot using an anti-phosphotyrosine antibody. −/− mice post-hoc Newman-Keuls, F3,28 = 346.2, ***p<0.001 Pyk2 siRNA+iono vs. Control siRNA+iono). The result confirmed the involvement of Pyk2 in ionomycin-induced neurotransmitter release.Pyk2 expression was inhibited by the introduction of Pyk2 siRNA in PC12 cells to investigate the function of Pyk2 in ionomycin-induced neurotransmitter release. Tyr-402, the major site of Pyk2 autophosphorylation upon activation, is essential for the induction of Pyk2 kinase activity, whereas Tyr-579, Tyr-580, and Tyr-881 further enhance Pyk2 kinase activity post-hoc Newman-Keuls, F5,18 =  = 8.017, *p<0.05). Transfection of Pyk2-WT and other mutants  could induce significant phosphorylation on Y402 compared with the Mock group (**p<0.01). These results indicate that Y579, Y580, and Y881 did not affect Y402 phosphorylation during the short duration of ionomycin stimulation  and the vector expressing wild-type Pyk2 (Pyk2-WT) were constructed to further investigate the role of Y402 in neurotransmitter release. The Pyk2 mutants and Pyk2-WT were individually transfected into PC12 cells. The transfection efficiencies exceeded 80% in PC12 cells . Under imulation .post-hoc Newman-Keuls, F5,42 = 43.02, **p<0.01), whereas Pyk2-Y402F transfection inhibited DA release compared with the mock (*p<0.05) and Pyk2-WT groups (###p<0.001). Other mutants performed similarly to Pyk2-WT, with no statistically significant difference. These results were consistent with the phosphorylation level of Y402.To investigate the function of different tyrosine sites of Pyk2 in ionomycin-induced DA release, the aforementioned vectors were transfected into PC12 cells. 2+-dependent neurotransmitter release from neuronal cells post-hoc Newman-Keuls, F2,21 = 22.12, *P<0.05 PP2 vs. Control, #p<0.05 PP2 vs. PP3). PC12 cells were transfected with empty vector (Mock) and Pyk2 mutants to determine the function of different tyrosine sites of Pyk2 in PP2-enhanced neurotransmitter release. post-hoc Newman-Keuls, F5,42 = 25.94, *p<0.05 PP2+Mock vs. Mock), whereas the enhancement was removed when PC12 cells were transfected with Pyk2-Y402F (#p<0.05 PP2+Pyk2-Y402F vs. PP2+Mock). By contrast, other mutants could further enhance PP2-induced DA release (##p<0.01 vs. PP2+Mock). This result indicates that Y402 of Pyk2 was essential for PP2-induced DA release, and other tyrosine sites were not equally important as Y402 in this process. The phosphorylation state of Pyk2 at various tyrosine sites was monitored after 2 min of stimulation with ionomycin. The result confirmed that the phosphorylation changes in Y402 between different groups were the associated factors in this process . The phosphorylation state of Src was also monitored. Src tyrosine kinase was activated at 4 min, and its activity was maintained until 6 min after ionomycin stimulation in the mock group; the added PP2 masked this tyrosine phosphorylation changes . Similarly, Src siRNA enhanced DA release in 2 min to 4 min , which indicates that Src inhibition could extend the duration of Pyk2 autophosphorylation and increase neurotransmitter release.To illustrate the relationship between Src and Pyk2 in ionomycin-induced neurotransmitter release, we used siRNA to inhibit Src expression. Pyk2 can perform several post-synaptic functions, such as enhanced post-synaptic NMDA-R phosphorylation 2+ triggers neurotransmitter release in at least two principal modes, synchronous and asynchronous release 2+ waves induced by action potentials 2+2+ ionophore, when added to the medium will spontaneously incorporate into the neuronal plasma membrane, which allows Ca2+ to enter nerve terminals directly, and triggers asynchronous transmitter release. Our study revealed that Pyk2 , among which Y402 was recognized as the main autophosphorylation site, and termed as the activated marker of Pyk2. To illustrate which site is involved in neurotransmitter release, site-specific phosphorylation antibody was used to test their phosphorylation degree individually. Our results demonstrate that ionomycin-induced calcium influx activated tyrosine phosphorylation of Pyk2 on Tyr-402 in PC12 cells, whereas tyrosine phosphorylation of the other three sites  did not cause any changes, at least during the monitor time . In addiWe constructed different vectors carrying Pyk2-WT and the four Pyk2 mutants  to verify the function of different tyrosine sites on ionomycin-induced neurotransmitter release. As shown in Based on our results, Pyk2-Y402 is essential in ionomycin-induced neurotransmitter release. However, Ohnishi To illustrate the relationship between Pyk2 and PP2 in neurotransmitter release, we added PP2 to the PC12 cells transfected with four kinds of mutants. The result shows that Y402 mutant could abolish the effect of PP2 compared with other mutants. Thus, PP2 could enhance neurotransmitter release, but that was Pyk2-Y402-dependent .2+-dependent neurotransmitter release, and this process associated with dephosphorylation of Src and its substrate Pyk2 and paxillin 2+ concentrations are increased. Activated Pyk2 then facilitates neurotransmitter release by interacting with or activating synaptic-related proteins. Later, the enhancement of intracellular Ca2+ concentrations contribute to the activation of Src. Src further activates other tyrosine sites of Pyk2 and paxillin, which possibly contributes to the polymerization of actin skeleton 2+-induced neurotransmitter release. An inhibitor of src family kinases, PP2 may contribute to neurotransmitter release by blocking this negative feedback . Animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, Chinese Academy of Medical Sciences and Peking Union Medical College, and the 1996 Guide for the Care and Use of Laboratory Animals .The following antibodies were used: rabbit polyclonal Pyk2 phosphorylation site-specific antibody  from Biosource International ; anti-phosphotyrosine monoclonal antibody (clone 4G10) from Upstate Biotechnology ; mouse monoclonal anti-Pyk2, actin antibodies, rabbit polyclonal anti-FAK, Src, actin, Fyn antibodies, horseradish peroxidase (HRP)-conjugated anti-mouse IgG, anti-rabbit IgG, rhodamine (TRITC)-conjugated anti-rabbit IgG, FITC-conjugated anti-mouse IgG, and Human Pyk2 transfected 293T whole cell lysate (sc-115595) were obtained from Santa Cruz Biotechnology .Dulbecco’s modified eagle medium (DMEM), and reduced serum medium Opti-MEM were purchased from Gibco . Lipofectamine LTX and Lipofectamine RNAiMAX were purchased from Invitrogen . Fetal bovine serum (FBS) and horse serum were obtained from Hyclone . Ionomycin was obtained from Sigma . Mouse Nerve Growth Factor for Injection (NGF) was obtained from Staidson . Polyvinylidene fluoride (PVDF) membrane was obtained from Millipore , and protein G-Sepharose was obtained from Amersham Pharmacia Biotech. Prolong antifade kit was purchased from Molecular Probes . Other materials were obtained from standard suppliers or as indicated in the text.5′-CCGGAATTCGCCACCATGTCCGGGGTGTCTGA-3′ (sense) and 5′-CCGCTCGAGTCACTCTGCAGGCGGGT-3′ (antisense).Pyk2 cDNA was amplified from RNA of rat pheochromocytoma (PC12) cells by PCR using the following composite primers encoding both ends of the rat Pyk2 open reading frame flanked with EcoRI and XhoI adapter as follows: The Pyk2 cDNA fragment was cloned into pMD18T vector, and then subcloned into the pcDNA3.1 vector using EcoRI and XhoI.The mutants of Pyk2  were generated by replacing tyrosine 402, 579, 580, and 881 with phenylalanine using site-directed mutagenesis, according to the manufacturer′s protocol . Primers designed for these mutants were as follows:TTGCAGAGATT-3′5′-AGCATAGAGTCAGACATCT (sense),Pyk2-Y402F: AAGATGTCTGACTCTATGCTACAGCTTTCT-3′5′- (antisense);TTTACAAAGCTT-3′5′-ACATTGAGGATGAGGACT (sense),Pyk2-Y579F: AAGTCCTCATCCTCAATGTACCGGGAGA-3′5′- (antisense);TCAAAGCTTCCG-3′5′-ATTGAGGATGAGGACTATT (sense),Pyk2-Y580F: AAATAGTCCTCATCCTCAATGTACCGGGAGA-3′5′- (antisense);TCCACAATGTCA-3′5′-ACAGGACTGATGACCTCGTGT (sense),Pyk2-Y881F: AACACGAGGTCATCAGTCCTGTCCAAGTT-3′5′- (antisense).The mutations were confirmed by sequence analysis.Pyk2 siRNA, Src siRNA, and their control siRNA were purchased from Santa Cruz Biotechnology, Inc. siRNA products generally consist of pools of three to five target-specific 19 nt to 25 nt siRNAs designed for knockdown of gene expression.5 cells/cm2, and maintained in DMEM with 5% heat-inactivated FBS and 5% horse serum at 37°C in a humidified atmosphere with 10% CO2.PC12 cells were plated on polyethylenimine-coated four-well plates  at a density of 5×105 cells/cm2. The cells were maintained in culture medium  containing 10% heat-inactivated FBS at 37°C in a humidified 10% CO2 atmosphere. Experiments were conducted after 19 d to 20 d of culture Cerebellar granule cells were dissected from seven-day-old Wistar rats , and incubated in 0.2% trypsin and 0.02% DNase I for 15 min at 37°C. The trypsinized tissue was triturated with a Pasteur pipette until no tissue aggregates were observed. The cells were then washed with DMEM containing 10% heat-inactivated FBS, and plated on polyethylenimine-coated four-well plates at a density of 5×105 cells/cm2. Thereafter cells received NGF (100 ng/ml), and were immediately plated on poly-L-lysine coated dishes. Cells undergoing differentiation were observed after 2 days PC12 cells were washed twice with PBS and resuspended in serum-free DMEM for 1 h at a concentration of 2×106 cells/well, and cultured for 24 h before transfection of plasmid with Lipofectamine or RNA oligonucleotides with Lipofectamine RNAiMAX according to the manual . The medium was changed after 6 h. Cells were used for the experiments after 48 h unless otherwise indicated in the text.Two days before the experiments, cells were plated on poly-L-lysine-coated four-well culture dishes at a density of 1.0×10Co-transfection was performed to investigate the transfection efficiency. According to the procedure above, PC12 cells were transfected with 4 μg of pcDNA3.1 and 0.4 μg of pEGFP-N1, which expresses green fluorescence protein (GFP), in each well of six-well culture dishes. The green fluorescence was tested by immunofluorescence microscopy after 48 h. A total of 700 to 800 cells were counted in six to eight representative fields to calculate the transfection efficiency.+ solution . After pretreatment in various conditions as indicated in the figure legends, the cells were incubated for 2 min in low-K+ solution. The release of neurotransmitters during this period represented the basal release (−2 min to 0 min). The buffer was immediately changed after 2 min with low-K+ solution containing 1 μM ionomycin to stimulate Ca2+ influx (0 min to 2 min). The buffer was changed every 2 min with low-K+ solution. Sample buffer solutions were immediately collected into microtubes on ice at the end of each incubation period. For DA detection, the solutions were transferred to a microtube containing 100 μl of 0.1 M perchloric acid (PCA) on ice. After the experiments, the cells were sonicated on ice in 500 μl of chilled 40 mM PCA and 0.1 mM EDTA. The samples were centrifuged at 15,000 rpm for 5 min at 4°C, and the supernatant was stored at −80°C until use. The secreted amount of DA was assayed by high-performance liquid chromatography (HPLC) using a C18 column  and an electrochemical detector system . For glutamate (Glu) measurement, samples were determined by reverse-phase HPLC on an Atlantis dC18 column  using pre-column derivatization with o-phthalaldehyde and fluorescence detection .The release assays were conducted at 37°C. Cells were washed thrice with low-K3VO4). Lysates were clarified by centrifugation at 12,000 rpm for 30 min at 4°C, and the supernatants were incubated overnight at 4°C with the indicated antibodies, followed by protein G-Sepharose incubation for 2 h to 3 h at 4°C, and three washes with lysis buffer. Total cell lysates or immunoprecipitated proteins were separated on 8% SDS–polyacrylamide gel electrophoresis, and transferred onto PVDF membranes with a semi-dry transblotting apparatus. The membranes were blocked in 3% (w/v) bovine serum albumin in Tris-buffered saline containing 0.05% Tween-20, which was incubated subsequently with primary antibodies, followed by HRP-conjugated secondary antibodies, and developed with enhanced chemiluminescence and a luminescent image analyzer . For reprobing, selected membranes were stripped off antibodies by incubation in restore stripping buffer at room temperature for 10 min, followed by washing, blocking, and antibody incubation as described above.Whole cells were lysed in ice-cold buffer  containing protease inhibitors  and phosphatase inhibitors .t test or one-way ANOVA analysis followed by pair-wise comparisons with Newman-Keuls test. A value of p<0.05 was considered significant. All statistical analyses were performed using SPSS13.0 software .All values are presented as means ± S.E.M. Each experiment was repeated at least thrice. Statistical analysis was performed by Student’s Figure S1Contrasting the morphology of treated PC12 cells. (A) PC12 cells were transfected with 100 nM control siRNA for 48 h. (B) PC12 cells were transfected with 100 nM Pyk2 siRNA for 48 h. (C) PC12 cells were differentiated with 100 ng/ml NGF for 48 h. Scale bar, 10 μm.(TIF)Click here for additional data file.Figure S2Transfection efficiency of pcDNA3.1 vector in PC12 cells. Co-transfection was performed in PC12 cells with 4 μg of pcDNA3.1 and 0.4 μg of pEGFP-N1 in each well of six-well culture dishes. The green fluorescence was tested by immunofluorescence microscopy after 48 h. A total of 700 to 800 cells were counted in six to eight representative fields to calculate the transfection efficiency. Scale bar, 10 μm.(TIF)Click here for additional data file.Figure S3Contrasting the effect of PP2 with PP3 on ionomycin-induced DA release. PC12 cells were washed thrice with low-K+ solution, and incubated for 20 min in low-K+ solution with or without (Control) 20 μM PP2 or PP3 for 20 min. The cells were washed thrice, and sequentially incubated for 2 min in low-K+ solution with 1 μM ionomycin. Sample buffer solutions were immediately collected into microtubes on ice after 2 min of incubation period. The amount of DA release in the medium was expressed as the percentage of the total cellular content. The values are expressed as means ± S.E.M. from four representative experiments, n = 8/sample .(TIF)Click here for additional data file.Figure S4Phosphorylation state of Fyn in section five. PC12 cells were transfected with 4 μg of empty vectors  or Pyk2-Y402F for 48 h. The cells were washed three times with low-K+ solution, and incubated for 20 min in low-K+ solution with or without (Mock) 20 μM PP2 for 20 min. The cells were washed thrice, and sequentially incubated for 2 min in low-K+ solution with 1 μM ionomycin. An equal amount of cell lysates harvested at different incubation periods was immunoprecipitated with anti-Fyn antibody, and immunoblotted with anti-phosphotyrosine antibody. The total amount of Fyn was used as an internal control.(TIF)Click here for additional data file.Figure S5Schematic diagram of the hypothesis about Pyk2-involved neurotransmitter release. Increased intracellular Ca2+ concentration causes Pyk2 Y402 autophosphorylation by dimerization. Activated Pyk2 facilitates neurotransmitter release potentially through interacting with or activating synaptic-related proteins. On the other side, increased intracellular Ca2+ concentration also activates Src, which further activates other tyrosine sites of Pyk2 and paxillin, and then contributes to the polymerization of actin skeleton. Neurotransmitter release was inhibited as the result. Thus, Src and its substrates form a negative feedback regulation of Ca2+ induced neurotransmitter release. PP2, the inhibitor of src family kinases, may contribute to neurotransmitter release though inhibiting this negative feedback.(TIF)Click here for additional data file.


RRs consist of a large subunit called RR1 (α), that contains two allosteric sites and one catalytic site, and a small subunit called RR2 (β), which houses a tyrosyl free radical essential for initiating catalysis. The active form of mammalian RR is an αnβm hetero oligomer. RR inhibitors are cytotoxic to proliferating cancer cells. In this brief review we will discuss the three classes of RR, the catalytic mechanism of RR, the regulation of the dNTP pool, the substrate selection, the allosteric activation, inactivation by ATP and dATP, and the nucleoside drugs that target RR. We will also discuss possible strategies for developing a new class of drugs that disrupts the RR assembly.Ribonucleotide reductase (RR) is a crucial enzyme in  Ribon1.1.2β2 and/or αn(β2)m . The reduction of the ribonucleoside diphosphate substrate occurs in the large α subunit, which also has two allosteric sites in addition to the catalytic site (C-site)  see  11,12].,12.2β2 aIn Classes Ia and Ib, the generation of the tyrosyl free radical (Y•) in the β subunit is oxygen dependent. Therefore, these enzymes are expressed under aerobic conditions. The Class Ic enzymes do not generate Y•, but the synthesis of active metal cluster in the β subunit requires oxygen. Here we will be briefly describing the Class Ia enzymes. Readers are encouraged to consult references , 2] and and2] annrdA locus and the β subunit is encoded by the nrdB locus  and enhancing the interactions in the spacer region. We showed from the structure of Cyc10 that some side-chains do not interact extensively with R1. Removing these groups may have little impact on binding, while reducing the molecular weight. Also, efforts will be made to enhance the interactions at subsite 1 by the introduction of hydrophobic groups at the N-terminal and altering the spacer region.In this study, a 2.7 Å resolution crystal structure of Cyc10 bound to ScRR1  99] sho sho99] sin vivo efficacies. Although the in vivo efficacies of the peptidomimetic/peptide-based inhibitors have not been reported in the literature, they have been shown to have reasonable LD50s . However, it should be noted that the best efficacies were obtained when the peptide-based inhibitors were conjugated by poly arginine containing peptides. These preliminary results are quite promising and encouraging for the development of a new class of anticancer agents.It is an interesting question as to if large drug molecules such as the peptide-based molecules described above that defy Lipinski's limits will be able to have Experiments conducted by the Cooperman group and our recent joint efforts show that the P-site is a good drug target for anti-cancer therapy. Molecules targeting the P-site will interfere with the quaternary structure of RR via specific protein-protein interactions, which enable the development of highly specific inhibitors that do not suffer the lack of specificity associated with nucleoside-based analogs that target the A-site, S-site and C-site of RR. Moreover, the P-site consists of two well formed subsites called site 1 and site 2. These two sites will be good candidates for the fragment-based drug design approach.4.3.Although the focus of this review is the inhibition of RR1, we would like to briefly describe some of the efforts involving RR2 inhibition. The inhibitors of the RR2 subunit are either radical scavengers or iron chelators. Hydroxyurea (HU) is the best characterized tyrosyl radical scavenger and has been used to treat various neoplastic and non-neoplastic conditions ,101. Its2dCDP in a wide variety of cancer cell lines [Another class of inhibitors currently under consideration is the iron chelators . Since tll lines . Futhermll lines . Currentll lines -114.5.2CDP [2CDP having low levels of RR1 had better survival rates than those patients who had undergone surgery alone. In contrast, the overexpression of the R2 subunit correlates to increased tumourogenic potential, enhanced invasiveness and celleular transformation [Recent pre-clincial and clinical studies suggest that the individual subunits of RR can be considered as potential biomarkers for predicting outcomes in cancer patients . RR1 sub2CDP ,121. Sev2CDP . Cancer ormation -125. Intormation . Therefo6.2dCDP targets the C-site. As human RR1 is thought to communicate with several protein molecules in the cell, new druggable sites are likely to emerge. In spite of the fact that human RR1 is a rich drug target, only a limited effort has been devoted to developing new drugs against it. As we begin to understand more about how this complicated molecule functions, a greater effort should be devoted to develop new classes of chemotherapies that target hRR1.Ribonucleotide reductase is a well-established cancer and antiviral target. It is emerging as a master enzyme due to its role in maintaining balanced dNTP pools. In a recent review, it has been noted that hRR is a useful biomarker in cancer therapy similar to BRCA1 , and rec


In this study, we found that recombinant Angptl3 treatment, together with the activation of Rac1, could cause F-actin rearrangement in podocytes. We also found that these effects could be blocked by an integrin αVβ3 inhibitor, implicating integrin αVβ3 as the Angptl3 receptor in its effects on actin cytoskeleton rearrangement. In addition, we studied the molecular pathway for this process. Our results showed that in podocytes, Angptl3 could induce actin filament rearrangement, mainly in lamellipodia formation, and that this process was mediated by integrin αVβ3-mediated FAK and PI3K phosphorylation and Rac1 activation. Our results might provide a new explanation for the effect of Angptl3 on increasing podocyte motility.Glomerular podocytes are highly differentiated cells whose foot processes, which are mainly maintained by the architecture of actin filaments, have a unique morphology. A rearrangement of F-actin in podocytes causes changes in their motility that involve foot process effacement and proteinuria in glomerular diseases. Members of the Rho family small GTPases, especially RhoA, Rac1, and Cdc42, are key molecules in the regulation of actin cytoskeleton rearrangement. Our previous study showed that angiopoietin-like 3 (Angptl3) can increase the motility of podocytes  Significant proteinuria is a characteristic manifestation of glomerular filtration barrier damage; moreover, long-term significant proteinuria itself is an important risk factor that can lead to chronic kidney disease and even to end-stage renal disease . The ultPodocytes in the glomerulus form the outer layer of the glomerular filtration barrier and play an important role in maintaining its permselectivity . PodocytThe special morphology of podocytes is based on an underlying network of dynamic and interconnected actin and microtubule polymers. Podocyte foot processes have a subcortical network of branched actin filaments as well as bundled filaments that run longitudinally through the process . In glomActin filaments can exist as networks or bundles that are highly regulated by more than 100 proteins. In podocytes, this dynamic regulation is mainly modulated by membrane proteins that are located in the glomerular basement membrane and slit diaphragm areas . IntegriAngiopoietin-like 3 (Angptl3) is a secreted protein that is mainly expressed in hepatocytes and is weakly expressed in normal kidney cells . Studiesin vitro increases podocyte motility [In our previous study, we found that Angptl3 expression was upregulated in nephrotic syndrome kidney tissue . In addimotility . αVβ3 and is mediated by FAK and PI3K phosphorylation.To explore Angptl3's function in podocytes, we investigated the effect of Angptl3 on the rearrangement of podocyte F-actin and the activation of small GTPases. In this study, we found that Angptl3 could lead to podocyte F-actin rearrangement and Rac1 activation, which is induced through integrin αVβ3 inhibitor echistatin α1 isoform was purchased from Tocris . FAK Inhibitor 14 was purchased from Santa Cruz Biotechnology . PI3K inhibitor LY294002 was purchased from Beyotime Biotechnology Corporation . Fluorescein isothiocyanate- (FITC-) labeled phalloidin was from Sigma-Aldrich . G-LISA RhoA, Rac1, and Cdc42 activation assay kits were purchased from Cytoskeleton Technology. Monoclonal antibodies to RhoA, Rac1, Cdc42, FAK, and PI3K were purchased from Cell Signaling Technology . The monoclonal antibody to phosphorylated FAK was purchased from BD . The polyclonal antibody to phosphorylated PI3K was purchased from Signalway Antibody . The monoclonal antibody to glyceraldehyde phosphate dehydrogenase (GAPDH) was purchased from KangChen Bio-tech . Horseradish peroxidase- (HRP-) conjugated secondary antibodies were purchased from Cell Signaling Technology.Recombinant mouse Angptl3 was obtained from R&D systems . Rac1 inhibitor NSC23766 was purchased from the Cayman Chemical Company . RhoA inhibitor CT04 was purchased from Cytoskeleton . Integrin μg/mL streptomycin. Podocytes were cultured by growth in medium containing 10 U/mL mouse INF-γ  at 33°C with 100% relative humidity and a 5% CO2 atmosphere, and they were induced to differentiate by culture at 37°C in medium without INF-γ for 10 to 14 days. Podocytes became large cells with plenty of small branches when well differentiated. All cell culture dishes were coated with type I collagen (Sigma-Aldrich), and cells never reached 90% confluency.The mouse podocytes used in this study were a conditionally immortalized cell line originated by Dr. Peter Mundel . Cells were cultured and differentiated as previously described . BrieflyThe cells were starved with 0.5% FBS culture media for 1 day when they reached 50–60% confluency and were then serum-depleted overnight, treated with or without inhibitors, and finally treated with 250 ng/mL Angptl3 for an appropriate time before immunofluorescent staining, western blot, or G-LISA assay.RhoA, Rac1, and Cdc42 activation in podocytes was determined with the G-LISA RhoA, Rac1 and Cdc42 activation assays, respectively. The G-LISA assay is a method that measures the quality of GTP-bound, or activated, small GTPases in cell or tissue samples. After treatment, cells were washed with PBS, resuspended in kit lysis buffer and harvested with a cell scraper. The total protein concentration in each lysate was determined by the Precision Red advanced protein assay reagent in the kits. The G-LISA kits contain a RhoA, Rac1, or Cdc42 GTP-binding protein that is immobilized on the provided microplates. Bound active small GTPases were treated with a specific primary antibody and then with horseradish peroxidase- (HRP-) conjugated secondary antibody and HRP detection reagent. The optical density was measured in a Tecan M200 plate reader  at 490 nm.Cells were seeded on 12 mm glass coverslips precoated with type I collagen in a 24-well cluster plate and treated as described above. Cells were then washed twice with PBS and fixed in 4% paraformaldehyde in PBS. The cells were then incubated in 4°C overnight with FITC-labeled phalloidin to stain F-actin and for 5 min with DAPI to stain the nucleus. Coverslips were mounted with antiquench mounting medium. Images were captured on an Olympus IX81 fluorescence microscope  at 400x magnification.Cellular lamellipodia which were defined as the projections that arise from the cell body, including well-defined typical membrane ruffles and finger-like protrusions in the shape of smooth bumps or spikes, were counted. Filopodia were not included in this count. In each group, 10 visual fields were randomly selected, and altogether, 100 cells were analyzed. The results are expressed as the number of lamellipodia per cell.3VO4 and 10 mM NaF), and protease inhibitors (1 mM PMSF), and then the lysates were quantified using the Enhanced BCA Protein Assay Kit . Equal amounts of lysates (30 μg) were loaded on a 10% SDS-PAGE gel and blotted onto PVDF membranes (Millipore Corp.). The samples were blocked in TBS-Tween (TBS-T)  with 5% nonfat dry milk, and the membranes were incubated with primary antibodies at appropriate dilutions in 5% milk TBS-T overnight at 4°C. Subsequently, the membranes were washed three times with TBS-T solution, followed by an incubation with HRP-conjugated secondary antibody (1 : 3000) in TBS-T and 5% milk. The ECL signals were captured using a CCD camera .The cells were washed with TBS and lysed in a buffer containing 50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, phosphatase inhibitors  and the N-terminal coiled-coil domain (CCD). As Camenisch et al.  have repn to GBM . Podocytmotility . We explαVβ3 inhibitor, echistatin α1 isoform, for 1 hour then followed by treatment with Angptl3. The podocyte F-actin rearrangement disappeared  is an intracellular nonreceptor tyrosine kinase and an important modulator of integrin-dependent focal cell contacts, thereby orchestrating well-known integrin-initiated outside-in signaling events. FAK colocalizes with us study  has showThe next experiment was therefore to examine whether the inhibition of FAK can block the effect of Angptl3 on F-actin rearrangement in podocytes. We treated cultured podocytes with FAK inhibitor 14, which can inhibit FAK activation by preventing the phosphorylation of FAK on Y-397 , for 1 hPhosphoinositide 3-kinase (PI3K) is a classic downstream target of FAK and is composed of two subunits, of which the phosphorylation of the p85 subunit is important to its activation . PI3K caWe then used the PI3K inhibitor LY294002 to block PI3K and investigate whether it interferes with the effect of Angptl3 on podocyte morphology and small GTPase activation. Our result showed that the lamellipodia formation process was stopped, but stress fiber formation was unaffected Figures . The G-LαVβ3 regulated the activities of RhoA or Rac1. Since Rac1 activation and lamellipodia formation were the main effects of Angptl3 expression in podocytes, we mainly studied the signaling pathway of this process. The inhibition of FAK or PI3K blocked Angptl3-induced Rac1 activation, and both factors were involved in this process.Next, we explored the possible signal pathway by which Angptl3- integrin α1 or FAK inhibitor 14, respectively, treated cells with Angptl3, and detected the phosphorylation of possible downstream molecules. We found that after the treatment with echistatin isoform α1, the phosphorylation of FAK and PI3K was totally inhibited, and after treating cells with FAK inhibitor 14, PI3K phosphorylation was inhibited. These results showed that cell signals pass through Angptl3, integrin αVβ3, and FAK to activate RhoA, and they may pass through Angptl3, integrin αVβ3, FAK, and PI3K to activate Rac1 in podocytes.We stimulated podocytes with Angptl3 and found that the levels of p-Y397 FAK and p-P85 PI3K both increased . We thenin vitro. Our results indicated that Angptl3 may bind integrin αVβ3 on the surface of podocytes and in turn cause the phosphorylation of downstream molecules FAK and PI3K to active Rac1, which leads to the formation of spikes and lamellipodia in podocytes. Simultaneously, the phosphorylation of FAK may also activate another Rho family small GTPase, RhoA, which can lead to stress fiber formation in podocytes. The stress fiber formation caused by Angptl3 may play a role in protecting podocytes from damage, but this effect is weak and short-lived. The following long-term lamellipodia formation caused by Rac1 stimulation can increase the motility of podocytes, which may aggravate foot process effacement and result in proteinuria.To summarize, our research provided an explanation for the phenomenon that Angptl3 can increase the motility of podocytes αVβ3 is the main receptor for Angptl3-mediated F-actin rearrangement, and Angptl3 binds to integrin αVβ3 through its FLD, this Angptl3 domain is the key component that causes Rac1 and RhoA activation and podocyte F-actin rearrangement. Our result may provide a new method of protecting podocytes and preventing F-actin rearrangement and foot process effacement in proteinuric kidney diseases.Since integrin


TRPA1, one of the transient receptor potential channels, has been reported to be involved in nociception and inflammatory pain, suggesting that this molecule could be a promising target for the development of analgesic agents. We screened several monoterpene analogs of camphor, which is known to inhibit human (h) TRPA1, to identify more effective naturally occurring TRPA1 antagonists. Borneol, 2-methylisoborneol, and fenchyl alcohol exhibited higher inhibitory effects on hTRPA1 activity than either camphor or 1,8-cineole. Our results revealed further that the S873, T874, and Y812 residues of hTRPA1 were involved in the inhibitory effects, suggesting that the hydroxyl group in the six-membered ring of the inhibitors may be interacting with these amino acids. Further research on these identified TRPA1 antagonists could lead to new pain therapeutics.The online version of this article (doi:10.1007/s12576-013-0289-0) contains supplementary material, which is available to authorized users. Transient receptor potential (TRP) channels respond to a wide variety of sensory stimuli, including temperature, nociceptive compounds, touch, osmolarity, and pheromones –3. TRPA1Previous studies showed that the inhibitory effects of AMG5445, a compound partially activating mTRPA1 and inhibiting hTRPA1 , and of  Cinnamomum camphora tree [Several TRPA1 antagonists have been reported: 1,8-cineole contained in eucalyptus oil , camphorora tree , HC-0300ora tree , AZ868 [ora tree , A-96707ora tree , and CMPora tree ). Among ora tree . Severalora tree . Given tora tree .We screened camphor analogs to identify more effective TRPA1 antagonists. From this screening, we found that borneol, 2-methylisoborneol, and fenchyl alcohol exhibited higher inhibitory effects than camphor and 1,8-cineole. In addition, we found that the S873, T874, and Y812 residues of TRPA1 were critically involved in the inhibitory effect of borneol.Full-length hTRPA1 was obtained from Life Technologies . cDNAs were cloned into the pcDNA3.1 vector.Camphor, borneol, fenchyl alcohol, and 2-methylisoborneol were obtained from Wako Pure Chemical Industries Ltd. . (−)-Fenchone, 1,8-cineole, camphorquinone, norcamphor, α,β-thujone, α-pinene oxide, (−)-limonene oxide, (+)-borneol, (−)-borneol, and (±)-isobornyl methyl ether were obtained from Sigma-Aldrich . Bornyl acetate, (±)-isoborneol, and 3-methylene-2-norbornanone were obtained from Tokyo Kasei Co. Ltd. . The compounds were used as a mixture of (+) and (−) isomers unless otherwise stated.l-glutamine  at 37 °C in 5 % CO2. For Ca2+-imaging, 1 μg of plasmid DNA containing hTRPA1 in pcDNA3 in OPTI-MEM medium (Life Technologies) was transfected into HEK293T cells using Lipofectamine Plus Reagent (Life Technologies). Following incubation for 3–4 h, cells were reseeded on coverslips and incubated further at 37 °C in 5 % CO2.Human embryonic kidney (HEK) 293T cells were maintained in DMEM  supplemented with 10 % fetal bovine serum , 100 U/mL penicillin (Life Technologies), 100 μg/mL streptomycin (Life Technologies), and 2 mM 2+-imaging was performed 1 day after transfection. HEK293T cells on coverslips were mounted in an open chamber and superfused with a standard bath solution . Cytosolic-free Ca2+ concentrations in HEK293T cells were measured by dual-wavelength fura-2  microfluorometry with excitation at 340/380 nm and emission at 510 nm. The fura-2 ratio image was calculated and acquired using the IP-Lab imaging processing system . Ionomycin was used to confirm cell viability in the vector-transfected cells.Ca2+-imaging experiments, and extracellular Ca2+ was removed and 5 mM EGTA added for the recording of AITC-, menthol- and FFA-induced current responses. The pipette solution contained 140 mM KCl, 5 mM EGTA, and 10 mM HEPES, pH 7.4 (adjusted with KOH). Data from the whole-cell voltage-clamp recordings were sampled at 10 kHz and filtered at 5 kHz for analysis . The membrane potential was clamped at −60 mV for all conditions. In some experiments, voltage ramp-pulses from −100 to +100 mV (500 ms) were applied every 5 s. All experiments were performed at room temperature.Whole-cell patch-clamp recordings were performed 1 day after transfection. The standard bath solution was the same as that used in the CaP values of <0.05 were considered to be significant. Statistical significance of the effects of borneol, 1,8-cineole, and camphor on hTRPA1 mutants were evaluated using Student’s t test. Dose-dependent curves were fit with a Hill equation.Data in all of the figures are shown as the mean ± standard error of the mean, and 2+-imaging method with hTRPA1-expressing HEK293T cells. In these experiments, changes in the fura-2 ratio (corresponding to cytosolic Ca2+ concentrations) induced by the test compounds and menthol were compared because menthol, which activates hTRPA1, and the test compounds are members of the monoterpene family. Borneol, 2-methylisoborneol, norcamphor, and fenchyl alcohol showed small changes in the fura-2 ratio, similar to 1,8-cineole and camphor , 2-methylisoborneol (1 mM), and fenchyl alcohol (1 mM) in a similar manner as 1,8-cineole and camphor  or FFA (100 μM)  of 0.20 ± 0.06, 0.12 ± 0.03, and 0.32 ± 0.06 mM, respectively, which are much lower than the IC50 of 1,8-cineole (3.43 ± 0.58 mM) and camphor (1.26 ± 0.32 mM)  Fig. . EnhanceμM) Fig. a–d, suggμM) Fig. 0 of 0.202+-imaging method and found that the effect of borneol was reduced with the Y812A mutant (data not shown). In terms of the effect on the AITC-activated hTRPA1 current, results from the patch-clamp studies revealed that borneol had a significantly lower inhibitory effect on the mutant (hTRPA1-Y812A) at two different concentrations than on hTRPA1-WT, whereas such differences between WT hTRPA1 and hTRPA1-Y812A were not observed for 1,8-cineole or camphor activity  effect in a Ca2+-imaging method, but failed to observe changes in the cytosolic Ca2+ concentrations . (n = 48-75)Supplementary material 1 (JPEG 324 kb) Supplementary Figure 2 Comparison of the inhibitory effects on AITC (20 μM)-induced hTRPA1 current among borneol isomers and (±) isoborneol. (n = 5-6)Supplementary material 2 (JPEG 126 kb) Below is the link to the electronic supplementary material.