MAPK signaling cascades are evolutionally conserved. The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK) and prevent activation by phosphorylation. YopJ is also able to block yeast MAPK signaling pathways using this mechanism. Based on these observations, we performed a genetic screen to isolate mutants in the yeast MKK, Pbs2, that suppress YopJ inhibition. One suppressor contains a mutation in a conserved tyrosine residue and bypasses YopJ inhibition by increasing the basal activity of Pbs2. Mutations on the hydrophobic face of the conserved G α-helix in the kinase domain prevent both binding and acetylation by YopJ. Corresponding mutants in human MKKs showed that they are conserved not only structurally, but also functionally. These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition. Yersinia pestis was the causal agent of three pandemics referred to as the Plague of Justinian, Black Death and the Third Pandemic Yersinia pseudotuberculosis and Yersinia enterocolitica are causal agents for gastroenteritis Yersinia species harbor a 70 Kb virulence plasmid that encodes a Type III Secretion System (TTSS) and effector proteins Yersinia outer proteins), from the bacterial cell into the infected host cell The bacterial pathogen Yersinia effectors, YopJ, is a 32 kD effector protein that disrupts the innate immune response and promotes apoptosis in infected cells by blocking the MAPK pathways and the NFκB pathway One of the Saccharomyces cerevisiae, including the mating and HOG pathways The mechanism YopJ uses to inhibit the MAPK pathways is evolutionarily conserved because YopJ was able to inhibit the MAPK pathways in Yersinia effector YopJ but also support predictions for mechanisms involved in the activation of the kinases that are sensitive to YopJ's inhibition.A genetic screen was performed to isolate suppressor mutations in Pbs2 that would allow growth on hyper-osmotic media in the presence of YopJ. We identified a novel mutation in conserved tyrosine that when mutated results in a constitutively active kinase. Other suppressor mutations in Pbs2 revealed a conserved binding site for YopJ on the MKKs. These studies not only provide mechanistic insight into the inhibitory activity of the We demonstrated that YopJ is able to block the HOG MAPK signaling pathway downstream of the negative regulator Sln1  [12]. AsTo further confirm whether YopJ inhibits the signaling pathway by preventing activation of the Pbs2 (yeast MKK) in the HOG pathway, phospho-mimic mutants of Pbs2, in which either the serine or threonine in the activation loop of the kinase were replaced with aspartate (Pbs2(S514D) or Pbs2(T518D)), were coexpressed with an empty vector, YopJ or YopJ-C/A mutant. We observed that both mutants grow in the presence of 1M sorbitol and YopJ . These oWe next assessed whether YopJ could bind to the yeast MKK Pbs2, using yeast two-hybrid analysis. As previously observed for mammalian MKK Recently, it was demonstrated that YopJ prevents the activation of MKKs by acetylating the serine and threonine residues in the MKK activation loop, thereby preventing their phosphorylation by upstream kinases pbs2Δ haploid strain to ensure that the suppressor mutations in Pbs2 contained the following two properties: (1) the mutants are enzymatically active, and (2) the mutants suppress growth inhibition induced by YopJ expression.YopJ inhibits growth of yeast in the presence of sorbitol , 2A [12]4 transformants screened. Pbs2 is a scaffolding kinase with domains that bind both the upstream MKKK and the downstream MAPK Hog1 We recovered eighty strains containing suppressor mutations in Pbs2 from the 9×10The three suppressor mutations in Pbs2 are due to changes in amino acids that are evolutionarily conserved in all the MKK family members . The mutWe predicted that the suppressor mutations in Pbs2 could inhibit YopJ by a variety of mechanisms. If the suppressor mutants had increased basal activity, they would bypass YopJ inhibition in a manner similar to that observed for Pbs2(S514D) and Pbs2(T518D) . TherefoFrom previous studies we have observed that YopJ inhibits only the kinases to which it binds  but not kinases it does not bind  in vitro acetylation reaction. We observed that the kinase domains of both wild type and P1 kinase domains were efficiently acetylated, but not that of P5 ; 2. inability to bind and be acetylated by YopJ, but unable to suppress YopJ in the presence of endogenous Pbs2 (P4); and 3. inefficient acetylation by YopJ and unable to suppress YopJ in the presence of endogenous Pbs2 (P5). Below, we delineate the properties and mechanisms utilized by each suppressor mutant to prevent YopJ inhibition of MAPK signaling.in vitro kinase assay. As seen in Based on growth assays on glucose, the P1 mutant would be expected to exhibit a higher basal kinase activity than the wild type Pbs2. To test this hypothesis, we immunopurified wild type and mutant forms of Pbs2 from yeast that were exposed to normal and hyperosmotic conditions and analyzed them in an pbs2Δ strain on hyper-osmotic media  demonstrates that YopJ cannot bind to the predicted interface of the helix if the corresponding residues are mutated or a phenylalanine is present on the APE loop .Yersinia strains express an effector protein, YopJ, that inhibits the activation of the second kinase, MKK, in these signaling pathways by acetylating residues that are normally phosphorylated by the upstream kinase. From our genetic screen, we discovered that a mutation in an evolutionarily conserved tyrosine residue is sufficient to increase the basal activity of MKKs. We found that the G α-helix is the binding site on MKKs for YopJ. If YopJ is unable to interact with this site, it cannot acetylate its substrate, MKK.MAPK pathways are conserved from yeast to man and each of the corresponding kinases in the cascade are activated by a similar mechanism. in vitro kinase assays and growth assays. We observe that mutation of mammalian MKK at this position also results in a kinase with high basal activity. We propose that mutation of the conserved tyrosine residue results in an activated kinase by a mechanism that is independent of phosphorylation. Therefore, although the P1 suppressor mutant can still bind to YopJ and be efficiently acetylated by YopJ, it bypasses the YopJ block and activates the HOG MAPK pathway.In the set of the evolutionarily conserved mutations, we isolated the P1 mutant, in which a conserved tyrosine residue is mutated to a histidine residue. The resulting suppressor mutant displays increased basal activity in the absence of stimulus as observed in both Recently, Rodriguez-Viciana and colleagues reported on the genetics of activating mutations in the mammalian MAPK pathway that cause Cardio-facial-cutaneous syndrome (CFC) Using the Pbs2 suppressor screen we discovered that the G α-helix is the binding site on MKKs for YopJ. Mutations along the side of the G α-helix that is opposite of the APE loop disrupt the interaction between YopJ and Pbs2p. Analogous mutations in the G α-helix of mammalian MKK1 also disrupt binding of YopJ, supporting the proposal that YopJ uses this region of the kinase as a binding site on all members of the MKK family. Based on the identification of the binding site for YopJ on MKKs, we have learned at least three characteristics about the mechanism of YopJ inhibition of the family of MKKs. The first and most obvious is that YopJ must bind to its substrate to acetylate and inhibit it.Second, we gained insight into why YopJ can inhibit some kinases but not others Finally, the third characteristic involves the orientation of the binding site on the kinase. P5 contains a mutation in a proline residue at the end of G α-helix and the proline is speculated to be a helix breaker. We hypothesize that the P5 suppressor mutant, Pbs2(P584L) contains a G α-helix that is extended by one amino acid and forms a structure that is not optimal for YopJ inhibition because it alters orientation of the YopJ binding site in Pbs2 and, as a result, we observe the P5 suppressor mutant is inefficiently acetylated by YopJ.Yersinia YopJ using yeast genetics. YopJ inhibits the MKK in the HOG MAPK pathway by acetylation, thereby preventing its activation by phosphorylation , together with YopJ or YopJ(C172A) mutant encoded by a galactose inducing vector pRS413-Gal1, by the lithium acetate procedure HA tagged Pbs2 and mutants were generated in the yeast expression vector pRS416-GPD1 and transformed into a 600 of 0.1 and serially diluting 5 times each step.Yeasts were cultured at 30°C on plates or in liquid media containing 2% glucose or 2% raffinose and 1% galactose in the presence or absence of 1 M sorbitol. Specific amino acids were excluded from the media for selection purposes. Dilution plating experiments were performed using 5 µl cells per spot, starting with an OD4 mutants) was generated with a Stratagene kit and cloned into the pRS416-GPD1 vector. The library was transformed into yeast strain BY4741 pbs2Δ strain together with pRS413-Gal1-YopJ and selected for growth on plates with 2% raffinose, 1% galactose and 1 M sorbitol. A total 80 positive clones were identified, 24 of which had a single mutation within the Pbs2 kinase domain.A Pbs2 random mutagenesis library  were purified using standard Ni++-NTA affinity chromatography purification protocols (Qiagen), while N-terminal GST tagged YopJ was purified as indicated before Constructs of recombinant proteins were transformed into In vitro acetylation assays were performed as described 14C-labeled acetyl-CoA for 1 hour at 30°C. Samples were separated by SDS-PAGE and analyzed by autoradiography.pbs2Δ strains co-expressing Pbs2 and YopJ were cultured in media containing 2% raffinose and 1% galactose, harvested, stimulated with 0.7 M sorbitol for 2.5 minutes, and lysed with 1% Triton X-100 in 25 mM Tris, pH 7.5 and 150 mM NaCl, supplied with protease and phosphatase inhibitors. HA tagged Pbs2 was pulled down with an anti-HA antibody (Covance) and Protein G Sepharose beads , which were washed with the same buffer with 500 mM NaCl to eliminate nonspecific binding. Kinase assays were performed as previously described 32P-γ-ATP, DTT and cold ATP at 30°C for 30 minutes, separated by SDS-PAGE, and then analyzed by autoradiography.Yeasts 6 cells/ml were transfected with either flag-tagged YopJ or YopJ C/A, HA-tagged ERK and various HA-tagged MKK1 mutants. Cells were lysed in HNT buffer containing protease inhibitors  and the HA-MKK was immunoprecipitated using an anti-HA antibody (Covance) and Protein G Sepharose beads (Roche). Total cell lysates and beads were separated using SDS-PAGE and probed with either anti-HA (Covance) or anti-phospho ERK antibodies .HEK293 cells plated at a density of 0.5×10YopJ wild type or C172A mutant cloned into the pLexAde vector (as bait) and Pbs2 or human MkK1 mutants cloned into the pVP16 vector (as prey) were transformed into yeast strain L40 using the same method mentioned above. Yeasts were grown on tryptophan- and leucine- deficient plates while two-hybrid interactions were tested using a colony-lift β-gal assay and visualized on nitrocellulose membranes Figure S1(0.68 MB TIF)Click here for additional data file.Figure S2(0.44 MB TIF)Click here for additional data file.


The cystatin superfamily comprises cysteine protease inhibitors that play key regulatory roles in protein degradation processes. Although they have been the subject of many studies, little is known about their genesis, evolution and functional diversification. Our aim has been to obtain a comprehensive insight into their origin, distribution, diversity, evolution and classification in Eukaryota, Bacteria and Archaea.in silico the full complement of the cystatin superfamily in more than 2100 prokaryotic and eukaryotic genomes. The analysis of numerous eukaryotic genomes has provided strong evidence for the emergence of this superfamily in the ancestor of eukaryotes. The progenitor of this superfamily was most probably intracellular and lacked a signal peptide and disulfide bridges, much like the extant Giardia cystatin. A primordial gene duplication produced two ancestral eukaryotic lineages, cystatins and stefins. While stefins remain encoded by a single or a small number of genes throughout the eukaryotes, the cystatins have undergone a more complex and dynamic evolution through numerous gene and domain duplications. In the cystatin superfamily we discovered twenty vertebrate-specific and three angiosperm-specific orthologous families, indicating that functional diversification has occurred only in multicellular eukaryotes. In vertebrate orthologous families, the prevailing trends were loss of the ancestral inhibitory activity and acquisition of novel functions in innate immunity. Bacterial cystatins and stefins may be emergency inhibitors that enable survival of bacteria in the host, defending them from the host's proteolytic activity.We have identified This study challenges the current view on the classification, origin and evolution of the cystatin superfamily and provides valuable insights into their functional diversification. The findings of this comprehensive study provide guides for future structural and evolutionary studies of the cystatin superfamily as well as of other protease inhibitors and proteases. The cystatin superfamily consists of a large group of cystatin domain-containing proteins, most of which are reversible and tight-binding inhibitors of the papain C1) and legumain (C13) families of cysteine proteases  and legu. On the Cysteine protease inhibitors are widely distributed in metazoans and angiosperms. They can function to protect cells from unwanted proteolysis and to control intra- and extracellular protein degradation -10. The Studies of the plant protease papain and human cysteine cathepsins were crucial for the discovery of the cystatins. The isolation and characterization of the chicken egg-white protein inhibitor of the plant cysteine proteases ficin and papain , of the Although stefins and cystatins differ considerably in their amino acid sequences, their tertiary structures are conserved and exhibit the cystatin fold that is formed by a five stranded anti-parallel β-sheet wrapped around a five-turn α-helix ,27. The The evolutionary analyses of the cystatin superfamily took place in the pre-genomic era, from the mid 80 s ,6,30 to Here we aim to obtain a comprehensive insight into the origin, distribution, diversity, evolution and classification of the cystatin superfamily in Eukaryota, Bacteria and Archaea. Such an analysis could not have been performed previously due to the small number of available completed eukaryotic and prokaryotic genomes. We traced the genesis and expansion of the cystatin superfamily through comparative genomic and phylogenomic analyses, using publicly available whole-genome information from more than 2100 prokaryotic and eukaryotic genomes, as well as from the numerous transcriptomic and proteomic databases. The results of this work underpin targeted functional and structural studies of the members of the cystatin superfamily.http://www.ncbi.nlm.nih.gov. In addition, we searched the Ensembl http://www.ensembl.org and the Joint Genome Institute (JGI) http://www.jgi.doe.gov databases. Taxon-specific genome databases were searched through the ENSEMBL and JGI websites, while diverse taxon-specific transcriptomic databases were searched for all eukaryotic lineages at NCBI. To detect all the available representatives of cystatin superfamily, database searches were performed iteratively. Comparisons were performed using the TBLASTN program ); 2Naegleria cystatin, (estExt_fgeneshNG_pg.C_180157 [Naegr1:79400]); Phytophthora infestans cystatin EPC2B (AAY21183); Trichomonas vaginalis cystatin (XP_001323421); Prototheca wickerhamii cystatin ; Cyanophora paradoxa cystatin ; Chlamydomonas cystatins estExt_fgenesh2_kg.C_150044 [Chlre3:183419]; Malawimonas californiana cystatin (EC715563); Reclinomonas americana cystatin (EC798377); 1 Homo sapiens stefin B (NP_000091) and 2 Homo sapiens cystatin C (CAA36497). Highly conserved QXVXG region is in bold.Click here for fileSupplementary Table 3. Distribution of the cystatin superfamily in the plant kingdom.Click here for fileSupplementary Table 4. Distribution of the cystatin superfamily in Holozoa (Metazoa plus Choanozoa).Click here for fileSupplementary Table 5. Loss of the cystatin superfamily representatives in Eukaryota.Click here for fileSupplementary Figure 2. Cystatin from Giardia resembles the most ancestral eukaryotic cystatin. The following protein sequences were used: Giardia lamblia (EAA37282) cystatin; Euglena gracilis stefin (EC675023); Naegleria cystatin, (estExt_fgeneshNG_pg.C_180157 [Naegr1:79400]); Phytophthora infestans cystatin EPC2B (AAY21183); Trichomonas vaginalis cystatin (XP_001323421); Reclinomonas americana cystatin (EC798377); and Homo sapiens cystatin C (CAA36497). Highly conserved QXVXG region is in bold.Click here for fileSupplementary Figure 3. Legumain binding motif in bifunctional cystatins is conserved in all land plants and in some green algae. The following protein sequences were used: Physcomitrella (estExt_gwp_gw1.C_2380025 [Phypa1_1:195387]); Oryza Oryzacystatin-12 (NP_001042702); Marchantia (BJ841987); Selaginella (BM402705); Zamia fischeri (DY036558); Pseudotsuga menziesii (CN639199); Ginkgo biloba (EX934790); Ceratopteris richardii (BE641752); Amborella trichopoda (CK755139) and green alga Scenedesmus obliquus cystatin (EC184546 + EC184713). Highly conserved QXVXG region and legumain binding motif (SNSL) are in bold.Click here for fileSupplemetary Figure 4. Gain of signal peptide in some eukaryotic stefins. The following protein sequences were used: Karlodinium micrum stefin ; Capsaspora owczarzaki stefin ; Hyperamoeba dachnaya stefin (EC853881), Nannochloropsis oculata stefin ; Euglena gracilis stefin (EC675023); Monosiga stefin (estExt_fgenesh2_kg.C_20002 [Monbr1:35345]); Dictyostelium discoideum (XP_629960) stefin; Reclinomonas stefin ; and Homo sapiens stefin B (NP_000091). Highly conserved G and QXVXG region are in bold. The names of the taxa where stefins gained signal peptide are in bold.Click here for fileSupplementary Table 6. Ancestral states for the cystatin superfamily in eukaryotic supergroups.Click here for fileSupplementary Figure 5. Functionally important structural motif of eukaryotic cystatins is conserved in the bacterial stefins and cystatins. Highly conserved QXVXG region is in bold. Eukaryotic cystatin (Giardia) and stefin (Euglena) have been included.Click here for file


Lag screw cut-out failure following fixation of unstable intertrochanteric fractures in osteoporotic bone remains an unsolved challenge. This study tested if resistance to cut-out failure can be improved by using a dual lag screw implant in place of a single lag screw implant. Migration behavior and cut-out resistance of a single and a dual lag screw implant were comparatively evaluated in surrogate specimens using an established laboratory model of hip screw cut-out failure.Five dual lag screw implants  and five single lag screw implants  were tested in the Hip Implant Performance Simulator (HIPS) of the Legacy Biomechanics Laboratory. This model simulated osteoporotic bone, an unstable fracture, and biaxial rocking motion representative of hip loading during normal gait. All constructs were loaded up to 20,000 cycles of 1.45 kN peak magnitude under biaxial rocking motion. The migration kinematics was continuously monitored with 6-degrees of freedom motion tracking system and the number of cycles to implant cut-out was recorded.The dual lag screw implant exhibited significantly less migration and sustained more loading cycles in comparison to the DHS single lag screw. All DHS constructs failed before 20,000 cycles, on average at 6,638 ± 2,837 cycles either by cut-out or permanent screw bending. At failure, DHS constructs exhibited 10.8 ± 2.3° varus collapse and 15.5 ± 9.5° rotation around the lag screw axis. Four out of five dual screws constructs sustained 20,000 loading cycles. One dual screw specimens sustained cut-out by medial migration of the distal screw after 10,054 cycles. At test end, varus collapse and neck rotation in dual screws implants advanced to 3.7 ± 1.7° and 1.6 ± 1.0°, respectively.The single and double lag screw implants demonstrated a significantly different migration resistance in surrogate specimens under gait loading simulation with the HIPS model. In this model, the double screw construct provided significantly greater resistance against varus collapse and neck rotation in comparison to a standard DHS lag screw implant. Operative treatment for hip fractures was introduced in the 1950s with the expectation of improved functional outcome and a reduction of the complications associated with immobilisation and prolonged bed rest .Since then a variety of different implants has been used either extramedullary or intramedullary in nature. The most commonly used extramedullary implant is the sliding hip screw (SHS) with side plate. It is currently considered the gold standard for fixation of extracapsular hip fractures as well as the implant that any new design should be compared with -6.The SHS has been shown to produce good results; however, complications are frequent, particularly in unstable fractures -9.Post-operative implant-related complications have been reported in a recent meta-analysis of 16 studies. Risks ranged from 0 to 23% (median 6%) in patients treated with intramedullary devices and from 0 to 7% (median 3%) in patients treated with SHS devices . The mosIntramedullary (IM) implants have been associated with an increased risk of intraoperative and postoperative femur fractures compared with sliding hip screws -16. ThisRecently, IM nails have been introduced that employ two small-diameter lag screws that enable a smaller diameter of the proximal nail segment ,21. The In a biomechanical comparison by Erik N Kubiak et al. between a dual, trochanteric antegrade nail (TAN) and a single lag screw implant (intramedullary hip screw IMHS), the two constructs showed equivalent rigidity and stability in all parameters. The dual screw implant had a significantly greater ultimate failure load . MickaelLarry Ehmke et al. developed a hip implant performance simulator (HIPS) that can reproduce the dynamic multi-planar hip forces seen during level walking. Their HIPS system evaluates lag screw migration in a pertrochanteric fracture model under more physiologic loading conditions. They chose the biaxial rocking motion (BRM) technology to simulate multi-planar forces with a loading protocol accounting for hip flexion-extension, ab-adduction and a double peak load history. The so-called BRM design is the most commonly used wear test device for prosthetic hip joints -28.To our knowledge, fixation strength of double-screw hip implants has not been tested in a laboratory cut-out simulator under dynamic multi-plantar loading to determine if an additional screw does provide significantly improved migration resistance as compared to a standard single screw implant. This study therefore investigated the migration behaviour and cut-out resistance of a novel double lag screw implant in comparison to a commonly used single lag screw implant under physiologic multi-planar loading in an established laboratory model ,30.As the gold-standard for single lag screw implants, five dynamic hip screws  made of stainless steel were tested. The DHS lag screws had a shaft diameter of 7.8 mm and an outer thread diameter of 12.5 mm Figure . To inve3) with 4 MPa compressive strength and 48 MPa elasticity modulus (E-modulus) to simulate mildly osteoporotic bone, as validated in a previous study [Lag screw fixation was tested in surrogate femoral head and neck specimens of 50 mm diameter machined from cellular polyurethane foam . These specimens had a density of 12.5pcf  . This coSpecimens were transferred for testing in the HIPS of the Legacy Biomechanics Laboratory . This moBRM, representative for level walking, was produced using concurrent axial loading and rotational displacement controlled by a biaxial material test system . A dynamic, double-peak loading regimen of 1.45 kN peak load approximating two times bodyweight was applied at 1 Hz to the steel shell over a polyethylene meniscus. The meniscus traced a path on the femoral head similar to the path of resultant force vectors during level walking. Concurrent flexion-extension and abduction-adduction motion were superimposed by sinusoidal rotation of a 23° inclined block affixed to the actuator Figure . This 23F), while the remaining two  describe the migration kinematics. The number of load cycles to implant failure, NF, was registered by the material test system. Cut-out failure was detected by means of electrical conductivity between the implant and the steel shell, which triggered an instantaneous stop of the test system to preserve the cut-out stage. Femoral head migration kinematics was analyzed in terms of varus collapse (αvarus) and rotation around the neck axis (αneck). These migration kinematics data were continuously recorded with an electromagnetic motion tracking system . To suppress distortion of motion tracking data by ferromagnetic interference, a non-ferrous experimental platform and actuator extension was implemented. Additionally for dual screws implants, the migration of lag screws in axial direction was assessed by measuring their position with a digital caliper before and after testing.Three dependent outcome variables were reported, one of which describes the cut-out resistance  for the inferior screw. However only one inferior screw exhibited cut-out by medial axial migration, which occurred after 10,054 load cycles. To prevent the "Z effect" in clinical practice, Jinn Lin  emphasizLimitations to the HIPS system must be recognized. Our neck constraint assumed that the fracture had undergone maximum collapse, but had not begun migration of the lag screw. This assumption has precedence in a study by Friedl and Clausen, which used neck constraints similar to those used in the HIPS model to simulate OTA 31-A3 pertrochanteric fractures . AdditioIn conclusion, double screw construct provided significantly greater resistance against varus collapse and neck rotation in comparison to the gold-standard DHS implant when tested in the HIPS model under conditions representative of an unstable fracture and mild osteoporotic bone.Financial support was provided by a grant from the Plus Orthopaedics Hellas S.A.GK, PG and MB have contributed to the conception/design, data interpretation, and drafting/revising of the manuscript. MS has contributed to perform the routine aspects of the study including, biomechanical tests, data capture and statistical analysis. PK has been involved in revising critically the manuscript. All authors approved the final manuscript.


Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce α−smooth muscle actin (α-SMA), type I collagen and CCN2 . The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies.Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc) patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and α−SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and α−SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766.Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc. Fibrosis is characterized by excessive deposition of scar tissue. Fibrosis is one of the largest groups of diseases for which there is no therapy. It has been estimated that nearly 45% of all deaths in the developed world are caused fibrotic conditions which include: cardiovascular disease, pulmonary fibrosis, diabetic nephropathy and liver cirrhosis Several different cytokines and extracellular proteins have been identified that contribute to myofibroblast formation  It is now appreciated that FA proteins can serve as a point of convergence for signals emanating from stimulated growth factor receptors The results described above strongly indicate that targeting myofibroblast action by pharmacological inhibition of Rac may represent a novel approach to inhibiting fibrosis. Dermal fibroblasts cultured from lesional areas of SSc patients retain their fibrotic phenotype for several passages in culture Dermal fibroblasts were isolated from by explant culture of 4 mm punch biopsies from the forearm of healthy individuals and those with diffuse cutaneous scleroderma (6 each) in DMEM, 10% fetal bovine serum (Invitrogen) as previously described 18) and random decamers (dN10) using M-MLV reverse transcriptase (Promega) for 1 h at 37°C. Primers for SMA, Col1A2, CTGF and Vinculin were as previously described Dermal fibroblasts were serum-starved for 18 h and treated in the presence or absence of 10 µM bosentan for 24 h. Total RNA was isolated using Trizol (Invitrogen), and the integrity of the RNA was verified by gel electrophoresis. Total RNA (10 µg) was reverse transcribed in a 20-µl reaction volume containing an oligonucleotide  were seeded into a collagen gel (First Link), floated in DMEM, 2% FBS, and forces generated across the collagen lattice were measured over a 24 hour period. Graphical readings are produced every 15 seconds providing continuous measurements of generated forces (Dynes: 1×10−5 N) Measurement of contractile forces generated in a tethered, floating fibroblast-populated collagen lattice (FPCL) was performed as described Cultured fibroblasts were cultured in 12-well plates. When cells were confluent, medium was removed, cells were rinsed with serum-free medium + 0.1% BSA and cultured for an additional 24 h in serum-free medium + 0.1% BSA. A uniform scratch was them made across the plate with a blue pipette tip. Cells were washed two times in PBS, and cultured in serum-free medium in the presence of mitomycin C .A rac-GTP pulldown assay was used, essentially as previously described . Briefly, fibroblasts were grown in 60 mm dishes and protein was harvested by lysis in buffer containing NP-40. Agarose beads to which a PAK-GSH fusion protein was conjugated was added and active Rac (Rac-GTP), which binds PAK-GSH, was recovered through repeated centrifugation and washing of the agarose beads. Bound Rac was identified by boiling beads in Laemmli buffer, and subjecting the resultant extracts to SDS-PAGE and Western blot analysis using anti-Rac antibody. When indicated, cell lysates were stimulated with GTPγS (100 µmol/L) as a control to establish maximal Rac activation.To extend our previous data showing that rac1 was required for bleomycin-induced skin fibrosis To provide a functional context for our experiments, we then assessed whether rac inhibition could alleviate the elevated ability of lesional SSc fibroblasts to contract a collagen gel matrix. First, we showed, as expected, that lesional SSc fibroblasts, relative to control dermal fibroblasts, showed an enhanced ability to contract a floating collagen gel matrix  and to gTo assess whether enhanced rac activity may contribute to this phenomenon, we cultured dermal fibroblasts isolated from SSc patients and healthy individuals until confluence. A uniform scratch wound was created across the cell layer, and migration monitored over 48 hours in the presence or absence of NSC23766. SSc cells (SScF) migrated faster than control fibroblasts (NF) . HoweverTo assess whether activation of rac was sufficient to generate a fibrogenic phenotype, we transfected into normal fibroblasts (NF) either an empty expression vector or an expression vector encoding constitutively active rac1 (NF+empty versus NF+caRac). We then performed western blot analysis on protein extracts prepared from cells 24 hours post-transfection. In parallel, western blot analysis was performed on extracts prepared from untransfected SSc fibroblasts (SScF). We found that, compared to empty expression vector, transfection of an expression vector encoding constitutively active rac1 into normal fibroblasts resulted in increased production of type I collagen, α−SMA, CTGF and vinculin protein .We then wished to examine the molecular mechanism underlying the ability of rac1 to promote a fibrogenic phenotype in fibroblasts. To begin to address this issue, we tested the hypothesis that rac might act through the activation of PI3kinase/Akt. We first used Western blot analysis to assess whether SSc fibroblasts displayed elevated phosphorylation of Akt. Cells were incubated overnight in serum free media in the presence or absence of NSC23766. Western blot analysis of the resultant protein whole cell extracts revealed that SSc fibroblasts possessed elevated phosphorylation of Akt, which was suppressed by the presence of NSC23766 . Thus SSWe then investigated if the ability of overexpressed active rac1 to activate profibrotic protein expression in fibroblasts was blocked by PI3kinase/Akt inhibition. To perform this expreriment, we repeated our transfection experiments, this time in the presence or absence of the JNK inhibitor SP600125, the p38 inhibitor SB203580, the MEK inhibitor U0126 or the PI3 kinase inhibitors wortmannin and Ly294002. We showed that, of these inhibitors, only wortmannin and Ly294002 blocked the ability of activated rac1 to increase profibrotic protein expression indicating that rac1 acted via PI3kinase .To extend these data, we showed that overexpression of rac1 could increase the ability of normal fibroblasts to contract a floating collagen gel matrix, and that this ability was blocked by wortmannin and Ly294002 but not JNK inhibitor SP600125, the p38 inhibitor SB203580 or the MEK inhibitor U0126 . FinallyFibrotic diseases are characterized by the persistence of myofibroblasts within lesions α (Akt1), PKBβ (Akt2), and PKBγ (Akt3), is activated in cells in response to a variety of stimuli including hormones, growth factors, and ECM Akt , a serine/threonine kinase consisting of three members termed PKBIn summary, our studies examining the involvement in Rac1 in dermal function may have profound implications for both homeostatic and pathological processes by contributing to our understanding of basic mechanisms regarding wound healing. As a consequence, our results may have future therapeutic implications for the treatment of non-healing or chronic skin wounds, and of fibroproliferative disease.


Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood.in vivo angiogenesis model of chicken chorioallantoic membrane (CAM).In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2; it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regulate VEGFR-2 function in a combinatorial manner by supporting both diversity of recruitment of angiogenic signaling proteins to VEGFR-2, and its ability to promote angiogenesis. VEGFR-2Y1173F knock-in allele died between E8.5 and E9.5 without any organized blood vessels in vitroActivation of vascular endothelial growth factor receptor-2  plays a pivotal role in mediating growth of blood vessels, angiogenesis in vitroThe recent study indicates that IQGAP1 associates with VEGFR-2 and its activity is required for proliferation of endothelial cells In this study, we uncovered a surprising correlation between phosphorylation of Y1057 and Y1173 and that phosphorylation of Y1057 plays a multitasking role in mediating VEGFR-2-directed angiogenic signaling events in endothelial cells. Phospho-Y1057 recruits c-Src kinase to VEGFR-2, and in part catalyzes phosphorylation of Y1173 via Src kinases. c-Src also bridges IQGAP1 to VEGFR-2 and directly phosphorylates IQGAP1 and promotes endothelial cell proliferation, a key step in angiogenesis.Although stimulation of VEGFR-2 in endothelial cells is known to promote activation of Src kinases in vitro dot blot assay. The result shows that SH2 domain of Src also directly interacts with phosphorylated Y1057 in a c-Cbl independent manner  domain t manner , suggestin vivoTyrosine 1173 functions as a multitasking residue on VEGFR-2 To further link Src kinase activity to phosphorylation of Y1173 of VEGFR-2, we tested the phosphorylation of Y1173 of VEGFR-2 in the triple knockout SYF  cells in vitro. Our analysis showed that mutation of Y1052 had no apparent effect on the ability of CKR to promote tube formation of PAE cells. However, mutation of Y1057 significantly reduced the ability of CKR to stimulate tube formation of PAE cells . Taken together, the data support the hypothesis that IQGAP1 directly associates with c-Src and undergoes c-Src-dependent tyrosine phosphorylation. Hence to further establish as to whether c-Src interacts with IQGAP1 we tested the ability of SH2 and SH3 domains of c-Src to associate with IQGAP1 in an in vitro pull-down assay. The result showed that SH2 domain of c-Src interacts with IQGAP1 independent of its tyrosine phosphorylation since SH2 domain of c-Src forms a complex with IQGAP1 prior to stimulation of cells with ligand  are absent, IQGAP1 is not tyrosine phosphorylated in response to activation of VEGFR-2, where re-introduction of c-Src rescued phosphorylation of IQGAP1 by VEGFR-2 . Our furh ligand . Additioh ligand . The datTo establish the biological importance of IQGAP1 in VEGFR-2 signaling, we initially examined its role in proliferation of endothelial cells. To this end, we employed siRNA-mediated knockdown strategy and analyzed proliferation of endothelial cells in response to VEGF stimulation. IQGAP1 siRNA significantly reduced expression of IQGAP1 in HMVE cells . We furtin vivo setting, we used chicken chorioallantoic membrane (CAM) angiogenesis assay where their expression were knocked down by siRNA strategy. The result showed that targeting IQGAP1, c-Src and b-Raf individually by siRNA suppresses the ability of VEGF to stimulate angiogenesis  were purchased from R&D Systems . Mouse monoclonal anti-phosphotyrosine antibody 4G10 and anti-IQGAP1 antibody were purchased from Upstate Biotechnology . Rabbit anti-phospho-VEGFR-2 (pY1052) and rabbit polyclonal anti-phospho-VEGFR-2 (pY1057) were made in collaboration with Upstate Biotechnology . Rabbit polyclonal anti-VEGFR-2 sera were raised against either a glutathione S-transferase-VEGFR-2 kinase insert domain fusion protein (1410) or a GST-VEGFR-2 carboxyl-terminus fusion protein (1412) 5′-AAACTCCCCTTG-CTCATGTAC-3′. The oligonucleotide siRNA used for IQGAP1was cloned into pSUPER.retro.puro vector as previously described CAUUGCCAAGGUCAGCGAU UU and AUGACGCCAC AGCGCAGGC UU, siRNA for avian b-raf were; UUGGCUGGGACACUGACAU UU and AGGAUAGGAUCUGGAUCAU UU; siRNA for avian IQGAP1 was; ACUCUGCAAGCCUUACA GAUU. Creation of mutant VEGFR-2s and other related methods are described in supplementary Porcine aortic endothelial (PAE) cells were grown in 10% FBS cells and lack endogenous expression of VEGFR-2 Angiogenesis assay was performed essentially as described Image J program (NIH).Cells were prepared for immunoprecipitation as described For statistical purposes the blots of three independent experiments were scanned and blots were quantified using NIH Image J program. Comparison of the different parameters for the blots was determined by repeated measures analysis of variance (ANOVA). Significant differences were assigned using Kruskal-Wallis post hoc test. The criterion for significance for all tests was set at p < 0.05. Specific software was used to assist in the data analysis .In vitro GST fusion protein binding experiments were performed as described 3VO4 and a protease inhibitor cocktail. Equal amounts of the appropriate immobilized GST fusion proteins were incubated with normalized whole cell lysates by rocking for 3 h at 4°C. The beads were washed four times in the presence of protease inhibitors and Na3VO4, and proteins were eluted and analyzed in Western blot analysis using appropriate antibody.Purified recombinant GST, GST-SH2-c-Src and GST-SH3-c-Src were eluted from glutathione-Sepharose beads and subjected to dot blot. Further information regarding this assay is provided in Supplementary 4 cells/well in 24-well plates and cultured overnight; the cells were then incubated in serum-free medium for 12 hours. Cells were stimulated with recombinant human CSF-1 or VEGF-A at different concentrations as indicated in figure legends, and after 48 hours they were washed with PBS, harvested by mild trypsinization, and counted with a hematocytometer. Experiments were performed in triplicate and 3 separate experiments were performed and where appropriate, values were presented as means of ± SD.Cell proliferation was evaluated by direct cell counting. Endothelial cells were seeded at a density of 2×10Statistical Analysis: Data represent mean ±SD of 10 CAM which includes three different fields from each CAM was used unless otherwise stated. Statistical significance was calculated by ANOVA soft ware program (prism v4.0b), considering p<0.05 as statistically significant. Tissue preparation for Western blotting: CAM tissue was also harvested for Western blotting. For this purpose CAM tissue was snap frozen in liquid nitrogen and samples were homogenized in a modified RIPA buffer. Five CAMs from each experimental group were homogenized in 1 ml of the RIPA buffer. Equal amounts of protein were subjected to Western blot analysis using desired antibody as described in the figure legends.Pathogen free grade fertilized chick embryos  were incubated for 10 days (37°C with 70% humidity). A hole was made with a drill over the air sac at the end of the egg as described Data S1(0.03 MB DOC)Click here for additional data file.Figure S1Effect of mutation of tyrosines 1052 and 1057 to phenylalanine: Equal number of serum-starved PAE cells expressing wild type CKR, F1052/CKR, F1057/CKR and F1052/F1057/CKR were either unstimulated (−) or stimulated with CSF-1 (+) for 10 minutes. Total cell lysates were subjected to Western blot analysis using an anti-phosphoY1052 specific-VEGFR-2 antibody (A), an anti-phosphoY1057 specific anti-VEGFR-2 antibody (B), an anti-phosphotyrosine antibody (C), and an anti-VEGFR-2 antibody (D). Equal number of serum-starved PAE cells expressing wild type CKR, F1052/CKR, F1057/CKR and F1052/F1057/CKR without stimulation were lysed and immunoprecipitated with an anti-VEGFR-2 antibody. The immunoprecipitated proteins were subjected to an in vitro kinase assay with increasing concentrations of ATP as indicated and described in (0.22 MB TIF)Click here for additional data file.Figure S2Effect of mutation of Tyrosines 1052 and 1057 to glutamic acid. Partial amino acid sequence of mouse VEGFR-2 containing Y1052 and Y1057 and the schematic presentation of VEGFR-2 are shown (A). Equal number of serum-starved PAE cells expressing wild type CKR, E1052/CKR, E1057/CKR and E1052/E1057/CKR were either unstimulated (−) or stimulated with CSF-1 (+) for 10 minutes. Total cell lysates were subjected to Western blot analysis using an anti-phosphoY1052 specific-VEGFR-2 antibody (B), an anti-phosphoY1057 specific anti-VEGFR-2 antibody (C), an anti-phosphotyrosine antibody (D), and an anti-VEGFR-2 antibody (E). Equal number of serum-starved PAE cells expressing wild type CKR, E1052/CKR, E1057/CKR and E1052/E1057/CKR without stimulation were lysed and immunoprecipitated with an anti-VEGFR-2 antibody. The immunoprecipitated proteins were subjected to an in vitro kinase assay with increasing concentrations of ATP as indicated and described in (0.24 MB TIF)Click here for additional data file.Figure S3GST-Src but not GST binds to VEGFR-2: Serum-starved PAE cells expressing CKR were either unstimulated (−) or stimulated (+) with CSF-1 for 10 minutes, lysed, and whole cell lysates were incubated with purified GST alone or GST-SH2-Src protein. The GST-SH2-Src bound proteins were subjected to Western blot analysis using an anti-VEGFR-2 antibody (A). Whole cell lysates were blotted with anti-VEGFR-2 antibody as a control (B).(0.09 MB TIF)Click here for additional data file.


Formation of chlorpyrifos oxon (CPO) in self-inflicted poisoning with chlorpyrifos (CPF).n = 74).Left: time course of plasma concentrations (single patient). Right: ratio of CPO/CPF 2–5 days after poisoning ( Chlorpyrifos (CPF) is a pesticide that causes tens of thousands of deaths per year worldwide. Chlorpyrifos oxon (CPO) is the active metabolite of CPF that inhibits acetylcholinesterase. However, this presumed metabolite has escaped detection in human samples by conventional methods  until now. A recently developed enzyme-based assay allowed the determination of CPO in the nanomolar range and was successfully employed to detect this metabolite. CPO and CPF were analysed in consecutive plasma samples of 74 patients with intentional CPF poisoning. A wide concentration range of CPO and CPF was observed and the ratio of CPO/CPF varied considerably between individuals and over time. The ratio increased during the course of poisoning from a mean of 0.005 in the first few hours after ingestion up to an apparent steady-state mean of 0.03 between 30 and 72 h. There was a hundred-fold variation in the ratio between samples and the interquartile range  indicated over half the samples had a 5-fold or greater variation from the mean. The ratio was independent of the CPF concentration and the pralidoxime regimen. CPO was present in sufficient quantities to explain any observed acetylcholinesterase inhibitory activity. The effectiveness of pralidoxime in reactivating the inhibited acetylcholinesterase is strongly dependent on the CPO concentration. Differences in clinical outcomes and the response to antidotes in patients with acute poisoning may occur due to inter-individual variability in metabolism. It may be estimated that it causes tens of thousands of deaths per year worldwide in vitro. It had been noted that BChE had extremely high reaction rates with CPO Until recently 22.1Serial blood samples stem from patients with acute chlorpyrifos poisoning who were enrolled in two randomised controlled studies (RCT1: ISRCTN02920054 and RCT2: ISRCTN55264358). The studies were performed in Sri Lankan hospitals in Anuradhapura and Polonnaruwa aimed at evaluating the efficacy of oral superactivated charcoal (RCT 1) or pralidoxime (RCT 2) in symptomatic organophosphorus pesticide poisoning. Additional patients were recruited in a prospective cohort study in Nuwara Eliya that was assessing changes in neuromuscular junctional function in organophosphorus pesticide poisoning. Ethics approval was obtained from the Faculty of Medicine Ethics Committee of Universities of Colombo and Peradeniya, and Oxfordshire Clinical Research Ethics Committee. Written informed consent was taken from each patient, or their relatives, in their own language. Study details are reported elsewhere Blood samples were collected into tubes containing EDTA prior to pralidoxime, then at 1, 4, 12, and 24 h after administration, then once daily until discharge or death. A 0.2 mL blood sample was diluted at the bedside immediately after collection with 4 mL of ice-cold saline and frozen to −23 °C until analysis of AChE activity. The remaining sample was promptly centrifuged and the aspirated plasma frozen at −23 °C until analysis of BChE activity, inhibitory activity, and the concentration of CPF and CPO 2.2in vivo) and after reactivation with supra-therapeutic obidoxime (AChE in vitro) to estimate the reactivatable fraction. These assays were performed using a modified Ellman assay as described Laboratory analyses were conducted in Munich, Germany. AChE activity was determined in native RBCs . The hexane phase was transferred in a borosilica vial and 10 μL of DMSO was added as keeper. Hexane was evaporated at approximately 40 °C for 30 min in a Speed-Vac-Concentrator. The residue was dissolved in 90 μL of methanol:water  and 50 μL were applied to LiChrosphere 100 RP-18, 5 μm, 125 mm × 4 mm  and eluted at a flow rate of 1 mL/min by gradient elution. The mobile phase started with 65% methanol in water and was linearly increased to 77% methanol between 4.5 and 7.5 min. Typically, fenitrothion was eluted after 3.3 min and chlorpyrifos after 11.5 min. The lower limit of quantification (LOQ) was 0.1 μM in plasma.CPO was determined by an enzyme-based assay as described recently ® were from Riedel-de Haen, Sigma, Taufkirchen, Germany; CPO was a generous gift from DowAgroSciences, Indianapolis, IN. Both compounds had purities ≥99%. Fenitrothion (95.5% pure) was obtained from Dr. Ehrenstorfer, Augsburg, Germany. All other reagents were from commercial sources at the purest grade available.Horse BChE (Cat. No C-1057) and CPF PestanalPralidoxime was determined by HPLC on reversed phase material with an ion-pairing reagent. Specifically, 100 μL plasma was mixed with 30 μL trichloroacetic acid (1 M) and spun down. Eighty microliter of supernatant was mixed with 40 μL of n-heptanesulfonate reagent  and 30 μL of a sodium formate buffer  to give a final pH ≥ 3.0. Fifty microliter was applied to a Purosphere Star RP-18 column  and eluted with a linear methanol gradient (0–15% within 7.5 min) in 5 mM PIC-B7 low UV reagent in water at a flow rate of 1.4 mL/min (20–25 °C) and detection at 293 nm. Typically, pralidoxime was eluted after 5.6 min. LOQ was 1 μM pralidoxime in plasma.Free EDTA in plasma samples was titrated with a standard calcium chloride solution (50 mM) in the presence of the calcium indicator eriochrome black T . Specifically, 50 μL plasma was diluted with 450 μL distilled water, 5 μL sodium hydroxide (2 M) and 1 μL indicator solution added and titrated with calcium chloride until formation of a pink coloration.2.3Data were included when CPF was ingested  with no other organophosphorus compound being detected, BChE was mostly inhibited  and AChE not significantly aged . Data were excluded when CPF and/or CPO were below the LOQs or when the time of ingestion or blood sampling was ambiguous. Data were also excluded when plasma pralidoxime was higher than 0.5 mM, indicating that blood was withdrawn proximally from the infusion site. In these cases significant post-sampling reactions had to be suspected.in vitro–AChE in vivo; kr is the first-order reactivation rate constant; KD is the dissociation constant of the complex EPOx; ki is the second-order inhibition rate constant; [P] is the oxon concentration; [Ox] is the oxime concentration.The relationship between measured and calculated (predicted) CPO concentrations was determined using equations as deduced elsewhere in vitro experiments using human red blood cells kr = 0.3 min−1; KD = 330 μM and ki = 6 × 106 M−1 min−1The constants for reactivation of human diethylphosphoryl-AChE by pralidoxime are known from E0 and CPO0 being the AChE and total CPO concentration at t = 0 .The fraction of active test AChE upon incubation with known amounts of CPO in plasma samples was calculated discretely in 1-min increments using the following equation:Calculations were done with Microsoft Excel 2004. Correlation analyses, Mann–Whitney U test and graphical presentations were performed with Prism4, GraphPad, San Diego, CA.33.1Formation of CPO occurred quite early after CPF ingestion. In one patient we detected significant amounts of CPO 70 min after CPF ingestion with BChE and AChE depressed to less than 2% and 10% of normal, respectively. p > 0.4; not shown). These data were indicative that in this setting CPO formation and decomposition did not show a saturation (dose-limiting) phenomenon.The variation of the ratio CPO/CPF was large, and some individuals showed a ratio below 0.01, while others had >0.05 in the period between 24 and 120 h, when a steady-state was reached cf. . The rat3.26 M−1 min−1p >0.2 and >0.5, respectively). These results pointed to competing side reactions of CPO during the incubation period.A large scattering of data was observed in the ratio of AChE-inhibitory activity of patients’ plasma and the CPO concentration, whether or not patients had received pralidoxime. As expected, the inhibitory activity was on average higher in the group who did not receive pralidoxime. Since BChE was essentially completely inhibited at even low concentrations of CPO we suspected that OP-hydrolases were degrading CPO in the inhibition assay. PON 1 is notorious for its high activity towards CPO 3.3Patient A received pralidoxime by the continuous infusion regimen while patient B received the intermittent lower dose scheme. In both cases, AChE activity was largely inhibited before pralidoxime. In case A, AChE reactivation was sustained and approached some 90% when pralidoxime was given for 5 days. In contrast, intermittent doses of pralidoxime for less than 48 h resulted in gradual re-inhibition of reactivated AChE, particularly since CPO concentrations were much higher than in patient A.The mutual dependence of the RBC–AChE activity on the pralidoxime and the CPO concentration was analysed according to the equation given in the Section 4Since CPO reacts more than 2 orders of magnitude faster with BChE than with AChE Using the standard assay for CPO determination ex vivo “inhibitory activity” and suggests that they may significantly and variably under-estimate the true extent of in vivo AChE inhibitory activity. As shown in ex vivo. Hydrolysis of CPO by PON 1 in the inhibition assay can be ruled out since EDTA in the plasma samples leads to complete inactivation of PON 1 Vmax of 0.9 nmol/min/mg protein with a Km value of 0.4 mM at pH 7.4 and 37 °C ki = 6 × 106 M−1 min−1, see above). It is clear that the  inhibition rate will be slowed down upon gradual inhibition of AChE, while covalent binding to albumin that has a large molar excess of about 150 μM still proceeds. This latter pseudo-first-order reaction explains the observation that the consumption of CPO was largely independent of the CPO concentration  but often in Chinese (32%), while the inverse relationship was found with the PON 1 QQ genotype Km values of about 1 and 30 μM, respectively, at comparable Vmax values. Since the former CYP is prone to polymorphism in protein expression Appearance of CPO in plasma lags behind CPF. This may be due to delayed formation or, more probably, due to sequestering reactions with limited capacity. CPO is rapidly bound to B-esterases, such as BChE, AChE and carboxylesterase. The latter enzyme has a high binding capacity, which in the rat was estimated to be 3 orders in magnitude higher than that of BChE Pralidoxime was able to reactivate inhibited RBC–AChE in CPF-poisoned patients as shown in In conclusion, this newly developed CPO assay gives reliable results that enable a better assessment of the mechanisms behind inter-individual variation in susceptibility in CPF poisoning. The study has shown that the steady-state ratios of CPO/CPF vary widely and probably mirror the activating and detoxifying capabilities. Thus, a large variation in the susceptibility for CPF- and CPO-mediated toxicities has to be taken into account when debating safety margins of CPF.


Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. However, Molecular mechanisms that regulate neuronal cell proliferation in the dentate gyrus (DG) following ischemic stroke insult are poorly understood. This study was designed to investigate the potential regulatory capacity of non-receptor tyrosine kinase Src on ischemia-stimulated cell proliferation in the adult DG and its underlying mechanism.Src kinase activated continuously in the DG 24 h and 72 h after transient global ischemia, while SU6656, the Src kinase inhibitor significantly decreased the number of bromodeoxyuridine (BrdU) labeling-positive cells of rats 7 days after cerebral ischemia in the DG, as well as down-regulated Raf phosphorylation at Tyr(340/341) site, and its down-stream signaling molecules ERK and CREB expression followed by 24 h and 72 h of reperfusion, suggesting a role of Src kinase as an enhancer on neuronal cell proliferation in the DG via modifying the Raf/ERK/CREB cascade. This hypothesis is supported by further findings that U0126, the ERK inhibitor, induced a reduction of adult hippocampal progenitor cells in DG after cerebral ischemia and down-regulated phospho-ERK and phospho-CREB expression, but no effect was detected on the activities of Src and Raf.Src kinase increase numbers of newborn neuronal cells in the DG via the activation of Raf/ERK/CREB signaling cascade after cerebral ischemia. It is well established that the generation of new neurons continues throughout adulthood in the DG in many species of vertebrates ,2. The nThe Src family kinases (SFKs) are a family of proteins that have been implicated in relaying signals as downstream of a wide variety of cell-surface receptors to regulate diverse cellular responses including proliferation, differentiation, survival changes in cellular architecture, and regulating cell adhesion and migration . Five meOne signaling cascade implicated in regulating the proliferative capacity of adult stem cells is mitogen-activated protein kinase (MAPK) . The extNon-receptor tyrosine kinase Src plays prominent roles in ischemic cerebral apoplexy . IschemiP < 0.05), the activity of Src remained in a high level until 24 h and 72 h reperfusion illustrated by strong dephosphorylation of Src at Tyr527 site compared with sham operated animals. It suggested that continuous Src kinase activation may be involved in triggering some pathological phenomena in the DG/CA3 region induced by ischemia and reperfusion. As has been well accepted, ischemia stimulates neurogenesis in the DG; thus we explored the possibility of Src being involved in the proliferation of adult hippocampal progenitor cells. SU6656, an inhibitor of Src kinases, was administered into cerebral ventricle before ischemia, and found to be effective in suppressing Src activity . At both spots the amount of p-ERK increased at least two-fold when compared with sham control group; while the elevated level of p-ERK lowered in the SU6656 - treated rats . Under a light microscope, the normal neurons showed round cell bodies and plain stained nuclei Fig. . After 5Cerebral ischemia insults have been shown to trigger proliferation of endogenous neural stem cells or progenitor cells located in the hippocampal dentate gyrus ,28, and 2+ concentration serve as a main contributing factor for activation of Src [in vitro [The molecular mechanism underlying Src kinase dependent neural cell proliferation stimulated by ischemia is complex. Signals that increase intracellular Can of Src , which, n of Src ,26, and n of Src -34, suggn of Src . While rin vitro ,36, in tin vitro . Our datin vitro -40. Morein vitro . Our resIn addition, ischemia insult may also trigger others molecular pathways, which may associate with altering proliferation of progenitor cells. The results in Fig.  showed tIt is generally accepted that the region of DG is relatively resistant to ischemic injury. Our result also validated that there is no ischemia - induced neuronal death in the DG fields Fig. . Thus we2+ influx to Raf/ERK activation, and then to CREB phosphorylation, which, in turn, triggers genes related to cell proliferation expression . The animals used in the present study were male Sprague-Dawley rats  weighing 250-300 g, maintained in individual cages with a 12-h light/dark cycle (lights on from 08:00-20:00). Transient global cerebral ischemia was induced using four-vessel occlusion as described previously [SU6656 -2,3- dihydro-1H-indole-5-sulfonic acid dimethylamide) is an inhibitor of the Src family tyrosine kinases . U0126 butadiene) is a potent inhibitor of MEK/ERK . 1 μl of dimethyl sulfoxide (DMSO) in 4 μl of phosphate-buffered saline  as vehicle or SU6656  or U0126  was administered using a microinjector into the left cerebral ventricle  over 5 min or its vehicle. After the injection, the injector was left in place for an additional 5 min to reduce any possible backflow of the liquid along with the injection void, and occlusion occurred 20 min post the injection. Bromodeoxyuridine , a synthetic nucleoside and an analogue of thymidine commonly used in the detection of proliferating cells in living tissues, was administered by intraperitoneal injections  twice daily during days 5 - 7 after 10 min ischemia or sham operation. The animals were put to death 24 h after the last injection of BrdU.et al., 2006). The separated brain regions were homogenized in 1:10 (w/v) ice-cold homogenization buffer A  supplemented with 1% mammalian protease inhibitor cocktail (Sigma-Aldrich Co.). Proteins in the cytoplasm and membrane were extracted by centrifugation at 800 × g at 4°C and those in the nucleus were obtained by centrifugation at 14000 × g at 4°C in homogenization buffer B  and then stored at -80°C until assayed. Protein concentrations were determined by a Bradford protein assay [For biochemical studies, rats were killed by decapitation at various times post the reperfusion . The hippocampi were quickly removed on ice in a cold room and the hippocampal CA3/DG subfields were dissected on ice as described previously  and transcardially perfused with 300 ml saline, followed by 500 ml 4% cold paraformaldehyde in PBS. Brains were removed, post-fixed overnight at 4°C in same solution and then sliced at 40 μm using a vibratome in a bath of physiological saline collected free-floating and stored in 30% ethylene glycol, 30% glycerol, and 0.1 M PBS at - 20°C until processed for immunostaining. Some sections were processed for staining with Toluidine blue (Nissl staining) for histological assessment of damage.2O2 for 30 min for the removal of endogenous peroxidases. After several rinses in PBS, sections were incubated in PBS/0.2% Triton X-100/5% goat serum (PBS-TS) for 30 min and then incubated with polyclonal sheep anti-BrdU (Biotin)  overnight at 4°C. After several rinses in PBS - TS, sections were incubated for 30 min with a streptavidin - HRP complex . BrdU positive (BrdU+) cells were labeled using DAB as chromogen . The labeling was imaged with a confocal laser - scanning microscope  and analyzed with Image-Pro Plus software .For the detection of BrdU immunoreactivity, DNA denaturation was conducted by incubating sections in 50% formamide and 2× sodium citrate solution for 2 h at 65°C, followed by incubation in 2 N HCl for 30 min. Then sections were incubated for 10 min in 0.1 M borate buffer. After being washed in PBS, sections were incubated in 1% HTo count BrdU - labeled cells after immunohistochemistry, the analysis was conducted using a modified version of the optical fractionator method, on every sixth section in a series of 40 μm coronal sections . All BrdTo quantitate the survival rate of hippocampal neurons after Nissl staining, cell counts were performed as described previously ,48. A reP < 0.05 were considered statistically significant.All data are expressed as mean ± SEM from animals. Statistical significance was assessed by one-way analysis of variance (ANOVA) with the least significant difference test using SPSS software version 10.0. Values of DG: dentate gyrus; BrdU: bromodeoxyuridine; ERK: Extracellular signal - regulated kinase; CREB: cyclic AMP response element-binding protein; MAPK: mitogen-activated protein kinase; SFKs: Src family kinases; PTK: protein tyrosine kinase; SU: SU6656; U0: U0126; 4VO: four-vessel occlusion; i.c.v.: intracerebral ventricular; BSA: bovine serum albumin; PAGE: polyacrylamide gel electrophoresis; Veh: vehicle; FGF: fibroblast growth factor; BDNF: brain-derived neurotrophic factor; NGF: nerve growth factor; ER: endoplasmic reticulum.HT, BH, JZ carried out the 4-VO model and sample preparation, participated in the western blot analysis, immunohistochemistry, statistical analysis and drafted the manuscript. XH participated in the i.c.v. infusion and cell counting. JG, LL conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.


P=0.031). In normal mucosa samples, the cystatin C level was influenced by the site of sampling: it was lower in nonlaryngeal tissue samples  than in laryngeal samples (P=0.004). The tumour cystatin C level correlated inversely with pN-stage (P=0.047), whereas a trend of lower cystatin C levels was observed in the group with extranodal tumour extension compared to those with no extranodal spread (P=0.069). In univariate analysis, the patients with low tumour cystatin C levels exhibited poor disease-free survival  and disease-specific survival . In multivariate analysis, the most powerful predictor of survival was pN-stage , followed by the cystatin C level . When comparing the prognostic strength of cystatin C to that of stefin A, another cysteine proteinase inhibitor, which emerged as the most significant prognosticator for survival in our previous study analysing the same cohort of patients, stefin A proved to be significantly more reliable predictor for both DFS and DSS than cystatin C. Our results indicate that cystatin C is implicated in the invasive behavior of SCCHN, and that there are variations in regulation of proteolytic pathways under nonmalignant conditions, inherent to individual subsites inside the upper aerodigestive tract. The correlation between high cystatin C levels and improved survival concurs with the concept of the protective role of high levels of cysteine proteinase inhibitors in tissue homogenates that has been previously suggested by the survival results in breast and lung carcinoma as well as SCCHN.To determine the role of the cysteine proteinase inhibitor cystatin C in the invasive behavior of squamous cell carcinoma of the head and neck (SCCHN), Cystatin C protein level was measured in 82 pairs of primary tumour tissue and adjacent noncancerous mucosa, using the enzyme-linked immunosorbent assay. The median level of cystatin C in tumour tissue was 1.18 times lower than that in corresponding mucosa ( CST3 gene, which is located on the short arm of chromosome 20. It is freely filtered in the glomeruli and almost completely reabsorbed and catabolised in the proximal renal tubular cells  has not been investigated so far. The aim of the present study was to determine the levels of cystatin C in the tissue homogenates of SCCHN and its surrounding noncancerous mucosa. As SCCHN is an extremely heterogenous disease, it is little surprise that no factor within the wide spectrum of biochemical and histological factors has yet been identified as reliably predicting the natural course of the disease or its response to therapy . This cohort of patients was the same as in our previous report, examining the prognostic significance of cysteine proteinases cathepsins B and L and their intracellular inhibitors A and B , It was delivered through two opposing lateral fields to the primary tumour and regional lymph nodes, with spinal cord shielded after a dose of 40–44 Gy. One anterior field was added to cover the lower neck lymphatics.Tumours were staged according to UICC pTNM classification after histopathological examination of surgical specimens . The hisM Na2HPO4, 1 mM monothioglycerol, and 10% v v−1 glycerol (pH 7.4). The suspension was centrifuged for 45 min at 100 000 × g to obtain a supernatant, that is homogenate, which was divided into aliquots and stored at −70°C until use.From operative specimen, two tissue samples of 200–500 mg, representing matched pairs, were obtained from the tumour and adjacent normal mucosa. Immediately after removal, they were immersed in liquid nitrogen until homogenate preparation. Fat and necrotic parts were carefully removed. Pulverisation was performed on the frozen tissue with a Mikro-Dismembranator  for 60 s at maximum power, and the resulting tissue powder was suspended in an extraction buffer consisting of 5 mEscherichia coli as described . Recombinant human cystatin C was expressed in −1 of total protein (ng mgp−1).Linearity and recovery of ELISA was tested as described  in the presence of hydrogen peroxide was added. The amount of degraded substrate, as a measure of bound immunocomplexed cystatin C, was visualised by absorbance at 450 nm. The cystatin C concentration was calculated from the calibration curve.Protein concentrations were determined according to the Bradford method . Bovine U test was used to test the relationship between the median values of the tumour tissue cystatin C concentrations in different groups of patients. The difference in distribution of pTNM-stages between the patients with laryngeal tumours and those with nonlaryngeal tumours was tested with a χ2 test.The results were analysed using a PC and the SPSS statistical package . The difference between the median concentrations of cystatin C in matched pairs of tumour and normal tissue samples was determined by the Wilcoxon signed-rank test. The Mann–Whitney Univariate analysis of the patients’ survival was carried out using the Kaplan–Meier product-limit method  and log-The study protocol was approved by the Medical Ethics Committee at the Ministry of Health of the Republic of Slovenia. All of the included patients gave their informed consent to voluntary participation in the study.P=0.041) decrease in inhibitor concentration in the total population of tumour samples compared to their control counterparts. Furthermore, the cystatin C levels in control samples in the group where an increase was observed was significantly lower than in the control group with decreased inhibitor (P<0.0001).The concentrations of cystatin C in 82 tissue homogenates of tumour and corresponding nontumorous mucosa are summarised in vs 24.0 ng mgp−1P=0.004). With regard to the cystatin C levels in tumour tissue or the distribution of downregulated and upregulated cases, no difference was found between these two major groups of tumours.In normal mucosa samples, the cystatin C level was influenced by the site of sampling, being lower in nonlaryngeal tissue samples  than in the laryngeal ones , whereas a trend of lower cystatin C levels was observed in the group with extranodal tumour extention compared to that with no extranodal spread .The data were further analysed with respect to the established clinical and histopathological parameters. No correlation was found between tumour cystatin C level on the one hand and, on the other hand, patients’ age and sex, histopathological tumour grade, pT-stage and the overall UICC pTNM-stage of the disease, and the presence of tumour emboli in the lymphatic vessels as determined in a histological examination of a resected tissue specimen from the neck. The tumour cystatin C level correlated inversely with pN-stage (pNP=0.029) decrease in tumour cystatin C concentration was found in the group of laryngeal tumours: compared to the normal tissue counterparts, the tumour cystatin C concentration decreased by a factor of 1.42. The inhibitor decreased significantly (P=0.0001), 2.92-fold in 55% of tumours, and was elevated 2.69-fold in 45% of cases .The analysis was repeated separately on laryngeal and nonlaryngeal tissue samples. In matched pairs of normal mucosa and tumour tissue, statistically significant (of cases . No corrAs of 31 December 2002 (close-out date), disease relapsed in 22 patients and 50 patients died: 20 due to disease recurrence and/or dissemination and 30 due to causes other than the treated malignant disease. In total, 32 patients were alive with no signs of the disease. The median follow-up period of all eligible patients was 4.3 years (range: 0.1–10.0 years), and was 6.5 years (5.2–10.0 years) for those alive at the last follow-up examination.+) and more advanced disease , and those in whom extranodal tumour spread or tumour emboli were determined in the lymphatic vessels, exhibited a significantly higher risk of relapse or disease-specific death  and cystatin C  were retained in the final model, and for DSS, pN-stage only . In the latter case, cystatin C turned out to be of marginal significance . As in the same cohort of patients stefin A has been identified previously as the most powerful prognosticator for both DFS and DSS, the standardised concentrations of the latter, calculated as described in our previous report, was also introduced in Cox model .In multivariate analysis, the prognostic value of cystatin C was compared to the values of pN-stage, UICC pTNM-stage, extranodal tumour spread and the presence of tumour emboli in the lymphatic vessels. Variables were eliminated singly in a backward manner with exclusion if P<0.0001, Figure 2In addition, to determine whether the combination of the two variables would increase the prognostic stratification of the patients, they were grouped as follows: high cystatin C and high stefin A – low-risk group (23 patients); high cystatin C and low stefin A, or low cystatin C and high stefin A – medium-risk group (41 patients); and low cystatin C and low stefin A – high-risk group (18 patients). We found statistically highly significant difference between the three prognostic groups for both DFS and DSS .The correlation between cysteine proteinases, their inhibitors and tumour malignancy is likely to be a qualitative rather than a quantitative one. Thus, the proteolytic activity in tumours might be affected not only by the alterations in synthesis but also in processing, subcellular localisation and secretion of the enzymes and their inhibitors. As a result, in breast cancer, the cathepsin B and L activities were found to be much more increased than the respective protein levels, suggesting that the increase in activity was not entirely due to the induction of the respective proteins  suggesteA large body of literature has been accumulated to suggest that cystatin C participated not only in the transformation of cells to a malignant state but also in tumour growth, invasion and metastasis. In cystatin C transfected B16 melanoma cells, the overexpression of inhibitor resulted in the inhibition of melanoma cell mobility and of the ability to penetrate artificial matrices by about 50% , as wellThere is good evidence that all nucleated cells constitutively express cystatin C. However, variability in the degree of cystatin C gene expression in different tissues, which could be further influenced by several factors, provides evidence for the differential regulation of cystatin C expression in different tissues . In the Concerning the results on survival in our study, two points should be stressed. To our knowledge, this is the only study evaluating the prognostic significance of cystatin C in SCCHN and no data are available at the moment for comparison. Second, the study population was quite homogenous in respect to the selection of the patients  and therapy. In multivariate analysis, only the prognostic power of the pN-stage of disease outweighs that of cystatin C. However, when comparing the prognostic strength of cystatin C with that of stefin A, which emerged as the most significant prognostic factor in our previous study analysing the same population of patient, cystatin C lost its significant prognostic power for both DFS and DSS. In addition, the combination of the two inhibitors, cystatin C and stefin A, could further stratify the risk of adverse event as was the case with stefin B and cathepsin B in colorectal cancer , the patients with high serum levels of cystatin C exhibited a significantly higher risk of death than those with lower levels of inhibitor, whereas a decreased metastatic spread was found in cystatin C deficient mice compared to wild-type mice (The correlation between high levels of tumour cystatin C and the longer survival of our patients concurs with the concept of protective role of high levels of cysteine proteinase inhibitors in tissue homogenates. This concept has been proposed following the survival results in the carcinoma of the breast (ype mice , so thatIn conclusion, our data indicate that cystatin C is implicated in the invasive behavior of SCCHN. The variations in regulation of proteolytic pathways seem to be the inherent characteristic of individual subsites inside the upper aerodigestive tract, which should be considered in further studies. Moreover, the protective role of high levels of cystatin C in tissue homogenates was suggested, as it had been proposed for some other cysteine proteinase inhibitors by the survival results in breast and lung cancer as well as in SCCHN. When comparing the prognostic strength of cystatin C to that of stefin A, the latter emerged as significantly more reliable predictor for survival.


The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1. The extracellular signal-regulated kinase5 (ERK5) signaling cascade is composed of MEKK2/3 at the MAP3K tier The molecular mechanism of activation of the ERK5 cascade is not fully elucidated yet. It seems that the cascade can be activated by more than one mechanism dependent on the extracellular stimulus. Thus, the protein tyrosine kinase c-Src As other MAPK cascades, the ERK5 cascade seems to function mainly through regulation of transcription. ERK5 was shown to directly phosphorylate, and thereby activate, members of the MEF2 group of transcription factors, including MEF2C, which consequently induces the transactivation of the genes like c-Jun 2O2 and fluid shear stress Here we studied the role of calcium, a critical and highly versatile second messenger EGF, BAPTA-AM, ionomycin, PP2, KN-62 and KN-93 were purchased from Sigma . Anti general ERK5, MEK5, HA, tubulin and GFP antibodies (Abs) were purchased from Sigma Israel . Anti phospho-ERK5 Ab was purchased from BioSource . Anti MEKK2, phospho-tyrosine (pY99) and Lad1 were purchased from Santa Cruz Biotechnology . Anti phospho-CREB Ab was purchased from Cell Signaling Technology .2, 1.5 mM DTT, 0.15 mM sodium orthovanadate, 3.75 mM EGTA, 0.3 mM ATP and 30 µM calmidazolium.Buffer A consists of 50 mM β-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM DTT and 0.1 mM sodium orthovanadate. Buffer H is Buffer A supplemented with 1 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin and, 2 µg/ml pepstatin-A. Radioimmune precipitation assay (RIPA) buffer consists of 137 mM NaCl, 20 mM Tris (pH 7.4), 10% glycerol, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate and 20 µM leupeptin. Kinase reaction buffer (x3) consisted of 75 mM β-glycerophosphate (pH 7.3), 30 mM MgClTGCAGGACTTTCCCTGAGG.rMEK5α-1 was a gift from Dr. J.D. Lee (Scripps Institute). MEK5-GFP was created by subcloning into pEGFP-N1 between HindIII and SacII sites. MEK5(K/A) was generated by replacing Lys194 to Ala. HA-ERK5 was a gift from Dr. J.S. Gutkind . ERK5(1–397)-GST fusion protein was generated by inserting into BamHI and XhoI sites in pGEX-N1(T4). WNK1 was a gift from Dr. M.H. Cobb . GFP-WNK1 was created by inserting into HindIII and BamH1 sites of the pEGFP-N1. GFP-Lad1 was prepared by inserting Lad1 into pEGFPC1 vector. GST-Lad1 was created by inserting into EcoRI and SalI sites of pGEX-4T. pMEF2FLuc was a gift from Dr. Ron Prywes (Columbia University). Lad1 shRNA was prepared in pSUPER targeting sequence HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). Sub-confluent cells were subjected to serum starvation (0.1% FBS) for 18 hours prior to stimulation. For transient overexpression, HeLa cells were transiently transfected with the different plasmids using Polyethyleneimine (PEI) 2, 2 mM EGTA, 150 mM NaCl and 0.1% Triton X-100.The cells were washed twice with phosphate buffered saline and once with Buffer A, scraped into Buffer H, and disrupted by sonication. For immunoprecipitation, the cell extracts were incubated with the proper Abs conjugated to protein A/G beads at 4°C for 2 h. The beads were washed once with RIPA buffer and twice with Buffer A. The immunoprecipitated complexes were then boiled in sample buffer and subjected to Western blot analysis using the indicated Ab. For coimmunoprecipitation, the beads were washed three times with buffer containing 20 mM HEPEs (pH 7.4), 2 mM MgClSamples were subjected to 8 or 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 2% bovine serum albumin (BSA) in TBS-T , and then incubated with the appropriate Abs. Membranes were developed with alkaline phosphatase (AP) or horseradish peroxidase (HRP)-conjugated anti mouse or anti rabbit Abs, followed by detection with BCIP/NBT color development substrate  or enhanced chemiluminescence (ECL) reagents , respectively. The intensity of the developed bands was well within the linear range of detection. The blots were scanned and the density of each band was analyzed with ImageJ. Each experiment was repeated at least three times and T-test was used to find statistic significance.32P]-ATP . For MEKK2 activity, endogenous MEKK2 was immunoprecipitated using anti MEKK2 Ab. MEKK2 activity was determined by the phosphorylating MEK5-K/A-GST.HeLa cells were transfected with MEK5-HA and were serum-starved for 18 hours. After different treatments, MEK5-HA was immunoprecipitated using anti HA Ab. MEK5 activity was determined by phosphorylating K/A-ERK5(1–397)-GST (2 mg/ml) in kinase reaction buffer with [γHeLa cells were grown in 24-well plates and transfected with pMEF2FLuc and pRL-TK with PEI. The cells were serum starved for 16 hrs. Three hours after the different treatments, luciferase activities were measured using Dual-Luciferase Reporter kit  according to the product manual.MEKK2 was immunoprecipitated from cell lysates and incubated with 2 µg GST-Lad1 for overnight in a buffer containing 50 mM β-glycerophosphate, 100 mM NaCl, 1 mM DTT supplemented with various concentrations of calcium. Each sample was washed four times with the buffer containing the same level of calcium.HeLa cells were plated on glass cover slips under standard culture conditions. After serum-starvation, the cells were treated with different stimuli and inhibitors as indicated, and the treatment was terminated by 3 washes with PBS. Then, the cells were subjected to fixation , permeabilization , and sequential incubation with the appropriate primary Abs  and secondary Abs (1∶100). The cells were visualized by fluorescence microscopy at 100X magnification.Calcium, as a major second messenger, influences the ERK1/2 cascade by inducing its activation We then further characterized the calcium effects, and found that changes in extracellular calcium levels following EGTA treatment had no effect, nor did it enhance the effect of BAPTA-AM when both were applied to the cells  and founWe further examined whether the effects of modulated calcium levels were transmitted to targets of ERK5, and MEF2 was selected for this purpose since it is a well-studied substrate of ERK5 ++/Calmodulin-dependent kinases (CaMK). We therefore undertook to examine the possible role of calcium as a cofactor that directly influences MEK5 activity towards ERK5. For this purpose, the phosphorylation of K/A-ERK5(1-397)-GST by MEK5 was assessed in vitro with different calcium concentrations in the reaction solution. We used the ATP binding site mutant which is catalytically inactive in order to eliminate the possible effect of feedback phosphorylation by ERK5 on its interaction However, 1 µM or 3 µM calcium in the reaction had essentially no effect on the activity of MEK5 towards ERK5 in vitro -GST). This construct was attached to glutathione beads and used for a GST-pulldown assay. Extracts of MEK5-HA transfected HeLa cells were then incubated with the beads in the absence of calcium (EGTA) or in the presence of different calcium concentrations. Following washing of the beads, levels of specifically bound MEK5-HA were assessed by Western blot. No effect on the interaction was detected when µM calcium concentrations were used, although higher (mM) concentrations slightly reduced the binding of MEK5 from both stimulated and unstimulated cells -GST  as a substrate. Indeed, MEKK2 from EGF-stimulated cells phosphorylated MEK5 much stronger than the phosphorylation by the protein precipitated from non-stimulated cells . These r–366) was overexpressed in the cells, ERK5 phosphorylation upon EGF treatment was much higher than in control transfected cells  15 min after stimulation. On the other hand, BAPTA-AM abolished the interaction between the two proteins almost completely. These results are in a good correlation with the effect of calcium modulation on MEKK2-ERK5 phosphorylation –2, ind–1 µM, which falls within the upper part of stimulated physiological levels. Above 1 µM, the binding was reduced to a comparable level to that of no calcium. Therefore, our results confirm that a narrow range of calcium concentrations is required for strong Lad1/MEKK2 interaction.In order to determine whether the calcium effect on MEKK2-Lad1 interaction is direct or mediated by other interacting proteins, we further examined the interaction by an in vitro binding assay. Thus, MEKK2 from HeLa cells treated with or without EGF was immunoprecipitated. After thorough wash, the precipitant was incubated with recombinant GST-Lad1 in the presence of various levels of calcium, and this was followed by three washes with buffers containing the same examined calcium concentrations. Overall, the interaction of Lad1 with MEKK2 was stronger when MEKK2 was immunoprecipitated from EGF-stimulated cells . More imIt was previously demonstrated that both Lad1 We next examined the Tyr phosphorylation of MEKK2 by immunoprecipitating the endogenous protein from treated HeLa cells that were transfected with either GFP-Lad1 or GFP alone. As expected, EGF stimulation of the control GFP-transfected HeLa cells induced a strong Tyr phosphorylation of the endogenous MEKK2, and this was inhibited by the c-Src inhibitor PP2 . Unlike We have previously shown that ERK5 and MEK5 are localized to the nuclei of resting, as well as EGF-stimulated cells, whereas MEKK2 is distributed all over resting cells and accumulates in the nucleus upon EGF stimulation The ERK5 cascade is a MAPK pathway that plays a role in the regulation of various cellular processes. However, the mechanism of activation and regulation of the ERK5 cascade are not well understood. One signaling mechanism that was shown to regulate the MAPK cascades is the elevation of calcium levels upon stimulation In order to identify the tier(s) in the ERK5 cascade that is directly responsive to the changes in calcium concentration we examined the effects of calcium modifiers on each of the cascade components. Our results clearly show that ERK5 phosphorylation by MEK5 is not affected by the calcium changes in vitro . On the After elucidating the signaling components upstream the ERK5 cascade, we examined which one of them may be responsible for mediating the reduced ERK5 phosphorylation upon changes in calcium concentrations. Our results show that a strong effect of varied calcium concentration was seen in MEKK2-Lad1 interaction , suggest–260 that were shown to be important for the MEKK2-Lad1 interaction The reduced MEKK2-Lad1 interaction upon pretreatment with BAPTA-AM and ionomycin  indicateAnother parameter examined here was the effect of calcium on the subcellular localization of the components of the ERK5 cascade. In a previous paper We found that both BAPTA-AM and ionomycin inhibited the nuclear translocation of MEKK2 upon EGF stimulation. These results were somewhat surprising since we expected that the prevention of interaction with the cytoplasmic Lad1 would release MEKK2 for a faster nuclear translocation. The fact that the translocation was inhibited might indicate either that the MEKK2-Lad1 interaction is important for the nuclear translocation of MEKK2, or that calcium plays a role in the nuclear penetration of this molecule. Since we found that calcium regulates ERK1/2 translocation In summary, this work presents novel regulatory aspects of EGF-induced ERK5 activation, mainly in regard to the role of intracellular calcium, as well as the process of nuclear translocation that accompanies this activation. We found that ERK5 activation by EGF is inhibited by both reduction and elevation of intracellular calcium levels. The changes in calcium were found to influence mainly the MEKK2-Lad1 interaction, and in vitro binding assays revealed that the proper calcium concentration is required for a direct MEKK2-Lad1 binding. The binding of these proteins is not affected by c-Src mediated phosphorylation on Lad1, but by itself slightly affects the Tyr phosphorylation of MEKK2, indicating a requirement for proper interaction to induce Tyr phosphorylation of MEKK2. In addition, we found that MEKK2 is distributed all over resting cells, and Lad1 appears mainly in the cytoplasm. Upon EGF stimulation MEKK2 accumulates in the nucleus where it can phosphorylate MEK5, while the localization of Lad1 remains unaffected. As changes in calcium levels inhibit EGF-induced MEKK2 translocation and nuclear accumulation, it is likely that this effect is due to a direct influence on its mechanism of translocation, independent of Lad1. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation by securing the proper interaction of the MEKK2 at the MAP3K level of this cascade with the upstream adaptor protein Lad1.