Protein kinases have evolved in eukaryotes to be highly dynamic molecular switches that regulate a plethora of biological processes. Two motifs, a dynamic activation segment and a GHI helical subdomain, distinguish the eukaryotic protein kinases (EPKs) from the more primitive eukaryotic-like kinases. The EPKs are themselves highly regulated, typically by phosphorylation, and this allows them to be rapidly turned on and off. The EPKs have a novel hydrophobic architecture that is typically regulated by the dynamic assembly of two hydrophobic spines that is usually mediated by the phosphorylation of an activation loop phosphate. Cyclic AMP-dependent protein kinase (protein kinase A (PKA)) is used as a prototype to exemplify these features of the PKA superfamily. Specificity in PKA signalling is achieved in large part by packaging the enzyme as inactive tetrameric holoenzymes with regulatory subunits that then are localized to macromolecular complexes in close proximity to dedicated substrates by targeting scaffold proteins. In this way, the cell creates discrete foci that most likely represent the physiological environment for cyclic AMP-mediated signalling. ThesecAMP was discovered by Sutherland as a hormone second messenger about the same time that Krebs and Fischer discovered protein phosphorylation as a regulatory mechanism ,6. PKA wcAMP is generated in response to the activation of G protein-coupled receptors (GPCRs) through the activation of Gαs, the activating G-protein subunit that stimulates adenylate cyclase . Over 302.The EPK superfamily is defined by a conserved kinase core. This core consists of a bi-lobal protein of approximately 250 residues that contains most of the essential machinery for catalysis and for scaffolding, the two functions that are essential for downstream signalling . ScatterThe N-terminal lobe (N-lobe) consists of a five-stranded anti-parallel beta sheet that is an essential part of the ATP-binding mechanism. Between β3 and β4 is the single conserved helix, the αC-helix. β-Strands 1 and 2 are joined by a glycine-rich loop, and this loop packs on top of the ATP with a backbone amide at the tip of the loop anchoring the γ-phosphate and positioning it for phosphoryl transfer. β-Strand 3 contains an essential Lys (Lys72 in PKA) that couples to a conserved Glu (Glu91 in PKA) in the αC-helix when the kinase is in an active conformation. The conserved helical subdomain in the N-lobe of PKA and other AGC kinases contains a short αB-helix and a long αC-helix. The αB-helix is not conserved in all kinases, but the αC-helix is an essential conserved feature of every protein kinase. When a kinase is in an active conformation, the N-terminus of the αC-helix typically interacts with the activation loop phosphate (see later text), while the C-terminus is part of the hinge at the base of the active site cleft.In contrast to the N-lobe, the C-terminal lobe (C-lobe) is mostly helical and quite stable conformationally. The αE-, αF- and αH-helices, based on hydrogen–deuterium exchange, for example, are very stable and do not exchange much with solvent even after several days . Resting3.Solving the structure of PKA allowed us to see the conserved sequence motifs of the EPK family in three-dimensions and gave functional significance to each motif ,22. HaviThe LSP method was then used to compare all protein kinases, and this revealed another hydrophobic ‘spine’ that runs parallel to the regulatory (R) spine. Unlike the R-spine, this second spine is completed not by the dynamic insertion of a hydrophobic amino acid side chain but rather by the adenine ring of ATP. This spine is therefore referred to as the ‘catalytic’ spine or C-spine. ATP binding thus positions the two lobes so that the catalytic residues are aligned optimally for catalysis. Both spines are anchored to the αF-helix. The catalytic loop is also firmly anchored through hydrophobic residues to the αF-helix. This hydrophobic core is a conserved feature of all EPKs and is quite distinct from the hydrophobic core of most globular proteins. Assembly of the R-spine in such a dynamic way is a fundamental feature of the ‘switch’ mechanism.Outliers of the EPK family are the phosphoinositide 3-kinases (PI-3 kinases) that share much of the catalytic machinery that is found in the EPKs and ELKs. However, the GHI subdomain is different as is the activation loop. These PI-3 kinases, nevertheless, do have the remnants of the two hydrophobic R- and C-spines .4.cis- or trans-autophosphorylation or by the action of a heterologous activating kinase (trans). This activation by phosphorylation is a characteristic feature of most EPKs, and in some cases, such as ribosomal S6 kinase (RSK), there are 4–5 kinases that contribute to the activation of a single kinase [The era of genome sciences has allowed us to delve deeply into the evolutionary history of proteins. From such genome-wide screens, we discover that EPKs evolved from much simpler ELKs that are abundant in prokaryotes . Many ofe kinase . A few kKm (ATP) and a decrease in kcat. The αH-helix is an allosteric regulatory site in the yeast homologue of PKA, tpk1, and provides a pathway for substrates to communicate with the active site [The GHI helical subdomain, which has co-evolved with the complex and dynamic activation loop in EPKs, is also missing in the ELKs. This subdomain provides docking sites for tethering protein substrates and also provides additional regulatory/allosteric control. Both the GHI helical subdomain and the activation loop are anchored firmly to the hydrophobic αF-helix and are linked to each other by a highly conserved and buried ion pair that is a hallmark signature motif of the EPKs. This ion pair consists of a conserved arginine (Arg 280 in PKA) that lies between the αH- and αI-helices and a conserved glutamate (Glu208 in PKA), which is at the end of the activation loop and part of the Ala-Pro-Glu (APE) motif. The segment that lies between the APE motif and the αF-helix is also a structurally conserved element; it serves as a hydrophobic anchor that locks the activation loop onto the αF-helix . Includeive site . This Glive site .5.Although the core that defines the protein kinase superfamily contains most of the essential catalytic machinery, it is surprisingly not usually sufficient to mediate optimal catalysis on its own. Instead, it is assembled into a fully active state by interactions with flanking regions or domains that are anchored to the core . In the The highly sophisticated ways in which the tails can contribute to both regulation and catalysis can perhaps best be appreciated by looking more closely at the PKA catalytic subunit and the AGC subfamily . PKA is cis and trans [The C-tail, however, is highly conserved in all members of the AGC subfamily and is also highly regulated by phosphorylation, both nd trans . The C-tnd trans . This isnd trans . The secnd trans . In the 6.The EPKs are highly regulated enzymes that function as molecular switches, and PKA once again serves as a template to understand how the dynamic features that are a requirement for a ‘switch’ are implemented. Nuclear magnetic resonance studies of the PKA catalytic subunit, for example, have begun to explore the dynamic properties of the different conformational states of the catalytic subunit. They define the apoenzyme as a catalytically ‘uncommitted’ state where there are few backbone movements is the ms/μs time frame, the range that is important for catalysis ,43. Addi7.Km values with efficient kcat values while EPKs have low Km values with relatively poor kcat values. Because of these drastic concentration differences, EPKs have evolved efficient mechanisms such as scaffolding, small protein : protein interaction domains and peptide-binding pockets in order to increase the effective concentrations of EPK–substrate encounter complexes.The EPKs are dynamic switches that are distinct from metabolic kinases such as hexokinases, which are molecular machines that turnover small molecules, often at or near rates limited only by diffusion and proportional to the concentrations of substrates. The other distinction between EPKs and metabolic kinases is the amount of substrates they encounter in the cell. There are usually millimolar concentrations of small molecules that get phosphorylated by other kinases, but EPKs and their protein substrates are present in submicromolar or even nanomolar concentrations. This substrate concentration difference between metabolic kinases and EPKs is one major reason why metabolic enzymes have high Km and kcat values are no longer relevant when concentrations of the enzyme and substrate are in the same range, sometimes with 1 : 1 stoichiometries. Thus, pre-steady-state kinetics with real numbers for the phosphoryl transfer step as well as Kd values are of critical importance to biophysically analyse EPKs. PKA was the first protein kinase to be comprehensively analysed by pre-steady-state kinetics [kcat (500 versus 20 s−1). Such kinetics are consistent with the sort of rapid and transient reactions that have been optimized for specific and local phosphoryl transfers to proteins involved in a pathway, while minimizing non-specific phosphorylation of off-target proteins. Unfortunately, pre-steady-state kinetics is not a routine tool for investigating kinase mechanism, but we suggest that it should be routinely used to reveal new details of EPK mechanisms, and steady-state kinetics must be used as a preliminary guide.The basic tenet of mechanistic enzymology, carried over from metabolic enzymes, does not apply to the true physiology of EPKs. By this, we mean that steady-state kinetics and the apparent kinetics , and thi2+ ions, it was found that the enzyme works on its specific substrate (neurexin) in the absence of Mg2+ [Appreciating that efficient catalysis is not a feature that is essential for an EPK allows us to go back and look more carefully at the members of the kinome that were predicted to be pseudo kinases or ‘dead’ kinases. These kinases were predicted to be inactive because they lacked one or more of the conserved catalytic residues. However, a number of these have subsequently proved to be functional kinases. WNK (with no lysine (K)), for example, which lacked the lysine in β-strand 3 was found to have another spatially conserved basic residue that occupies the same space in the structure and serves the same functional role . In the  of Mg2+ . Vaccini of Mg2+ . This ki of Mg2+ ,49. It i8.Most of the EPKs are themselves phosphoproteins, and PKA serves as a template to understand how phosphorylation contributes to regulation. There are two sites of phosphorylation in the PKA catalytic subunit, Thr197 in the activation loop and Ser338 in the C-tail. Each contributes in unique ways to creating an active enzyme.Escherichia coli, so until recently, all of the PKA structures have been of the fully phosphorylated and active enzyme. By mutating the P-3 Arg in the activation loop, Arg194, we were able to purify a dephosphorylated enzyme that could then be phosphorylated in vitro by PDK1, which is a universal activating kinase for the AGC subfamily of EPKs. The C-subunit that lacks the phosphate on its activation loop is unstable and is also highly dynamic based on hydrogen deuterium exchange mapping by mass spectrometry (HDXMS), and in each case, the properties of the wild-type protein are restored by simply adding back the phosphate [Km values for ATP and peptide are both increased. However, the most profound effect on catalysis is seen when one examines the pre-steady-state kinetics. PKA, such as a number of EPKs, shows an initial burst of activity that then levels off to give a steady-state rate of catalysis. The kcat is regulated by the off-rate of MgADP. In the absence of the Thr197 phosphate, the pre-steady-state burst is virtually eliminated [Phosphorylation of the activation loop is essential for optimal PKA activity. As seen in hosphate . The catiminated .Figure The crystal structure of the PKA C-subunit lacking its activation loop phosphate shows the profound effect that this phosphate has on the assembly of the active enzyme . It democis-autophosphorylation site and occurs co-translationally as in the case of Akt [In almost every AGC kinase, there are two critical sites, in addition to the activation loop site, that are regulated by phosphorylation . Betweene of Akt , wherease of Akt . With the of Akt . The HM e of Akt . As the In PKA, the C-tail site is most likely phosphorylated prior to the activation loop; however, once the two sites are phosphorylated they are very resistant to removal by phosphatases . This is9.While we have learned much about the structure and function of PKA from studying the free C-subunit, in cells, it is assembled as a holoenzyme complex with inhibitory regulatory (R) subunits. Ultimately, it is the tetrameric holoenzyme complex that reflects the basal physiological state of the enzyme. In all mammals, there are four functionally non-redundant R-subunits , which all have a stable dimerization domain at the N-terminus. This is followed by a flexible linker that is classified as an intrinsically disordered region. Embedded within the linker is an inhibitor site that resembles a substrate and docks to the active site cleft in the holoenzyme. At the C-terminus are two CNB domains. In the absence of cAMP, two fully phosphorylated C-subunits bind to the R-subunit dimer rendering it inactive. The very stable helical dimerization domain is multi-functional. In addition to creating a stable dimer, it serves as a docking site for AKAPs. These are polyvalent scaffold proteins that are characterized by an amphipathic helix that binds with high affinity to the dimerization/docking (D/D) domain of the R-subunits . Other sOur understanding of PKA signalling has grown significantly as new structures were solved that included not only the free C-subunit but also complexes between the R- and C-subunits . We did 10.Clearly, one needs to now consider the entire system if one is to truly appreciate the complexity and beauty of how the eukaryotic cell has evolved to regulate its biological events by a highly dynamic process such as protein phosphorylation. One also needs to revisit the established paradigm that PKA signalling requires full dissociation of the R- and C-subunits. If activation occurs on a macromolecular complex that is anchored to a channel, for example, and co-localized with other signalling proteins such as phosphodiesterases, cyclases and/or phosphatases, it is not difficult to envision PKA signalling as an oscillatory process where full dissociation is not required. Such oscillatory circuits for PKA signalling have been reported recently for cAMP and calcium . We know


A small molecule can enhance the memories of rats and mice by blocking the integrated stress response in these animals. Related research article Sidrauski C, Acosta-Alvear D, Khoutorsky A, Vedantham P, Hearn BR, Li H, Gamache K, Gallagher C, Ang KK-H, Wilson C, Okreglak V, Ashkenazi A, Hann B, Nader K, Arkin MR, Renslo AR, Sonenberg N, Walter P. 2013. Pharmacological brake-release of mRNA translation enhances cognitive memory. eLife2:e00498. doi: 10.7554/eLife.00498Image Immunofluorescence images of cells treated with ISRIB, a small molecule that enhances the memory of mice and rats by blocking a cellular pathway that controls protein synthesisMrs Frisby and the rats of NIMH, written by Robert C. O’Brien in 1971, Mrs Frisby is a field mouse aided by super-intelligent rats. In the story readers learn that scientists experimenting at the National Institute of Mental Health (NIMH) have injected rats and mice with chemicals to improve their memories. The chemicals work so well that the animals learn how to read and, ultimately, escape from the laboratory… Now Peter Walter of the University of California at San Francisco and colleagues, including Carmela Sidrauski of UCSF as first author, report that they have identified a small molecule that enhances the memory of mice and rats by blocking a highly-conserved pathway for the control of protein synthesis  that attenuates ATF4 induction without altering the IRE1 or ATF6 responses. Subsequent tests convincingly showed that this molecule does not prevent the activation of PERK or the phosphorylation of eIF2α. This means that ISRIB—unlike GSK2606414, a recently identified small molecule that inhibits PERK —is not aHaving shown that ISRIB can inhibit one of the most conserved translational control pathways in eukaryotic cells, a remaining challenge is to identify the molecule that it targets. The prime candidate is eIF2B, which is normally inhibited by eIF2α phosphorylation, and Sidrauski et al. suggest that ISRIB might either boost eIF2B activity or reduce its sensitivity to eIF2 phosphorylation. Both are plausible ideas, but other explanations cannot yet be excluded. Genetic studies using yeast have identified many mutations in eIF2 and eIF2B that permit eIF2 phosphorylation but prevent the downstream signalling responses . The impThe control of protein synthesis is important in many contexts, including the establishment of long-term memories. Experiments in which the genes involved in the integrated stress response are manipulated revealed that the modulation of this pathway influences learning and memory. For example forebrain-specific knock-out of PERK function in mice reduces both the phosphorylation of eIF2 and the expression of ATF4, leading to impaired behavioural flexibility . By contSidrauski et al. now show that administering ISRIB to mice and rats results in the enhancement of their spatial and fear-conditioning memories. These results are consistent with previous work by Nahum Sonenberg of McGill University, who is part of the UCSF-McGill-Genentech collaboration: Sonenberg and co-workers replaced the serine 51 residue that the kinases bind to with an alanine residue within one of the two eIF2α gene copies present in every cell in a mouse model, and showed that eIF2α phosphorylation signalling was reduced, but not eliminated, and that memory was enhanced in a range of tests .Taken together with other studies, it is clear that signalling through eIF2α is finely balanced, and that the chronic loss or stimulation of a single pathway can have highly deleterious effects. This is also observed in human disease. Mutations in the gene that codes for PERK cause Wolcott-Rallison Syndrome, a recessive and severe form of diabetes with bone abnormalities and mental retardation, and mutations in the genes that code for eIF2B cause VWM disease , a disorder that affects the formation of white matter within the brain . It has It is tempting to speculate that ISRIB, or a derivative of this compound, may have beneficial effects for patients with diseases where memory is impaired. However eIF2α phosphorylation affects multiple tissues and pathways, not just the brain: Wolcott-Rallison Syndrome, for example, also affects the bones, kidney and liver, and eIF2 phosphorylation is also known to affect metabolism . ReducedIt is clear that ISRIB will be an excellent experimental tool to help provide deeper understanding of the role of the integrated stress response, but it is likely that further advances will be needed to develop ISRIB, or a derivative, into a therapeutic that could treat memory disorders without unwanted side effects.


Initiation is the rate-limiting phase of protein synthesis, controlled by signaling pathways regulating the phosphorylation of translation factors. Initiation has three steps, 43S, 48S and 80S formation. 43S formation is repressed by eIF2α phosphorylation. The subsequent steps, 48S and 80S formation are enabled by growth factors. 48S relies on eIF4E-mediated assembly of eIF4F complex; 4E-BPs competitively displace eIF4E from eIF4F. Two pathways control eIF4F: 1) mTORc1 phosphorylates and inactivates 4E-BPs, leading to eIF4F formation; 2) the Ras-Mnk cascade phosphorylates eIF4E. We show that REN and NCI-H28 mesothelioma cells have constitutive activation of both pathways and maximal translation rate, in the absence of exogenous growth factors. Translation is rapidly abrogated by phosphorylation of eIF2α. Surprisingly, pharmacological inhibition of mTORc1 leads to the complete dephosphorylation of downstream targets, without changes in methionine incorporation. In addition, the combined administration of mTORc1 and MAPK/Mnk inhibitors has no additive effect. The inhibition of both mTORc1 and mTORc2 does not affect the metabolic rate. In spite of this, mTORc1 inhibition reduces eIF4F complex formation, and depresses translocation of TOP mRNAs on polysomes. Downregulation of eIF4E and overexpression of 4E-BP1 induce rapamycin sensitivity, suggesting that disruption of eIF4F complex, due to eIF4E modulation, competes with its recycling to ribosomes. These data suggest the existence of a dynamic equilibrium in which eIF4F is not essential for all mRNAs and is not displaced from translated mRNAs, before recycling to the next. Extracellular signals control the translational machinery, inducing phosphorylation and changes in the activity of translation factors. However, the rapidity by which the translational machinery responds to extracellular signaling, regulating rate and mRNA selection has not been systematically addressed. Initiation of translation is the rate-limiting step of translation, and is controlled by initiation factors (IF) 1) Formation of 43S through the recruitment of the ternary complex, formed by eIF2-GTP-tRNA(i)Met, on the small 40S ribosomal subunit. Inhibition of eIF2 activity is mediated by phosphorylation of eIF2α subunit by either one of four eIF2α kinases, PERK, Gcn2, HRK, PKR switched on by several conditions, including ER stress, aminoacid deprivation, lack of heme, double strand RNA 7Gppp cap structure 2) Formation of 48S after the binding of mRNA complexed to eIF4F, and scanning to the AUG start codon. The 5′ end of mRNA binds 43S, mediated by eIF4F. eIF4F is a multiprotein complex: initiation factors eIF4E, eIF4G, and eIF4A assemble on the mIn principle, eIF4F assembly depends from signaling pathways. Prominently, the phosphoinositide 3-kinase (PI3K) signaling pathway, one of the most frequently deregulated oncogenic pathways, converges on the serine/threonine kinase Akt, and to the mammalian target of rapamycin (mTOR) kinase 3) The third regulating step of initiation is the formation of elongating 80S, upon recruitment of free 60S subunits and release of eIF2, assisted by eIF5B To further dissect the signaling pathways leading to translational machinery regulation, we screened for cells where the mTORc1 and Ras/ERK pathways were active and studied the effect of their inhibition on translation. We focused on short term treatment with inhibitors, in order to analyze direct effects on the translational machinery. We found that in REN and NCI-H28 cell lines, initiation of translation was insensitive to the treatment with mTOR inhibitor, with exception for translation of TOP mRNAs. Sensitivity to inhibition of the mTORc1 pathway could be reestablished only by 4E-BP1 expression or eIF4E downregulation. These data lead to a model in which the rate of rapamycin sensitivity can be regulated transcriptionally. The lack of effects on global translation by short term inhibition of the main pathways suggests that initiation factors can recycle to a new mRNA efficiently without being regulated by mTORc1 or Ras signaling.We screened for cell lines with constitutively activated mTOR-Akt pathway in order to test the hypothesis that activated pathways lead to deregulated translation, which can be targeted by pharmacological treatment. We found a malignant mesothelioma cell line REN, able to grow under starving conditions that was further characterized. Most mesothelioma cells Mesothelioma cells are actively secreting cells Taken together, these data indicate that REN cells are highly sensitive to ER stress-induced block of protein synthesis, i.e. 43S formation, but are independent from growth factor stimulation. These data suggest that REN cells have constitutive activation of the growth-factor activated pathways, and translation should be repressed by inhibiting signaling pathways.Two main pathways transduce the growth factor stimulation to the translational machinery: PI3K-mTORc1 and Ras-MAPK-Mnk. These two pathways are always deregulated in solid tumors, and converge on the translational machinery . We wantIt has been reported that mTORc1 inactivation by rapamycin results in a feedback activation of ERK1/2 It is established that rapamycin blocks mTORc1 complex, but not mTORc2 complex. mTORc2 phosphorylates Akt, accounting for the activation of a pathway that may impinge on survival and on the translational machinery These data indicate that in REN cells translation rates are insensitive to the inhibition of growth factor signaling pathways.A known readout of growth factor stimulation is the assembly of the cap complex, which is dependent on mTOR signaling. Data in Thus, we asked whether inhibition of mTORc1 and MAPK signaling affected translation of specific mRNAs. Polysomal profiles of cells treated with inhibitors showed small changes in 80S/polysomal ratio, confirming the minimal reduction of initiation when the pathways are blocked . FractioWe then asked what is the rate-limiting event of cap dependent translation of REN cells. Thus, we formulated the hypothesis that 4E-BP1 is not sufficient to sequestrate eIF4E. REN cells were infected with adenovirus overexpressing 4E-BP1 wt, 4E-BP1 LM/AA, protein mutant unable to bind eIF4E Next, we downregulated eIF4E, by interfering with specific siRNA. Downregulation was confirmed by western blot  and rapaSo far we have shown that REN cells have an impairment of cap complex formation, but not global reduction of translation, upon mTOR inhibition. Next we evaluated whether this phenomenon is present in other cells. Global translation of ME-180 cells, that have constitutively activated PI3K Here we conclude that cap complex inhibition does not match the global translation, and that global translation is not necessarily reduced by mTOR inhibition in all cellular types.in vivo, at steady-state recycling of eIF4E from a processed mRNA to a new preinitiation complex is either kinetically or sterically favored respect to binding of eIF4E to 4E-BP.We show that in the absence of 4E-BP1 overexpression or eIF4E downregulation, the efficient pharmacological inhibition of mTOR pathway does not change, in short term, the efficiency of the translational apparatus. These data indicate that part of the regulation of translation rate, at least in REN cells, may occur transcriptionally through the abundance of eIF4E. In addition, rapamycin does not depress translation, in spite of reducing cap complex formation. It follows that, Translational control is becoming an attractive target in cancer cells The transcriptional regulation of initiation factors is an emerging concept, that is becoming relevant in cancer research in vivo, is delayed, indicating that eIF4E actively engaged in eIF4F, recycles to the mRNA, rather than binding to free 4E-BPs. This interpretation is backed up by the observation that increasing 4E-BP1 or lowering eIF4E results in a more rapid inhibition of translation by rapamycin; 2) in principle, oncogenic activation by Ras, PI3K or Myc may alter by a transcriptional mechanism the sensitivity of the translational machinery to pharmacological inhibition.The marginal effect of the inhibition of signaling pathways on the translational machinery can reflect the fact that we employed short-term treatments. This led to an interesting observation: the temporal disjunction between the efficient inhibition of the pathway and the effect on translational machinery. First, we found that in REN cells rapamycin treatment blocked mTOR dependent assembly of cap complex, without a significative reduction of metabolic rate. Moreover, when we blocked TOR kinase activity with PP242, leading to a complete inhibition of both mTORc1 and mTORc2, even at 100 µM, where PP242 has been reported to aspecifically block PI3K/Akt pathway An early event observed after rapamycin treatment is the decrease of translation of TOP mRNAs. This indicates that TOP mRNA translation is highly sensitive to cap complex inhibition as shown in several laboratories , cycloheximide, MEK inhibitor U0126 and mTOR inhibitor PP242 were from Sigma. RNasin® was from Promega. Mnk1 inhibitor 4-Amino-5-(4-fluoroanilino)-pyrazolopyrimidine and mTORc1 inhibitor rapamycin were purchased from Calbiochem. All other chemicals were from Sigma.35S-labeled methionine (PerkinElmer Life Sciences) for 45 min, in the tested conditions. Cells were lysed in 50 µl of RIPA buffer without SDS . The lysate was cleared by centrifugation. Aliquots of 10 µl of extracts were TCA-precipitated and counted with scintillation fluid in a β-counter (Packard). Values were normalized on sample protein content by the bicinchoninic acid (BCA) protein assay (Pierce). Each sample was performed in triplicate and expressed as mean ± SEM. Statistical analysis was performed with T Student test. * is P<0.05. ** is P<0.01.Cells were pulsed with 33 µCi per plate of Promix 2, 10 mM Tris–HCl, pH 7.3, 1% Triton X-100, 1% Na-deoxycholate, 1 mM DTT, 5 mM NaF, 2 mM Na3VO4, 40 U/ml RNasin® (Promega), protease inhibitor cocktail. The crude cell extract was clarified at 4°C at 15,000g for 10 min and the amount of protein in the supernatant was quantified by the bicinchoninic acid (BCA) protein assay. Equal amount of proteins was analyzed. Extracts were resolved on SDS–PAGE, transferred to Immobilon-P membranes (Millipore), and probed with appropriate antibodies. The following antibodies were used: rabbit anti-phospho-eIF2α (Ser 51), anti-eIF2α, anti-phospho-4E-BP1 (Ser 65), anti-4E-BP1, anti-phospho-rpS6 (Ser 235/236), anti-rpS6, anti-phospho-p44/42 MAPK (ERK1/2) (Thr 202/Tyr204), anti-p44/42 MAPK (ERK1/2), anti-phospho-eIF4E (Ser 209), anti-eIF4E, anti-phospho-Mnk1 (Thr 197/202), anti-Mnk1, anti-phospho-Akt (Ser 473), anti-Akt, anti-eIF4G and anti-c-myc from Cell Signaling; mouse anti-β Actin from Sigma; mouse anti Caspase 3 from Alexis. Densitometric analysis was performed by ImageQuant 5.0 software (Molecular Dynamics).Cells were lysed with buffer containing 10 mM NaCl, 10 mM MgClCap column pull-down was performed on 400 µg of total extracts as described 6) that had been washed once with phosphate-buffered saline buffer , were lysed directly on the plate with 300 µl of lysis buffer  and transferred into a microcentrifuge tube. After 5 min of incubation in ice with occasional vortexing, the lysate was centrifuged for 10 min at 10,000 rpm  at 4°C. The supernatant was frozen in liquid nitrogen and stored at −70°C to be analyzed later, or immediately layered in a 15–50% (w/v) sucrose gradient containing 30 mM Tris-HCl pH 7.5, 100 mM NaCl and 10 mM MgCl2, and centrifuged in a Beckman SW41 rotor for 110 min at 37,000 rpm .Polysomal profiles were performed as previously described Analysis of the polysome/mRNP distribution of mRNAs were performed essentially as previously described Adenovirus: Lac Z control, 4E-BP1 WT and 4E-BP1 LM/AA, were a kind gift of Pr C. Proud. Virus stock were amplified and titer were determinated using Adeno-X TM rapid titer kit . Multiplication of infection (M.O.I) of 50 for 4E-BP1 WT and of 100 for 4E-BP1 LM/AA and control LacZ were used (as reported in 5′-GGUAUACAAGGAAAGGUUAtt-3′ (sense) and 5′-UAACCUUUCCUUGUAUACCct-3′ (antisense). Identical not shown results were obtained with siRNA ID #4577, 5′-GCUAAUUACAUUGAACAAAtt-3′ (sense) and 5′-UUUGUUCAAUGUAAUUAGCca-3′ (antisense). The negative control siRNA was purchased from Ambion (negative control siRNA #1).The siRNA targeted to human eIF4E was purchased from Ambion. The sequence is as follows: siRNA ID #4578,5) were seeded per plate (35 mm diameter) the day before transfection. Cells were transfected with 20 nM oligonucleotide using N-TER™ Nanoparticle siRNA Transfection System (Sigma) according to manufacturer protocol. Protein expression was assessed by Western blotting at 48 h post-transfection and densitometric analysis was performed. A metabolic assay was carried out at 48 h post-transfection as described above.REN cells  Densitometric analysis of phospho-eIF2α normalized to total eIF2α after thapsigargin (Tg) treatment is reported from four independent experiments. (B) Total extracts from (TIF)Click here for additional data file.Figure S2ER stress inducing drugs do not cause Caspase 3 activation at the analyzed timepoint. (A) REN cells were infected with HA-4E-BP1 WT, 4Ala (unable to be phosphorylated) or with empty control retrovirus pBABE. Infected cells were treated with rapamycin and pulsed with 35S-methionine. Methionine incorporation in newly translated proteins was measured in triplicate. 4Ala-4E-BP1 transfected cells are unable to sense the rapamycin treatment. (B) REN cells extracts from (TIF)Click here for additional data file.Figure S3C-Myc and initiation factors protein expression in REN and mesMM98 cell lines. Total protein extract from malignant mesothelioma cell lines REN and mesMM98 were analyzed with WB for eIF4E, eIF4G, 4E-BP1 and c-Myc protein expression. Densitometric analysis of eIF4E, eIF4G, 4E-BP1 and c-Myc levels normalized to β-Actin are reported. REN cell express more c-Myc and eIF4E proteins.(TIF)Click here for additional data file.


While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs.Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation.Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products. G6PD deficiency affects more than 400 million people worldwide, with a prevalence varying from 10 to 25% in most areas where malaria is endemic. This genetic defect provides partial protection against malaria, but may lead to severe hemolytic episodes after the administration of some drugs , the ingestion of fava beans (favism) or infection − RBCs) display a failure of the protective response to oxidant stress, which leads to irreversible oxidation of glutathione − individuals While the molecular biology of G6PD deficiency has been extensively studied − RBCs, such as the oxidation and clustering of membrane proteins, the binding of hemichromes to the internal face of the membrane, the destabilization of the membrane and the release of micro-vesicles − RBCs infected by malaria parasites − RBC destruction. The dearth of information concerning the mechanisms of red cell lysis represents a practical drawback which impedes both any prediction about the hemolytic activity of drugs and the understanding of the large individual susceptibility even in presence of the same G6PD mutation Some membrane alterations have been described in GThe authors, as well as others have shown that band 3 red cell membrane protein (AE1) displays a marked tendency to become tyrosine phosphorylated in G- RBCs after –SH group oxidation or GSH depletion by 1-chloro-2,4-dinitrobenzene (CDNB) or diamide − RBCs. Syk kinase inhibition largely prevents red cell membrane lysis and vesiculation, strongly suggesting a functional role of AE1 tyrosine phosphorylation in the red cell membrane destabilization.In the present study we have demonstrated that following –SH group oxidation induced by diamide (–SH group oxidant) and divicine, an oxygen reactive compound held responsible for favism − RBCs than in control RBCs − deficient subjects − RBCs were not easily predictable as, although the G− RBC samples used in our experiments had low G6PD levels , the hexose monophosphate shunt activity in these cells presented normal activity and could be 2–3 fold further activated following oxidant treatments Previous work has described how oxidant treatments induce more intense AE1 tyrosine phosphorylation in G− RBCs reduced GSH further declined and reached un-measurable levels within 2 hours of incubation  was oxidized within 5 minutes and the pre-treatment levels were restored within 45 minutes in control RBCs. Conversely, in Gcubation . Indisti− RBCs, AE1 phosphorylation progressively increased during the course of incubation (10 hours). In control RBCs AE1 phosphorylation was completely reverted in approximately 1 hour. Syk kinase inhibitors markedly reduced the rate of AE1 phosphorylation both in control and G− RBCs. AE1 oxidation (disulfide cross-linking) parallels its tyrosine phosphorylation both in control and G− RBCs  by ultra-centrifugation and then characterized by confocal analysis of hemichrome auto-fluorescence, western blotting and mass spectrometry. Confocal microscopy analysis was suggestive of large amounts of hemichromes in isolated vesicles . The pre− RBCs than in control RBCs following their treatment with diamide but, after a short incubation time, we did not observe any functional consequence. Hemolytic crises in G− subjects occur many hours after the intake of pro-oxidant compounds − RBCs with diamide (an -SH group oxidant reagent) or divicine (a compound extracted from fava beans held responsible for severe hemolytic crises in G− deficient subjects) In a previous study − and control RBCs but after 2–3 hours of incubation, striking differences were observed between the two types of red cells. While control RBCs returned to the pre-treatment conditions, G− RBCs showed a time dependent modification of several parameters: i) the GSH levels and -SH groups of AE1 continued to decrease; ii) Syk tyrosine kinase became irreversibly associated with the membrane; iii) AE1 tyrosine phosphorylation steadily increased over time, iv) hemichromes became progressively bound to the membrane; v) phosphorylated AE1 formed large clusters; vi) these clusters were exovesiculated and this phenomenon was associated with lysis.The treatment with these compounds caused similar short term effects in G− RBCs, AE1 hyper-phosphorylation may play a role in the progressive membrane damage induced by oxidant treatments.We have observed that Syk inhibitors consistently reduced the amount of vesiculation and lysis. The protective effect of Syk inhibitors suggested that, in G− RBCs revealed their high content of hemichromes and phosphorylated AE1.This hypothesis is supported by some additional findings: i) o-vanadate (tyrosine phosphatase inhibitor) further increased AE1 phosphorylation and the rate of vesicle release, ii) we have isolated membrane protein complexes which contain both hemichromes and phosphorylated AE1, iii) confocal microscopy confirmed the co-association between hemichromes and phosphorylated AE1, iv) vesicles isolated from G− subjects undergoing hemolysis are currently available, but the presence of high levels of hemoglobin in the plasma strongly suggests the occurrence of intravascular hemolysis due to red cell membrane instability The present findings are in accordance with previous results which have demonstrated that Syk kinase binds preferentially to oxidized AE1, inducing its tyrosine phosphorylation − RBCs with oxidant compounds  in presence of 5 mM glucose (PBS-glucose) to obtain packed cells. To simulate oxidative stress, RBCs at 30% hematocrit were treated for different incubation times (0–600 min) at 37°C in PBS-glucose containing 0.25 mM of diamide (Sigma) or divicine . Divicine was activated by β-glucosidase treatment as previously described Venous blood was drawn from 5 healthy and 5 G6PD deficient volunteers . Written informed consent was obtained from each blood donor to allow: “The use of 10 ml of their blood donation for research use. In particular, to study the causes of hemolytic episodes in G6PD deficient subjects”. Ethical approval to perform the present study was obtained from the “Ethical Committee of the ASL 1 – Sassari”. The data were analyzed anonymously and all clinical investigation was conducted according to the principal expressed in the Declaration of Helsinki. None of the donors showed clinical and laboratory evidence of hemolysis and all presented hemoglobin levels within the normal range. G6PD deficiency assessment was performed as previously described For the inhibition of tyrosine phosphatases, RBCs were suspended at 30% hematocrit in PBS-glucose and incubated with 1 mM of o-vanadate in presence or absence of oxidants.For the inhibition of Syk kinase, RBCs were suspended at 30% hematocrit in PBS-glucose and pre-incubated with 10 µM Syk inhibitors II and IV  for 1 h at 37°C before oxidant treatments. The inhibitors were not washed prior to the oxidant treatment.Each reaction was stopped by washing 3× in PBS-glucose, and membranes were prepared as described below. For all the protocols described above, untreated controls were processed identically, the only difference being the deletion of the stimulant/inducer from the incubation.Standard hypotonic membranes were prepared at 4°C on ice as previously described The supernatants of the samples treated as described above, were collected and centrifuged at 25000× g for 10 min at 4°C to eliminate spontaneously formed red cell ghosts. Lysis was quantified by measuring hemoglobin absorbance at 405 nm and expressed in nmoles/ml. After the addition of phosphatase infhibitors, supernatants were centrifuged for 3 hours at 100000× g on a refrigerated ultracentrifuge (Beckman) to isolate vesicles. Protein vesicles were identified by MALDI TOF MS Membrane proteins were solubilized in Laemmli Buffer 2EDTA, 5 mM dithiothreitol, 2 mM MnCl2.Proteins separated by SDS/PAGE were transferred to nitrocellulose membranes as previously described N-ethylmaleimide, 1 mM EDTA, 1 mM PMSF, 1% triton-X100, pH 7.4), gently shaken for 10 min at 20°C and centrifuged at 13000 rpm for 5 min in an Eppendorf microfuge. The supernatant was applied to a 40×1 cm column filled with Sepharose CL-6B equilibrated with a solution containing 10 mM Hepes, 50 mM NaCl, 0.1% triton-X100 (pH 7.4), at a flow rate of 1 ml/min. Constant flow was maintained using an HPLC pump. The effluent was collected in 1 ml fractions. Eosine maleimide fluorescence was quantified as described AE1 was labeled with eosine maleimide and red cell membranes were fractionated as previously described Control and G6PD deficient RBCs were pelleted and washed twice in PBS 1× containing 5 mM glucose and then fixed for 5 min in 0.5% acrolein in PBS. Cells were rinsed three times then permeabilized in PBS containing 0.1 M glycine (rinsing buffer) plus 0.1% Triton X-100 for 5 min and again rinsed 3× in rinsing buffer. To ensure complete neutralization of unreacted aldehydes, the cells were then incubated in rinsing buffer at room temperature for 30 min. After incubation, all nonspecific binding was blocked by incubation again for 60 min in blocking buffer . Staining of fixed, permeabilized RBCs was performed by using specific antibodies diluted in blocking buffer. After labeling, resuspended RBCs were allowed to attach to cover slips coated with polylysine, and the cover slips were mounted by using Aqua-Mount . The auto-fluorescence of hemichromes was visualized by exciting at 488 nm and observing their emission in the 630–750 nm range. Samples were imaged with a Bio-Rad MRC1024 (Bio-Rad) confocal microscope equipped with a 60×1.4 numerical aperture oil immersion lens.Hemichromes were quantified by measuring heme absorbance at 560, 577 and 630 nm GSH estimations were performed using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)


Phosphosites are annotated with specific protein kinases and/or phosphatases, along with the condition(s) under which the phosphorylation occurs and/or the effects on protein function. We report here an updated data set, including nine additional high-throughput (HTP) mass spectrometry studies. The version 2.0 data set contains information on 20 177 unique phosphorylated residues, representing a 4-fold increase from version 1.0, and includes 1614 unique phosphosites derived from focused low-throughput (LTP) studies. The overlap between HTP and LTP studies represents only ∼3% of the total unique sites, but importantly 45% of sites from LTP studies with defined function were discovered in at least two independent HTP studies. The majority of new phosphosites in this update occur on previously documented proteins, suggesting that coverage of phosphoproteins in the yeast proteome is approaching saturation. We will continue to update the PhosphoGRID data set, with the expectation that the integration of information from LTP and HTP studies will enable the development of predictive models of phosphorylation-based signaling networks.PhosphoGRID is an online database that curates and houses experimentally verified Database URL:http://www.phosphogrid.org/ We have also documented the effects of known specific regulatory subunits for protein kinases and phosphatases, which regulate phosphorylation or dephosphorylation, respectively. Information on any substrate, protein kinase or phosphatase in PhosphoGRID can be obtained through the web-based search interface. Phosphosites are highlighted as red text on protein substrates in a sequence-based viewer. A summary of evidence for the phosphorylation event, as well as the specific condition(s) it occurs under, appears on mouse-over of the phosphosite. Consensus sequences for a limited number of protein kinases (Saccharomyces Genome Database (SGD)  analyses of individual proteins or complexes. Salient features of the database include documentation of each  kinases , which o kinases , the Sacse (SGD)  and the se (SGD) , and hypse (SGD) . All of with SGD , Yeastrawith SGD , CYGD-MIwith SGD  and BioGwith SGD .N-acetyltransferase expression  included phosphosites annotated from articles published before the end of 2009, based on a standard PubMed query to retrieve candidate publications , 11, Cdkpression  or disrupression . We alsoThe updated data set in version 2.0 contains information on 20 178 unique phosphorylated residues, representing a 4-fold increase from version 1.0 . The vasin vivo, we have recorded each of the residues where mutations were made in attempts to deduce functional phosphorylation sites. For potential phosphorylation sites from these types of studies, we have included a mouse-over note on the protein sequence that describes the effect(s) produced by the multiple-site mutation. For example, a Rec8 mutant with 24 point mutations of potential casein kinase 1 (CK1) and Cdc7 kinase (Dbf4 dependent kinase (DDK)) sites eliminated detectable phosphorylation of Rec8 protein and produced a discernable phenotype WGG(Q/A), on which phosphorylation has been detected by anti-phosphopeptide antibodies and MS  of phosphosites characterized in LTP studies have an assigned function, often deduced from a mutant phenotype . AnnotatIn contrast, a much smaller fraction of sites identified in HTP studies (∼3%) have a defined function by virtue of overlap with the LTP data set . Based oSupplementary Table S2), and of these, only 15 have been examined for function , Grant Numbers 1R01RR024031-01 and R24RR032659 from the NIH National Center For Research Resources , Grant Number BB/F010486/1 from the Biotechnology and Biological Sciences Research Council (M.T.) and Grant Number HOP-120237 from the Canadian Institutes for Health Research (I.S.). Funding for open access charge: Canadian Institutes for Health Research (I.S.) Grant Number HOP-120237.This work was supported by Grant Number Conflict of interest. None declared.


RP induction depends on binding of GABAAR with GABAAR associated protein (GABARAP) which is regulated by Ca2+/calmodulin-dependent kinase II (CaMKII). Whether RP is induced or not is determined by the balance between phosphorylation and de-phosphorylation activities regulated by intracellular Ca2+ and by metabotropic GABA and glutamate receptors. Recent studies have revealed that the subunit composition of CaMKII has significant impact on RP induction. A Purkinje cell expresses both α- and β-CaMKII, and the latter has much higher affinity for Ca2+/calmodulin than the former. It was shown that when the relative amount of α- to β-CaMKII is large, RP induction is suppressed. The functional significance of RP has also been studied using transgenic mice in which a peptide inhibiting association of GABARAP and GABAAR is expressed selectively in Purkinje cells. The transgenic mice show abrogation of RP and subnormal adaptation of vestibulo-ocular reflex (VOR), a type of motor learning. Thus, RP is involved in a certain type of motor learning.Purkinje cells receive both excitatory and inhibitory synaptic inputs and send sole output from the cerebellar cortex. Long-term depression (LTD), a type of synaptic plasticity, at excitatory parallel fiber–Purkinje cell synapses has been studied extensively as a primary cellular mechanism of motor learning. On the other hand, at inhibitory synapses on a Purkinje cell, postsynaptic depolarization induces long-lasting potentiation of GABAergic synaptic transmission. This synaptic plasticity is called rebound potentiation (RP), and its molecular regulatory mechanisms have been studied. The increase in intracellular Ca The cerebellum consists of cortex and nuclei, and is involved in motor control Figure . There aClimbing fibers are thought to code error signals . CaMKII activity is necessary for RP induction . GABARAP has a binding site for GABAAR γ2 subunit  corresponding to the intracellular region of γ2 subunit that mediates the binding to GABARAP . It was revealed that down-regulation of PKA activity decreases the amount of phosphorylated dopamine- and cyclic adenosine monophosphate (cAMP)-regulated phospho-protein 32 kDa  of GABAergic inhibitory synaptic transmission which is called RP. Induction of RP depends on CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MAPK pathway, while MK5 can be activated by the atypical MAPK ERK3 and ERK4, protein kinase A (PKA), and maybe p38MAPK. MK2, MK3, and MK5 can phosphorylate the common substrate small heat shock protein 27 (HSP27), a modification that regulates the role of HSP27 in actin polymerization. Both stress and cAMP elevating stimuli can cause F-actin remodeling, but whereas the in vivo role of p38MAPK-MK2 in stress-triggered HSP27 phosphorylation and actin reorganization is well established, it is not known whether MK2 is involved in cAMP/PKA-induced F-actin rearrangements. On the other hand, MK5 can phosphorylate HSP27 and cause cytoskeletal changes in a cAMP/PKA-dependent manner, but its role as HSP27 kinase in stress-induced F-actin remodeling is disputed. Therefore, we wanted to investigate the implication of MK2 and MK5 in stress- and PKA-induced HSP27 phosphorylation.Classical mammalian mitogen-activated protein kinase (MAPK) pathways consist of a cascade of three successive phosphorylation events resulting in the phosphorylation of a variety of substrates, including another class of protein kinases referred to as MAPK-activating protein kinases (MAPKAPKs). The MAPKAPKs MK2, MK3 and MK5 are closely related, but MK2 and MK3 are the major downstream targets of the p38MAPK or the catalytic subunit of PKA trigger HSP27 phosphorylation, and specific inhibitors of p38MAPK and PKA prevent this phosphorylation. Depletion of MK2, but not MK3 and MK5 diminished stress-induced HSP27 phosphorylation, while only knockdown of MK5 reduced PKA-induced phosphoHSP27 levels. Stimulation of the p38MAPK, but not the PKA pathway, caused activation of MK2.Using HEK293 cells, we show that MK2, MK3, and MK5 are expressed in these cells, but MK3 protein levels are very moderate. Stress- and cAMP-elevating stimuli, as well as ectopic expression of active MKK6 plus p38Our results suggest that in HEK293 cells MK2 is the HSP27 kinase engaged in stress-induced, but not cAMP-induced phosphorylation of HSP27, while MK5 seems to be the sole MK to mediate HSP27 phosphorylation in response to stimulation of the PKA pathway. Thus, despite the same substrate specificity towards HSP27, MK2 and MK5 are implicated in different signaling pathways causing actin reorganization. MAPK pathways, while the atypical MAPK pathways include the less studied ERK3, ERK4, and ERK7 [MAPK pathway, which is induced by cellular stress stimuli and cytokines, can activate MNK1/2, MSK1/2 and MK2, 3 and 5 [The mitogen-activated protein kinase (MAPK) pathways control crucial cellular processes such as proliferation, differentiation, cell survival, apoptosis, gene regulation, and motility ,2. The tand ERK7 . The claand ERK7 ,2. Basedand ERK7 . Activat 3 and 5 . MK5 is  3 and 5 .-/- mice on a mixed 129 × C57BL/6 genetic background appear normal, but are more susceptible to chemically-induced skin cancer [in vitro and in vivo [MAPK [in vivo and is sufficient to cause reorganization of the cytoskeleton [MAPK-MK2, while MK5, but not MK2, is implicated in PKA-triggered HSP27 phosphorylation. These results indicate that although MK2 and MK5 may play a role in a common cellular process such as F-actin reorganization, they operate in different pathways to do so.MK2, MK3 and MK5 are closely related, but MK2 and MK3 are more identical to each other than to MK5 . Cell cun cancer ,12, whiln cancer . Despite in vivo . However in vivo . The relvo [MAPK -17, and vo [MAPK ,18-23. Wvo [MAPK . Furtherskeleton ,25. MK2 skeleton ,25, suggMAPK inhibitor SB203580 was purchased from Enzo Life Sciences. The PKA inhibitor H89 was from Bioaffin GmbH&Co KK . Sodium arsenite was obtained from Sigma-Aldrich. Recombinant MK2, MK3 and MK5 were purchased from Invitrogen Corporation , while purified Recombinant HSP27 with a C-terminal six histidine tag was obtained from R&D Systems . Purified HSP27 with an N-terminal GST tag was from SignalChem . Anti-HSP27 antibodies were purchased from Millipore  and Santa Cruz Biotechnology . MK2 and MK3 antibodies were from Cell Signaling Technology , while PRAK antibodies were from Santa Cruz (cat. no. sc-46667). Phosphospecific antibodies against phosphorylated HSP27 at serine 78, and 82 were purchased from Millipore (cat. no. 04-447 and 04-448), Epitomics (cat. no. 1543-1 and 1118-1), and Cell Signaling Technology (cat. no. 2405 and 2401). PhosphoSer-15 Hsp27 antibody was from Cell Signaling technology (cat. no. 2404) and Upstate Biotechnology . The phosphoMK2 Thr222 and Thr334 were both from Cell Signaling Technology . Expression plasmids for the catalytic subunit Cα of PKA, EGFP-MK5, Flag-tagged HSP27, Flag-tagged HSP27-3A, MKK6, and p38MAPK have been previously described [We used the adenylate cyclase activator forskolin from both Enzo Life Sciences  and from Sigma-Aldrich , while the p38escribed ,31.HEK293 cells (ATCC CRL-1573) were purchased from the American Type Culture Collection  and maintained in Eagle's minimum essential medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (110 units/ml), and streptomycin (100 μg/ml).2; 0.05 mg/ml BSA; 2.5 mM dithiothreitol; and 0.15 mM cold ATP in a total volume of 40 μl at 30°C for 30 min. The reactions were stopped in 4xLDS sample buffer and the proteins were denaturated at 70°C for 10 min. The samples were then analyzed by polyacrylamide gel electrophoresis (PAGE) and phosphorylation was monitored by western blotting using phospospecific antibodies (see below).Phosphorylation of recombinant HSP27 by MK2, MK3, and MK5 was performed in 25 mM Tris.HCl, pH 7.5; 10 mM MgCl5 cells were seeded out per well in a six-well plate. The next day, cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Scrambled and siRNA directed against MK2, MK3 and MK5 were purchased from Ambion, Applied Biosystems .The day before transfection, 3×10For detection of specific (phospho)proteins, samples were analyzed by ClearPAGE 4-12% BisTris SDS-PAGE  or NuPage (Invitrogen Life Technologies) according to the manufacturer's protocol and blotted onto a 0.45-μm polyvinylidene difluoride membrane . Incubation of the membrane with antibodies was performed as previously described . Briefly® RNA II kit from Macherey-Nagel  according to the manufacturer's instructions. The RNA concentration and purity were checked by spectrometry using a Nanodrop ND-1000 . Two μg of RNA was reversed transcribed using the iScript cDNA synthesis kit  and subjected to PCR. To amplify MK2, MK3, and MK5 cDNA following primers were used: MK2 forward: GAATCTGTACGCAGGGAGGAAG; MK2 reverse: CATACTGGCCCATTCGGAT; MK3 forward GTTGTCCAAGCAGGTGCTGGG, MK3 reverse: TTAAGCACTGCGTCTTTCTCC; MK5 forward: CCCTACACTTACAACAAGAGCTGTG, and MK5 reverse: CTTTATCTGTGAATCCACGACCATTC. The primers for rRNA have been previously described [RNA was isolated from HEK293 cells using total RNA isolation NucleoSpinescribed . The PCRDensitometry was performed using a BioRad Model GS-700 Imaging Densitometer and the Multi-Analyst version 1.1 Software.MAPK pathway induces phosphorylation of HSP27 and that p38MAPK downstream targets MK2, MK3, and MK5, are genuine HSP27 kinases [MAPK- and PKA-induced HSP27 phosphorylation.Previous studies have shown that activation of the p38 kinases . Moreove kinases . The aimin vitro phosphorylated HSP27. Recombinant GST-HSP27 or His-tagged HSP27 were incubated with purified active MK2, MK3, or MK5, respectively, and phosphorylation of Ser-15, -78, and -82 was monitored by phosphospecific antibodies. Increased in vitro phosphorylation, although with different stoichiometry, could be detected at Ser-15, Ser-78 and Ser-82 in the presence of MK2, MK3, and MK5 , indicating that the stimuli were functional  after p38MAPK activation [MAPK or PKA signaling pathways. The group of Gaestel found lower expression levels and activity of MK3 compared to MK2 in all cells tested [Although HSP27 was reported to be a good MK3 substrate, at least tivation . This ins tested . We alsoin vitro, although with different stoichiometry (Figure in vivo phosphorylation of this residue by MK5 [in vitro results indicate that HSP27 may be a better substrate for MK2 or that the intrinsic kinase activity of MK2 is higher than MK5. In agreement with this assumption, the group of Matthias Gaestel reported that immunoprecipitated MK5 from arsenite-treated MEF cells had a 6-times lower activity towards Praktide than MK2 immunoprecipitated from arsenite-treated cells [in vitro [in vivo [MAPK.Both MK2 and MK5 phosphorylated HSP27 at Ser-78 and Ser-82 y Figure . While Me by MK5 . The in ed cells . Our stuin vitro , while w[in vivo ,29. Howe[in vivo . The lowMAPK pathway has been shown to be necessary for activation in vitro [in vivo interaction between p38MAPK and MK5 remains controversial [-/- MEF cells, but not in MK5-/- MEFs. Moreover, they were unable to phosphorylate recombinant HSP27 with immunoprecipitated MK5. Our siRNA studies confirmed that MK5 is not involved in arsenite-induced HSP27 phosphorylation (Figure MAPK-induced HSP27 phosphorylation is that MK5 is not a bona fide substrate for p38MAPK [MK2 does not participate in PKA-induced HSP27 phosphorylation in HEK293 cells, an assumption that is underscored by our previous finding that PKA did not phosphorylate MK2 . Phosphoin vitro ,31, but oversial . These an Figure . The obvin vitro PKA phosphoacceptor site and that ectopic expression of the phosphomimicking MK5 S115D mutant resulted in potent in vivo phosphorylation of HSP27, while the MK5 S115A mutant was unable to do so [Our results demonstrate that MK5 is required, whereas MK2 and MK3 appear dispensable for PKA-induced HSP27 phosphorylation. The selectivity of PKA for MK5 can be explained as follows. We have recently demonstrated that Ser-115 is an to do so . Interesto do so . Hence MMAPK-MK2-HSP27 pathway in F-actin rearrangements is well illustrated [Both stress and cAMP elevating signals can cause F-actin rearrangements . Phosphoustrated . The PKAustrated  may explMAPK and the PKA pathway can cause HSP27 phosphorylation at Ser-78 and Ser-82. We did not test phosphorylation at Ser-15 because the antibodies we used gave weak signals for any of the three kinases using in vitro phosphorylated HSP27. We were previously unable to detect HSP27 phosphorylation at Ser-15 in cells transfected with an active MK5 mutant, although increased phosphorylation on Ser-78 and Ser-82 was observed [in vivo or whether the failure to detect phosphorylation was the result of poor antibody quality.We also showed that both the p38observed . At presMAPK pathway, MK5 seems to be involved in phosphorylation of HSP27 triggered by PKA (Figure In conclusion, we suggest that both MK2 and MK5 are genuine HSP27 kinases that are engaged in different pathways. While MK2 mediates phosphorylation of HSP27 in response to stress-induced activation of the p38HSP27: heat shock protein 27; PKA: cAMP-dependent protein kinase or protein kinase A; MAPK: mitogen-activated protein kinase; MAPKAPK: MAPK-activated protein kinase; MK: MAPKAPK;The authors declare that they have no competing interests.AS, GD, and UM designed the experiments, interpreted and discussed the results. AS, GD and UM performed the experiments. UM wrote the first draft of the manuscript. AS and GD contributed with writing the manuscript. All authors read and approved the final manuscript.


Measurements of intrinsic fluorescence showed that D-(−)-arabinose induced obvious tertiary changes to tyrosinase . This strategy of predicting tyrosinase inhibition based on specific interactions of aldehyde and hydroxyl groups with the enzyme may prove useful for screening potential tyrosinase inhibitors.Tyrosinase is a ubiquitous enzyme with diverse physiologic roles related to pigment production. Tyrosinase inhibition has been well studied for cosmetic, medicinal, and agricultural purposes. We simulated the docking of tyrosinase and D-(−)-arabinose and found a binding energy of −4.5 kcal/mol for theup-formof D-(−)-arabinose and −4.4 kcal/mol for thedown-form of D-(−)-arabinose. The results of molecular dynamics simulation suggested that D-(−)-arabinose interacts mostly with HIS85, HIS259, and HIS263, which are believed to be in the active site. Our kinetic study showed that D-(−)-arabinose is a reversible, mixed-type inhibitor of tyrosinase ( Tyrosinase EC 1.14.18.1) is a ubiquitous enzyme with diverse physiologic roles related to pigment production. It plays a central role in melanin synthesis in skin .14.18.1 , the broThe tyrosinase mechanism is complex, and this enzyme can catalyze multiple reactions. Despite several reported crystallographic structures of tyrosinase, the 3D structure and architecture of the active site are not well understood , 23. MecIn the current study, we investigated the mechanism of tyrosinase inhibition by D-(–)-arabinose using computational simulation and kinetic analysis. We hypothesized that the aldehyde and hydroxyl groups of D-(–)-arabinose may block L-DOPA oxidation by binding to tyrosinase. Previous findings have shown the importance of aldehyde , 28 and Km of 0.29 ± 0.11 mM (Vmax = 0.15 ± 0.03 mmol · min−1) using a Lineweaver-Burk plot. All kinetic reactions and measurements in this study were performed in 50 mM sodium phosphate buffer (pH 6.9) at the temperature of 25°C.Tyrosinase (M.W. 128 kDa) and L-DOPA were purchased from Sigma-Aldrich. D-(–)-arabinose was purchased from Tokyo Chemistry Industry. When L-DOPA was used as a substrate in our experiments, the purchased tyrosinase had a Agaricus bisporus tyrosinase (PDB ID: 2Y9X). The original structure of D-(–)-arabinose was derived from the PubChem database (ID: 66308), named -2, 3, 4, 5-tetrahydroxypentanal. At room temperature, D-(–)-arabinose exists in a ring structure because the terminal alcohol and aldehyde group interact with each other. Because the structure has an ambiguous chiral center in the ring, we generated two chemical forms (up and down). We used the Pck software to find the binding pocket  is linearly fit, assuming a single inhibition site or a single class of inhibition site, as shown in The μM ANS for 30 min prior to measurement. An excitation wavelength of 390 nm was used for the ANS-binding fluorescence, and emission wavelength ranged from 420 to 600 nm. The final concentrations of tyrosinase was 6.6 μg/mL.Fluorescence emission spectra were measured using a Hitachi F-2500 fluorescence spectrophotometer with a 1 cm path-length cuvette. An excitation wavelength of 280 nm was used for the tryptophan fluorescence measurements, and the emission wavelength was between 300 and 420 nm. Changes in the ANS-binding fluorescence intensity for tyrosinase were studied by labeling with 40 K) and number of binding sites (n):F0 and F are the relative steady-state fluorescence intensities in the absence and presence of quencher, respectively, and [Q] is the quencher (D-(−)-arabinose) concentration. The values for the binding constant (K) and number of binding sites (n) can be derived from the intercept and slope of a plot based on :F0F0−F=1nWe searched for the D-(−)-arabinose binding pocket  and commNext, the 10 ns molecular dynamics simulation was performed to examine structural changes during simulation. We verified the root mean square deviation (RMSD) of the alpha carbon, and the results showed that the structure was first rearranged and then stabilized . PlausibIn our studies, the identified plausible residues for docking and the molecular dynamics simulations were consistent.50) was estimated to be 0.61 ± 0.02 M (n = 3), and tyrosinase was almost completely inactive at 1.75 M D-(−)-arabinose (v) versus enzyme concentration [E] resulted in a decrease in the slope of the line, indicating that the inhibition of D-(−)-arabinose on the enzyme was reversible (We assayed tyrosinase activity changes in the presence of D-(−)-arabinose. Tyrosinase activity was inactivated by D-(−)-arabinose in a dose-dependent manner . The inhrabinose . When D-rabinose . The difversible .Next, time courses for tyrosinase inhibition at different concentrations of D-(−)-arabinose were performed . The resVmax and the Km, indicating that D-(−)-arabinose induced a mixed-type of inhibition (Y-intercept versus D-(−)-arabinose concentration were linearly fit , and the Ki was 0.21 ± 0.19 M (n = 3).The kinetics of tyrosinase in the presence of D-(−)-arabinose was studied using double-reciprocal Lineweaver-Burk plots. The results showed changes in the apparent hibition . The sec Figures –3), the, theVmaK = 1.58 ± 0.02 M−1) and the binding number (n = 1.49 ± 0.06) according to -arabinose were determined from measurements of the enzymatic intrinsic fluorescence changes. We found that the intrinsic fluorescence of tyrosinase was significantly changed. This was accompanied by a quenching effect that gradually decreased, with a significant shift of the maximum peak wavelength from 332 nm to 349 nm Figures . At a 2 Next, we monitored changes in tyrosinase hydrophobicity in the presence of D-(−)-arabinose . UnfortuPrevious studies have identified compounds with aldehyde and hydroxyl groups to have a potent inhibitory effect on tyrosinase –40. The in silico docking, we predicted that D-(−)-arabinose could bind directly with amino acid residues inside the tyrosinase active site. Further molecular dynamics simulation showed a specific binding site consisting of HIS85, HIS259, and HIS263 for the up- and down-forms of D-(−)-arabinose. We found that the enzymatic activity of tyrosinase decreased as the concentrations of D-(−)-arabinose increased. D-(−)-arabinose inhibited tyrosinase in a reversible way and induced changes in the apparent Vmax and the Km, consistent with a mixed-type of inhibition through interactions with residues at or close to the active site. The second plots of Slope versus D-(−)-arabinose concentration and Y-intercept versus D-(−)-arabinose concentration were linearly fit. These results were consistent with previous computational simulation. Using As conformational changes at or near the active site could influence access by the enzyme substrate, the conformational changes of tyrosinase induced by D-(−)-arabinose were detected by spectrofluorometry. Obvious changes were observed which indicated that the structure of the tyrosinase active site had been altered to reduce the access of the substrate L-DOPA. Changes in catalytic rate were not detectable over time, which indicated that D-(−)-arabinose binding to the enzyme and decreased enzyme activity occurred very quickly. These results indicated that the overall tertiary structural changes might not be synchronized with D-(−)-arabinose-induced inhibition of tyrosinase due to rapid achievement of equilibrium states. Our study had several principal findings: (1) D-(−)-arabinose binding to tyrosinase caused a mixed-type of inhibition; (2) D-(−)-arabinose inhibition of tyrosinase caused gross changes in tertiary structure, detected as unfolding; (3) putative D-(−)-arabinose-binding residues HIS85, HIS259, and HIS263 were predicted using computational simulation. Further molecular dynamics simulation predicted the specific binding site of HIS85, HIS259, and HIS263 for the up- and down-forms of D-(−)-arabinose.Ki = 0.22 ± 0.07 mM) [Compared with other inhibitors, D-(−)-arabinose is a poor inhibitor of tyrosinase . D-(−)-a0.07 mM) . TherefoThe inhibitory mechanism of D-(−)-arabinose resembles that of a copper chelator but differs in that D-(−)-arabinose does not directly bind to the copper atoms in the active site. Our study provides fresh insight into the role of active site residues in tyrosinase catalysis, and presents useful information regarding the 3D structure of tyrosinase. Minor change in the configuration of arabinose can lead to major difference of the inhibition effect on tyrosinase. These findings provide important information in the study of tyrosinase inhibitors in the development of hyperpigmentation treatment. A combination of computational modeling and inhibition kinetics may facilitate the testing of potential tyrosinase inhibitors and the prediction of their inhibitory mechanisms.


Chlamydomonas raudensis is an important primary producer in a number of ice-covered lakes and ponds in Antarctica. A C. raudensis isolate (UWO241) from Lake Bonney in the McMurdo Dry Valleys, like many other Antarctic algae, was found to secrete ice-binding proteins (IBPs), which appear to be essential for survival in icy environments. The IBPs of several Antarctic algae  are similar to each other (here designated as type I IBPs) and have been proposed to have bacterial origins. Other IBPs (type II IBPs) that bear no resemblance to type I IBPs, have been found in the Antarctic Chlamydomonas sp. CCMP681, a putative snow alga, raising the possibility that chlamydomonad IBPs developed separately from the IBPs of other algae. To test this idea, we obtained the IBP sequences of C. raudensis UWO241 by sequencing the transcriptome. A large number of transcripts revealed no sequences resembling type II IBPs. Instead, many isoforms resembling type I IBPs were found, and these most closely matched a hypothetical protein from the bacterium Stigmatella aurantiaca. The sequences were confirmed to encode IBPs by the activity of a recombinant protein and by the matching of predicted and observed isoelectric points and molecular weights. Furthermore, a mesophilic sister species, C. raudensis SAG49.72, showed no ice-binding activity or PCR products from UWO241 IBP primers. These results confirm that algal IBPs are required for survival in icy habitats and demonstrate that they have diverse origins that are unrelated to the taxonomic positions of the algae. Last, we show that the C. raudensis UWO241 IBPs can change the structure of ice in a way that could increase the survivability of cells trapped in the ice.The green alga  Chlamydomonas sp. CCMP681 Chlamydomonas raudensis UWO241 (this study), and the snow algae Chloromonas brevispina and Chlamydomonas augustae ) showed that each had IBP activity. Together, these findings led to the question whether the IBPs of chlamydomonad algae were fundamentally different from those of other algae, possibly with a separate origin. Characterizing the IBPs of other chlamydomonad algae would help to answer this question.Ice-binding proteins (IBPs) are secreted by many polar unicellular algae. They appear to be essential for survival because every alga from icy environments examined so far has been found to produce them, while mesophilic algae do not C. raudensis UWO241, a species that was isolated from a permanently ice-covered Antarctic lake C. raudensis UWO241 by sequencing the transcriptome, confirmed that they encode IBPs, and compared the ice-binding activities of C. raudensis UWO241 and a mesophilic sister species. C. raudensis UWO241 was found to have many IBP genes, but not the expected type II genes. Our results confirm the association of IBPs with icy habitats and provide further evidence that the IBP genes of polar algae have diverse origins.To characterize the IBPs of a second chlamydomonad alga, we selected Chlamydomonas raudensis strain UWO241  at 8°C.Chlamydomonas raudensis Ettl (SAG49.72) cells were originally isolated from a meadow pool in the Czech Republic Chlamydomonas sp. CCMP681 Many attempts to PCR-amplify IBP genes similar to those in the snow alga 6 molecules/µl. Emulsion-based clonal amplification and sequencing on a 1/4 plate on the 454 Genome Sequencer FLX + system was performed according to the instructions of the manufacturer . Signal processing and base calling were performed using the bundled 454 Data Analysis Software v2.6.To prepare cDNA, cells were grown under the conditions stated above except that the growth temperature was 1°C. Cell pellets were shipped on dry ice to UNLV where RNA was extracted with Trizol (Invitrogen) following the manufacturer's instructions. Normalized cDNA was prepared and sequenced at the Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign. Briefly, messenger RNA was isolated from 50 µg of total RNA with the Oligotex kit . The messenger RNA-enriched fraction was converted to a 454 cDNA library and normalized as previously described The sequences were screened for sequences resembling known IBP genes. Those resembling known IBP-like sequences were aligned manually. The sequences of three complete IBP isoforms were confirmed by PCR using DNA as template. The presence of N-terminal signal peptides was predicted with SignalP v. 3.0 E. coli codons  was sent to GenScript  for expression of recombinant protein. The nucleotide sequence was optimized for i codons , modifieetic gel  showed aetic gel . The larUWO241 cells were grown at 8°C at Miami University and transported to UNLV, where the culture supernatant was confirmed to have ice-binding activity. An ice-binding fraction was ice-affinity purified by eight cycles of freezing, centrifugation, and thawing, as described previously Ice-binding activity was estimated by observing growth of an ice single crystal  submerged in the culture supernatant from cells grown at various temperatures as described previously −1 . The solutions were cooled to −4.0°C, allowed to freeze slowly for an hour, frozen overnight at −5.9°C, and photographed. Under these conditions, about 41% of the water (2.42/5.9) would be in the liquid state.The effect of the IBPs on the macrostructure of ice was determined as described previously C. raudensis UWO241 culture grown at 8°C showed strong ice-binding activity in the form of both irregular dendrites growing from the prism faces and pitting on the ice basal plane , showed no activity . Instead, dozens of sequences resembling type I IBPs were found. These were assembled into about 12 isoforms, 3 of which (GenBank acc. nos. KC012985-KC012987) were complete  showed clear ice-binding activity . The product appeared as a single band of approximately 24 kDa (the expected size) on an electrophoretic gel, although part of the product appeared to aggregate and not enter the gel. A solution of 318 µg mlactivity , confirmC. raudensis, four primer pairs that amplified sequences containing isoforms 1 and 2 in C. raudensis UWO241 either did not amplify similar bands or amplified weak bands of different sizes in C. raudensis SAG49.72  is widely distributed in Antarctic lakes and ponds C. raudensis DNA was recovered from a mixed culture grown from an exposed cyanobacterial mat embedded in the Lake Bonney ice cover, which suggests that viable cells are present in the frozen mats . The mats presumably work their way to the surface by surface ablation Curiously, C. raudensis SAG49.72 is a mesophilic alga isolated from a meadow pool in the Czech Republic C. raudensis UWO241 based on cell morphology and internal transcribed spacer (ITS1 and ITS2) and 5.8S rDNA sequences C. raudensis UWO241 and other polar algae probably serve to increase freezing tolerance, possibly through the preservation of a liquid environment as water freezes. During the summer, liquid water inclusions can make up 40% of the ice cover volume of Dry Valley lakes, which form nutrient-rich habitats for diverse microbial consortia Melosira arctica created convoluted ice-pore morphologies in sea ice, potentially increasing its habitability and primary productivity Chlamydomonas sp. CCMP681 and Phaeocystis antarctica have similar effects on the structure of sea ice P. antarctica, when frozen in sea ice, has an unusually high freeze-thaw tolerance The IBPs of C. raudensis IBPs is unclear). By contrast, the type II IBPs of Chlamydomonas sp. CCMP681 have numerous introns. The differences in sequence and gene structure suggested that chlamydomonad IBPs might have a different origin. However, the present findings show that this is not the case, i.e., that the C. raudensis UWO241 IBPs are type I IBPs that are most closely related to bacterial proteins that cluster separately from other bacterial IBP genes (Stigmatella aurantiaca (ZP-0146295) with 54% identity and 67% similarity over a 214-amino acid region  that have been implicated in ice-binding In the case of polar algae with type I IBPs , a number of observations suggest a polyphyletic origin of their IBP genes, with the donors likely being bacteria BP genes . The clod region . As in td region  bear almd region , providiC. raudensis UWO241 are consistent with the findings in two other Antarctic ice-associated algae, the diatom Fragilariopsis cylindrus  and the prymnesiophyte Phaeocystis antarctica , both of whose genomes contain dozens of IBP and IBP-like genes. The high number of isoforms provides further evidence that the IBPs have an important role in survival under icy conditions.The large number of IBP isoforms in C. raudensis are thus an additional tool in its repertory of adaptations for survival under harsh conditions of low light, high salinity and low temperature. We next plan to compare the transcriptomes of C. raudensis UWO241 and its mesophilic sister species SAG49.72 to better define the features that make UWO241 an Antarctic species.The ice-binding proteins of Figure S1E. coli optimized codons for Chlamydomonas raudensis UWO 241 IBP isoform 1, without N-terminal signal peptide.(TIF)Click here for additional data file.Figure S2Analysis of recombinant Chlamydomonas raudensis IBP by GenScript . A, SDS-PAGE gel. Lane 1, BSA; lane 2, recombinant protein. B, Western blot using anti-HIS antibody.(TIF)Click here for additional data file.Figure S3Two-dimensional gel showing proteins purified from C. raudensis culture medium by ice-affinity. Arrow indicates spot that matches pI and MW of sequenced ice-binding proteins. The spot above it may be a dimer. Y-axis, MW in kDa; x-axis, pI.(TIF)Click here for additional data file.Figure S4Alignment of a putative ice-binding protein from Stigmatella aurantiaca and three isoforms of ice-binding proteins from Chlamydomonas raudensis UWO241. Underlined sequences at N-terminus are predicted signal peptides. Isoform 3 also has a 117-amino acid sequence that goes between the DD residues marked by asterisks, which was removed to reduce the size of the figure.(TIF)Click here for additional data file.Figure S5Agarose gel showing PCR products amplified from DNAs from the Antarctic Chlamydomonas raudensis UWO241 and the mesophilic Chlamydomonas raudensis SAG49.72, using primers for the following genes. Lanes 1, IBP isoform 1; lanes 2–4, IBP isoform 2; lanes 6, RUBISCO (control); lanes 7, 18S rRNA (control). Right lane, DNA ladder.(TIF)Click here for additional data file.Figure S6Neighbor-joining tree constructed from 18S rRNA sequences of ice-associated algae. Tree is rooted on the 18S sequence of the rhodophyte Cyanidioschyzon merolae. Numbers at nodes indicate bootstrap values for 500 replications. Values less than 50 are not shown. Colors: Light green, diatoms; dark green, chlorophytes; brown, haptophyte; red, rhodophyte.(TIF)Click here for additional data file.


T674I FIP1L1-PDGFRα in a subset of chronic eosinophilic leukemia (CEL) is a gatekeeper mutation that is resistant to many tyrosine kinase inhibitors (TKIs) , similar to T315I Bcr-Abl. Therefore, novel TKIs effective against T674I FIP1L1-PDGFRα are needed. Ponatinib (AP24534) is a novel orally bioavailable TKI against T315I Bcr-Abl, but it is not clear whether ponatinib is effective against T674I FIP1L1-PDGFRα. The purpose of this study was to examine the effect of ponatinib on T674I FIP1L1-PDGFRα.in silico was performed. The effects of ponatinib on PDGFRα signaling pathways, apoptosis and cell cycling were examined in EOL-1, BaF3 cells expressing either wild type (WT) or T674I FIP1L1-PDGFRα. The in vivo antitumor activity of ponatinib was evaluated with xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice models.Molecular docking analysis 50: 0.004–2.5 nM). It induced apoptosis in CEL cells with caspase-3-dependent cleavage of Mcl-1, and inhibited tyrosine phosphorylation of β-catenin to decrease its stability and pro-survival functions. In vivo, ponatinib abrogated the growth of xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice.Molecular docking analysis revealed that ponatinib could bind to the DFG (Asp-Phe-Gly)-out state of T674I PDGFRα. Ponatinib potently inhibited the phosphorylation of WT and T674I FIP1L1-PDGFRα and their downstream signaling molecules . Ponatinib strikingly inhibited the growth of both WT and T674I FIP1L1-PDGFRα-carrying CEL cells (ICPonatinib is a pan-FIP1L1-PDGFRα inhibitor, and clinical trials are warranted to investigate its efficacy in imatinib-resistant CEL. Platelet-derived growth factor receptor α (PDGFRα) is a class III receptor tyrosine kinase with an extracellular domain, a single transmembrane domain, and a cytoplasmic tyrosine kinase domain . Upon liTo search for novel TKIs to overcome imatinib resistance, midostaurin, EXEL-0862 and sorafenib have been investigated both in vitro and in vivo in cells harboring T674I FIP1L1-PDGFRα ,13. ThusThere has been exciting recent advance in third-generation TKIs  efficacious against the gatekeeper mutants -18. In vin silico. In vitro and in vivo study then confirmed that ponatinib is a potent inhibitor of CEL cells bearing wild-type (WT) or T674I FIP1L1-PDGFRα.We investigated the molecular docking of ponatinib to T674I PDGFRα To gain insights into the structural basis for ponatinib to bind to the ATP-binding site of T674I PDGFRα, we performed computer simulations of molecular docking between ponatinib and T674I PDGFRα: ponatinib bound to native or mutated PDGFRα with the same orientation in the DFG-out state ; levels of phosphorylated and total PDGFRα were measured by immunoblotting. The phosphorylation of T674I FIP1L1-PDGFRα was altered by sorafenib but not imatinib , which is consistent with a previous report . In contWe next examined signal transduction downstream of PDGFRα after ponatinib treatment. The phosphorylation of Stat3, Stat5, Akt and Erk1/2 were measured by immunoblotting with phospho-specific antibodies. Treatment for 24 h with ponatinib reduced the phosphorylation of Stat3, Stat5, Akt and Erk1/2 in cells expressing WT or T674I PDGFRα at 0.3-30 nM .We examined the effect of TKIs on cell viability (MTS assay). The three lines of FIP1L1-PDGFRα-expressing cells were incubated with or without increasing concentrations of ponatinib, sorafenib, or imatinib for 72 h; log concentration-response curves are shown in Figure 50 value 0.6 nM for BaF3-WT FIP1L1-PDGFRα and 2.8 nM for BaF3-T674I FIP1L1-PDGFRα cells.In another independent set of experiments, we evaluated the impact of ponatinib on clonogenicity of the two lines of BaF3 cells. Cells were exposed to increasing concentrations of ponatinib for 24 h, then equal numbers of treated cells were seeded in methylcellulose medium. Ponatinib concentration-dependently inhibited the number of clonogenic BaF3-WT or -T674I FIP1L1-PDGFRα cells  revealed increased degradation of Mcl-1 level in ponatinib-treated CEL cells as compared with controls   pyridazine core that occupies the pocket for adenine in the enzyme, whereas the methylphenyl group occupies the hydrophobic pocket behind the gatekeeper residue of the enzyme -ribose) polymerase (PARP), X-linked inhibitor of apoptosis (XIAP), caspase-3, active caspase-3, cytochrome c (clone 6H2.B4), survivin, and C-terminal β-catenin ; phospho-β-catenin (Y654) ; antibodies against phospho-PDGFRα (Y1018), phospho-Erk1/2 (T202/Y204) and Erk1/2 ; antibodies against phospho-Stat3 (Y705), phospho-Stat5A/B (Y694/Y699), Stat3, Stat5, Bcl-2 and PDGFRα ; anti-Bim ; anti-actin . Anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G horseradish peroxidase-conjugated antibodies were from Pierce Biotechnology .Immunoblotting involved use of whole cell lysates prepared in RIPA buffer ,9,40. An5/ml and exposed to ponatinib treatment, then underwent cell death assay and immunoblotting.EOL-1 cells were transfected with plasmids or siRNA duplexes with use of Nucleofector  by use of the Cell Line Nucleofector Kit T (Amaxa) and program O-17 . At 24 h5) were transfected with TOPflash or FOPflash plasmid (0.5 μg) and pEFRenilla-luc (10 ng) by electroporation. At 24 h, cells were incubated with or without ponatinib for 24 h. Luciferase activity was then measured with the dual-luciferase assay kits  as described , and imatinib was dissolved in sterile double-distilled water. Mice in each group were treated once daily by oral gavage with ponatinib, imatinib or the same amount of vehicle. The body weight, feeding behavior and motor activity of each animal were monitored as indicators of general health. Tumor xenografts were immediately removed, weighed, stored and fixed after animals were killed. All animal studies were conducted with the approval of the Sun Yat-sen University Institutional Animal Care and Use Committee.Male 2O2 in 50 mM Tris–HCl (pH 7.6), and slides were counterstained with hematoxylin [Formalin-fixed BaF3-T674I FIP1L1-PDGFRα-cell xenografts were embedded in paraffin, sectioned (4-μm thick), then immunohistochemically stained by using the anti-Ki67 MaxVision kit . Color was developed with 0.05% diaminobenzidine and 0.03% Hatoxylin .α sequence  and the site to be mutated by use of CLUSTAL X [The kinase domain sequence was identified from the Human Kinome database  by sequeLUSTAL X .Prime module in Maestro  was chosen to build homology models for the native kinase domain sequence and the mutated sequence. We performed a BLAST search against the PDB database to choose a suitable template; the 1.6 Å X-ray structure of KIT kinase (PDB code: 1 T46) in complex with imatinib was chosen  . After SThe compound ponatinib was prepared by the Ligprep module, then the Glide module. Then docking simulations were performed to test binding of ponatinib to the native kinase model and the mutated one with the same default parameter sets. The grid-enclosing box was centered on the centroid of the aligned ligand (imatinib) and defined to enclose residues located within 20 Å around the ATP binding site; a scaling factor of 1.0 was set to van der Waals (VDW) radii of the receptor atoms with the partial atomic charge < 0.25. The Extra-Precision (XP) mode of Glide was used to dock ponatinib into the ATP binding site with default parameters, and the top 10 docked poses were reserved for the binding mode analysis.P < 0.05 was considered statistically significant.All experiments were performed at least 3 times, and results are reported as mean ± 95% confidence intervals, unless otherwise stated. GraphPad Prism 5.0  was used for statistical analysis. A PDGFRα: Platelet-derived growth factor receptor α; (FIP1L1)-PDGFRα: FIP1-like 1-PDGFRα; CEL: chronic eosinophilic leukemia; EMSA: electrophoretic mobility shift assay; TCF/LEF: T cell factor/lymphoid enhancer factor; CHX: cycloheximide; APC: adenomatous polyposis coli; GSK3β: kinase glycogen synthase kinase 3β.The authors declare that they have no competing interests.YJ designed, performed experiments and analyzed data; KD synthesized ponatinib; HL and MX performed molecular docking analysis; XS and CW performed experiments of apoptosis. JP designed, performed research, analyzed data, directed the whole study and wrote the manuscript. All authors read and approved the final manuscript.Computer-simulated binding of ponatinib to the native or mutated PDGFRα kinase in DFG(Asp-Phe-Gly)-out state. The explanation was described in the fulltxt.Click here for file