Recent work shows that cooling protection after mouse cardiac arrest and cardiomyocyte ischemia is mediated by Akt activation. The PI3K p85 subunit can either augment or inhibit Akt activation depending on its binding to p110 or PTEN respectively. To further clarify the role of PI3K p85 in cardioprotection, we studied novel TAT-p85 fusion proteins that selectively inhibit PI3K p85 binding. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-ΔPTEN p85) would enhance Akt phosphorylation to afford cardioprotection. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) would decrease Akt phosphorylation and abrogate cardioprotection. Microscopy and Western blot analysis demonstrated that TAT fusion protein was transduced into cardiomyocytes within 5 min and remained more than 2 h. Inhibition of PI3K/Akt by TAT-Δp110 p85 significantly increased cell death from 44.6±2.7% to 92.5±3.4% after simulated ischemia and reperfusion. By contrast, PTEN inhibition using TAT-ΔPTEN p85 decreased cell death to 11.9±5.3%, a similar level of cardioprotection seen with past cooling studies. Additional studies with the small molecule PTEN inhibitor VO-OHpic confirmed that PTEN inhibition was highly protective against cell death induced by ischemia and reperfusion. We conclude that blockade of p85-PTEN interaction and PTEN inhibition may be promising strategies for rescuing the heart from ischemia and reperfusion injury. Sudden cardiac arrest (SCA) is a leading cause of death that is related to both global and focal ischemia/reperfusion (I/R) injury of the heart. While there are currently no drugs available that improve SCA survival, therapeutic hypothermia is a strategy that appears to improve heart function and survival after cardiac arrest. Our work and others suggest that therapeutic hypothermia protection is significantly mediated by enhanced phosphatidylinositol 3-kinase (PI3K) -Akt activation Altered regulation of the PI3K signaling pathway contributes to many human diseases including cancer, diabetes, obesity, autoimmunity as well as I/R. A critical aspect of PI3K regulation that has recently emerged is the paradoxical ability of its p85α regulatory protein to bind and directly regulate not only the p110 catalytic subunit required for the stabilization and localization of p110-PI3K activity, but also the PTEN phosphatase that works simultaneously to deactivate the PI3K/Akt pathway For this study we constructed a TAT fusion protein, TAT-ΔPTEN p85, that contains the TAT protein transduction domain fused to a p85 subunit of PI3K lacking a PTEN binding site. The N-terminal SH3-BH region of p85α (amino acids 1–313) has been identified as the region that mediates PTEN binding and regulation. Cellular expression of the ΔPTEN binding site of p85 results in a substantially increased magnitude and duration of p-Akt levels in response to growth factor stimulation To further confirm the role of PTEN inhibition in cardioprotection, we investigated whether the small molecule PTEN inhibitor VO-OHpic The investigation conforms to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health . The procedure for cardiomyocyte isolation was approved by the University of Illinois at Chicago's Institutional Animal Care and Use Committee (Permit Number: 11-198).Escherichia coli was obtained from Novagen . pGEX vectors containing wild type p85 and Δp110-p85 cDNA were gifts from Dr. M. Kasuga . pTAT and pTAT-green fluorescent protein (GFP) were gifts from Dr. S. Dowdy .The PTEN inhibitor VO-OHpic was obtained from Sigma . Propidium iodide (PI) for measuring cell death was obtained from Invitrogen (Carlsbad CA). The primary antibodies, including Akt, pAkt-S473, pAkt-T308 were purchased from Cell Signaling Technology . The secondary antibodies include horseradish peroxide conjugated anti-mouse and anti-rabbit antibodies from Amersham . Polyclonal anti-p85 subunit of PI3K was purchased from Upstate Biotechnology . TAT-Δp110 p85, a dominant-negative form of the class IA PI3K adaptor subunit with the deletion of 35 amino acids  of its p110 binding site, was produced in our laboratory as described previously 5′- CAC ACC GGT CCC ACT ACT GTA GCC AAC AAC GGT ATG –3′; antisense, 5′- CAC GAA TTC TCA TCG CCT CTG CTG CGC GTA CAC -3′). The PCR products have AgeI and EcoRI cutting sites plus 3 extra bases for efficient cutting before and after ΔPTEN p85 cDNA, respectively. PCR was carried out for 35 cycles using 0.5 min of denaturation at 95°C, 0.5 min of annealing at 62°C, and 1.5 min of extension at 72°C. The amplified PCR products were digested with AgeI/EcoRI, purified and ligated into an AgeI/EcoRI digested pTAT vector using T4 ligase. The final sequence was verified by an Automatic Sequencer.To construct the pTAT-ΔPTEN p85 plasmid, a cDNA fragment encoding ΔPTEN p85 (deletion of amino acids 1–313 from p85) was amplified by PCR from the wild type p85 cDNA in pGEX with the following primers  plus protease inhibitor cocktail  and centrifuged, and the resulting supernatant was loaded onto a Ni-ProBond resin column . The column was washed with 100 mL TBS containing 75 mM imidazole, and 1 L of TBS containing 0.1% Triton X-114 to remove endotoxin Purification of TAT fusion proteins was performed as previously described 2+ and Mg2+ (pH 7.4). The first cell suspension was discarded, whereas the subsequent suspensions were added to trypsin inhibitor solution in cold HBSS with Ca2+ and Mg2+ (pH 7.4) until all cardiac cells were isolated (5 to 6 cycles). To remove fibroblasts, the isolated cells were preplated for 90 min at 37°C. The resulting supernatants were then centrifuged and plated at a density of 0.6×106 on laminin-coated coverslips with MEM supplemented with 10% fetal bovine serum , 50 U/ml penicillin, and 1.5 µM vitamin B12 . Myocyte purity was ∼90% as determined by immunofluorescent staining for α-sarcomeric actin and myosin heavy chain . Experiments were performed on 6–8 day cultures.Primary cultures of mouse ventricular cardiomyocytes were prepared from hearts of 1 to 2-day-old neonatal C57BL6/J mice  as described previously 3, 0.76 mM MgSO4, 1.0 mM NaH2PO4, 1.21 mM CaCl2 and 5.6 mM Glucose, then subjected to simulated ischemia for 90 min in a ischemia solution that was placed in a hypoxic chamber  followed by 3 h reperfusion at 37°C. Ischemia for 90 min and reperfusion for 3 h was selected for these studies because it was the optimum time to induce ∼50% death of cardiomyocytes +], and bubbled with 80% N2 - 20% CO2 for 45 min to produce BSS with 3–5 Torr PO2, 144 Torr PCO2, and pH 6.8. After the ischemic challenge, cells were incubated with normoxic BSS  to simulate reperfusion Synchronously contracting cardiomyocytes on glass coverslips were equilibrated for 30 min in a Balanced Salt Solution (BSS) containing 117 mM NaCl, 4 mM KCl, 18 mM NaHCOCell death was assessed under a fluorescent microscope  with the exclusion dye propidium iodide  as measured at 540 nm excitation and 590 nm emission 3VO4, 200 mM PMSF, and 1× protease inhibitor cocktail. Protein quantification was performed with a Bradford assay . Protein lysate (30 µg/lane) was loaded and resolved on a 10% SDS-PAGE gel and was then transferred to a nitrocellulose membrane. After the membranes were blocked in 5% fat-free milk in TBS-Tween 20, they were probed with antibodies against phosphorylated Akt (Thr308 and Ser473) and total Akt. Signals were amplified and visualized with horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. Band quantification was performed using Image J software  and normalized to loading controls.The cells were lysed in buffer containing 1% Triton X-100, 20 mM Tris, 137 mM NaCl, 2 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 50 mM NaF, 1 mM NaCardiomyocytes were incubated with 1 µM FITC-conjugated TAT for up to 120 min at 37°C, and then were washed three times with BSS buffer. Cells were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, washed three times in PBS. Cells were examined under a fluorescent microscope  with ×400 magnification using Smart capture VP imaging software .P<0.05 was considered statistically significant.Results were expressed as means ± SEM. A field of ∼500 cells was observed in each experiment for cell death measurement. For comparison among the different treatment groups, one-way ANOVA were used with post hoc examination by Fisher's least significant difference test. To assess the efficacy of TAT protein transduction in cardiomyocytes, FITC-conjugated TAT was used to determine the localization of TAT protein under fluorescence microscope. At 5 min, TAT protein was mostly associated with the plasma membrane . By 30 m2O2 in a concentration-dependent fashion. Akt phosphorylation was reduced at 100 nM and blocked completely with 300 nM TAT-Δp110 p85  significantly increased Akt phosphorylation . Treatmep<0.01 vs. I/R alone, We next examined the functional consequence of Akt inhibition by TAT-Δp110 p85 in cardiomyocytes exposed to I/R. Cardiomyocytes were pretreated with 300 nM TAT-Δp110 p85 or TAT-GFP before exposed to I/R. I/R induced significantly cell death from 2.3±0.4% of baseline control to 44.6±2.7%. Compared to I/R alone, TAT-Δp110 p85 increased cell death to 92.5±3.4%  was used in order to test for possible enhancement of Akt activation. Enhanced Akt phosphorylation was observed at 0.5 µM and further increased with 1.7 µM TAT-ΔPTEN p85. By contrast, 1.7 µM TAT-GFP had no effect on Akt phosphorylation caused by H2O2. These results demonstrated that TAT-ΔPTEN p85 enhanced Akt phosphorylation after transduction into cardiomyocytes.Akt activation is a specific marker for PTEN inhibition and has been used to indicate the inhibitory potency of PTEN inhibitors We next examined whether TAT-ΔPTEN p85 enhanced phosphorylation of Akt under I/R conditions. Compared to BSS equilibration buffer control, I/R (ischemia for 90 min followed by 30 min reperfusion) significantly increased Akt phosphorylation . TAT-ΔPTp<0.01). TAT-GFP has no effect on cardiomyocyte death compared to I/R alone. These results supported a dual role for p85 binding during cardiomyocyte I/R, with decreased p110 binding associated with cell death and decreased PTEN binding (i.e. PTEN inhibition) associated with improved heart cell survival.We then examined the functional consequence of Akt activation by TAT-ΔPTEN p85 on cardiomyocyte survival after exposed to I/R. Cardiomyocytes were pretreated with 1.7 µM TAT-ΔPTEN p85 or TAT-GFP immediately before exposed to I/R. I/R caused cell death from 2.3±0.4% of baseline control to 47.8±1.4%. Compared to I/R alone, TAT-ΔPTEN p85 attenuated cell death to 11.9±5.3% , p<0.01.2O2. VO-OHpic at 1–5 µM augmented phosphorylation of Akt induced by H2O2 .We next examined the functional consequence of Akt activation by VO-OHpic in cardiomyocytes. Cardiomyocytes were treated with 1 µM VO-OHpic before simulated I/R. I/R increased cell death from 2.3±0.4% of baseline control to 48.7±2.6%. VO-OHpic decreased I/R induced cell death to 24.4±1.3% , p<0.01.The present study demonstrated that 1) inhibition of p85 binding to PI3K catalytic subunit p110 by TAT-Δp110 p85 resulted in decreased Akt phosphorylation and increased cell death after I/R, 2) inhibition of p85 binding to PTEN by TAT-ΔPTEN p85 in cardiomyocytes increased Akt activation and attenuated I/R induced cell death, and 3) compared to TAT-ΔPTEN p85, chemical inhibition of PTEN by VO-OHpic has a similar protective effect on I/R injury. This data supports a dual role for p85 in regulating Akt activation during I/R via binding either to the PI3K catalytic subunit p110 versus the phosphatase PTEN. Blocking p85 binding to p110 results in decreased Akt phosphorylation and enhanced I/R injury of cardiomyocytes, while blocking its binding to PTEN increased Akt activation and decreased death of cardiomyocytes exposed to I/R . In summ2Considerable research has been undertaken to investigate the possibility of increasing cell survival and reducing cell death following ischemia and reperfusion. Studies have shown the essential role of PI3K in activating Akt with downstream activation of pro-survival kinases and suppression of pro-apoptotic pathways 2 and PIP3 level by regulating PTEN As the key negative regulator of the PI3K/Akt pathway, PTEN is an important target for cardioprotection in I/R injury. Previous studies found that PTEN inactivation in neonatal cardiomyocytes by overexpression of dominant negative PTEN H123Y mutant activates the Akt prosurvival pathway, reduces apoptosis, and increases survival PI3K isoforms convey distinct roles in cardiac physiology and development. Class IA PI3K  regulates cardiomyocyte growth and apoptosis, whereas class 1B PI3Kγ downregulates cardiac contractility through inhibiting cyclic adenosine monophosphate production 2O2 or I/R (In this study, we also showed that pharmacological inhibition of PTEN with VO-OHpic ameliorated reperfusion injury and reproduced the cardioprotective effects obtained by TAT-ΔPTEN p85. The VO-OHpic action on PTEN is confirmed by the increased phosphorylation of Akt, a downstream target of PTEN-dependent signaling, caused by H2 or I/R  and 6. TIt is important to recognize some limitations of our findings. We used neonatal cardiomyocytes that may not entirely reflect the response of adult cardiomyocytes in the intact heart following I/R. Neonatal cardiomyocytes were chosen over adult cardiomyocytes in this study because of their low rates of baseline apoptotic activity and the availability of sufficient protein to assess cellular signaling events. The use of neonatal cells to study molecular cardiac events is well established in the literature, and similarities between neonatal and adult cardiomyocyte response to I/R injury have been observed In summary, these results support the dual functions of p85 in modulating I/R injury. Modulating PTEN binding to 85 or PTEN phosphatase activity could have therapeutic implications in the survival of heart cells following I/R injury.


Cell division is controlled in part by the timely activation of the CDK, Cdc28, through its association with G1 and G2 cyclins. Cdc28 complexes are regulated in turn by the ubiquitin conjugating enzyme Cdc34 and SCF ubiquitin ligase complexes of the ubiquitin-proteasome system (UPS) to control the initiation of DNA replication. Here we demonstrate that the nutrient sensing kinases PKA and Sch9 phosphorylate S97 of Cdc34. S97 is conserved across species and restricted to the catalytic domain of Cdc34/Ubc7-like E2s. Cdc34-S97 phosphorylation is cell cycle regulated, elevated during active cell growth and division and decreased during cell cycle arrest. Cell growth and cell division are orchestrated to maintain cell size homeostasis over a wide range of nutrient conditions. Cells monitor changes in their environment through nutrient sensing protein kinases. Thus Cdc34 phosphorylation by PKA and Sch9 provides a direct tether between G1 cell division events and cell growth. The ubiquitin proteasome system (UPS) controls cellular functions through the targeted degradation of key regulatory proteins. The covalent attachment of ubiquitin often serves as a signal for the degradation of these regulatory proteins by the 26S proteasome  monoubiquitylate Sic1 with kinetics comparable to wild type Cdc34, but extend ubiquitin chains at a negligible rate in vitro Cdc34 autoubiquitylation or histone ubiquitylation assays which do not require RING finger containing proteins, Cdc34Δ12 mutants function as well as, if not better than, Cdc34 Δ12 mutants are nearly inviable, as are cells harboring Cdc34S97D or Cdc34S73K/S97 mutants tm) shows only subtle defects on cell growth in vivoin vitro SCFCdc4 dependent Cdc34tm polyubiquitylation of Sic1 is defective similar to Cdc34Δ12tm expressing cells show key differences from wild-type cells. Importantly, Cln1 and Cln2 proteins are more stable, while Sic1 has a decreased t1/2 and Far1 becomes undetectable. Further, the steady state level of the Ace2 and Swi5 transcription factors, as well as the abundance of their transcriptional targets, is altered. A subsequent Synthetic Gene Array (SGA) screen revealed that CDC34tm cells depend on the expression of RAD23, RPN10 and several other regulators of the UPS The Cdc34/Ubc7 family of ubiquitin conjugating enzymes is defined by a conserved motif within the catalytic domain that consists of two serines and a twelve amino acid acidic loop, which lie in close physical proximity to the catalytic cysteine. In contrast, the majority of E2s, of which Rad6 is a classic example, have a lysine and aspartic acid residue in lieu of these serine residues and lack the acidic loop. This motif allows the Cdc34/Ubc7 family to catalyze both monoubiquitylation and ubiquitin chain extension This study demonstrates that the Cdc34/Ubc7 specific motif is also a key target of signaling pathways coordinating the regulation of cell growth in response to changes in environmental conditions such as nutrient levels. Here we demonstrate that Cdc34-S97 can be directly phosphorylated by either cAMP-dependent protein kinase or the AKT protein kinase homolog Sch9. We also demonstrate that Cdc34-S97 phosphorylation is elevated under conditions of active cell growth and division while it is decreased under conditions causing cell cycle arrest. These results provide a direct link between the growth regulating protein kinases PKA and Sch9 and early cell division events through post-translational modification of the ubiquitin-conjugating enzyme Cdc34.A partial sequence alignment of the yeast ubiquitin conjugating enzymes and Cdc34 orthologs is shown in S73K/S97D or Cdc34S97D enzymes are non-functional both in vivo and in vitro and the Cdc34Δ103–114 mutant supports growth much more poorly than Cdc34 tm) mutations is viable, even when introduced at the normal CDC34 chromosomal locus under the CDC34 promoter S97D can be rescued by combining this mutation with the Δ103–114 mutation  nor CDC34S97D retain Cdc34 function at 35°C, each can suppress the growth defect of the other. This intragenic suppression suggests that multiple Cdc34 proteins work together during ubiquitylation events. On the other hand, CDC34S97T, a mutation that ostensibly permits phosphorylation at this residue, complements both the cdc34-2 temperature sensitive and cdc34Δ strains in spot dilution assays . This antibody, termed α-pS97, recognizes a protein from whole cell yeast lysate which migrates at the same position as Cdc34  is an important determinant of Cdc34-S97 phosphorylation, since the level of Cdc34-S97 phosphorylation is dramatically reduced on COOH-truncated Cdc34  prior to α-factor induced arrest and remain sensitive to α-factor when returned to a permissive temperature Cdc34 in conjunction with SCF complexes has a plethora of substrates which are targeted for proteasome mediated degradation. The known substrates whose degradation is required for timely cell cycle progression are all ubiquitylated and degraded in the G1 phase of the cell cycle. Thus, we hypothesized that Cdc34-S97 phosphorylation is regulated through the cell cycle. To test this hypothesis, cells were synchronized in late G1 with α-factor. After three hours, >95% of the cells were arrested with a mating projection, or shmoo, morphology. The α-factor was washed out; cells were resuspended in fresh medium; and cells were then collected at the indicated times . At the in vivo. There are also two MAP kinase kinases, Mkk1 and Ste7, which when over-expressed noticeably reduce the level of Cdc34-S97 phosphorylation  responsible for this event. We began our screen for candidate Cdc34-S97 kinases by an in vivo over-expression assay. This was done to avoid overlooking candidate protein kinase genes that are essential as would have been the case if we had begun our analysis with a collection of yeast containing viable protein kinase deletion mutants. To identify the kinase(s) responsible for phosphorylating Cdc34-S97, we utilized a genomic ordered array of 124 yeast strains, each expressing a unique GST-kinase fusion under the control of a copper inducible promoter. Construction and utilization of the yeast ORF collection has been described previously rylation . While wMEC1 or MPS1 as these genes are essential. Based on this analysis, the level of S97 is reduced in strains lacking Gcn2, Mkk2, Sch9, Snf1, and Vps15/Vps34  does not noticeably reduce the level of S97 phosphorylation . While tFor several reasons, we also chose to examine specifically whether the Tor kinases controlled Cdc34-S97 phosphorylation for several reasons. First, as shown above, several other nutrient sensing kinases alter Cdc34-S97 phosphorylation. A function of the TORC1 complex  is as a sensor of nitrogen availability in eukaryotic cells. Secondly, both Vps15, a kinase which activates TORC1, and Sch9, a kinase activated by TORC1, increase Cdc34-S97 phosphorylation when over-expressed and strains lacking either of these kinases have reduced Cdc34-S97 phosphorylation. Thus TORC1 could be part of a Vps15/Vps34-TORC1-Sch9 cascade leading to Cdc34-S97 phosphorylation. Finally, neither Tor1 nor Tor2 are part of the collection of copper inducible kinases used in the initial screen; however, Tor activity can be inhibited by the natural compound rapamycin. As predicted, Cdc34-S97 phosphorylation is reduced when cells are treated with the Tor kinase inhibitor rapamycin .RAS2, genetically interacts with CDC34RAS2 or by growing cells on a non-fermentable carbon source which reduces intracellular cAMP levels, inhibits the growth of cdc34-2 temperature sensitive strains at otherwise permissive temperatures TPK2 is overexpressed led us to test whether S97 phosphorylation levels are affected in other circumstances where PKA activity is altered. There are three isoforms of the catalytic subunit of PKA, Tpk1–3  implicated in Cdc34-S97 phosphorylation directly phosphorylate Cdc34 on S97. Protein Kinase A has a strong preference for arginine residues at positions −2 and −3 and a preference for an amino acid residue with a hydrophobic side chain at the +1 position relative to the phosphorylation site −6-X-X-R−3-X-X-(S/T0)-B+1 with X referring to any amino acid residue and B referring to an amino acid residue with a hydrophobic side chain in vitro. Indeed, Cdc34 can be phosphorylated by bovine PKA while the Cdc34S97A mutant is phosphorylated to a lesser degree  SCH9 acts as a multi-copy suppressor of PKA signaling defects and activation of PKA eliminates the slow growth defects of sch9Δ cells −6-X-X-R−3-X-X-(S/T0)-B+1 with X referring to any amino acid residue and B referring to an amino acid residue with a hydrophobic side chain Sch9 and the PKA catalytic subunits (Tpk1–3) are members of the AGC (Protein Kinase A/G/C) kinase family which consists of the cAMP dependent protein kinases, the cGMP dependent protein kinases and Protein Kinase C. Sch9 and PKA may function in redundant pathways as suggested by the findings that in vivo and in vitro activity of Cdc34; however, an S97T mutation is functional. There are published genetic interactions between the cAMP/Ras/PKA pathway and Cdc34 suggesting that PKA stimulates Cdc34 activity in vivo. We hypothesize that Cdc34-S97 phosphorylation increases the ubiquitin conjugating activity of Cdc34. The mechanism is unclear but one possible working model is as follows. Self-association is required for Cdc34 function and Cdc34S97D mutants do not self-associate S97D mutant restores its ability to polyubiquitylate a natural substrate S97D can interact with an SCF complex in vivo immunoprecipitation of FlagCdc34S97D showing that it co-precipitates Cdc53. Wild-type Cdc34 enzyme was present in this assay and it is possible that wild-type Cdc34 dimerizes with Cdc34S97D, allowing Cdc34S97D to interact with SCF. Our discovery that the Cdc34S97D mutant can complement a cdc34 temperature sensitive strain but not a null strain suggests that the residual activity of the temperature sensitive enzyme is supplying a component of the necessary activity to the Cdc34S97D mutant which the Cdc34S97A mutant cannot utilize. In light of the above discussion, it is possible that the Cdc34S97D mutant and wild type enzymes form functional heterodimers and supply the necessary Cdc34 activity to the cell. In a physiological setting, this model could be extended to a scenario where two Cdc34 molecules dimerize after one has been phosphorylated on S97  and associated protocol. Complementary forward and reverse primers, approximately 25 nucleotides in length, encoding the desired mutation(s) were used to amplify the appropriate plasmid. Following PCR amplification, 1 µl of Standard rich (YPD) and defined minimal (SD) media were prepared as described previously. To transform yeast with plasmid DNA, a quick method was utilized. Briefly, 0.5 µg of the plasmid DNA, 20 µl of 2 mg/ml carrier DNA and four volumes of PEG/Li-acetate/TE solution  were combined in an microfuge tube along with a small swap of cells from a fresh plate or overnight culture. The mixture was quickly vortexed and left at room temperature for 24 hours at which time 100 µl was plated on selective media.For spot dilution assays, cells were grown overnight in the indicated media, diluted to equal densities and then 3 µl of each culture and tenfold serial dilutions were spotted onto the indicated plates.6XHis and Cdc34ΔC(S97A)6XHis proteins, encoding the first 244 amino acids of Cdc34 fused to a 6XHis tag at their COOH-terminus, were expressed and purified for these studies. BL21(DE3) bacterial cells  harboring plasmid AD002 (Cdc34ΔC6XHis) or pRC004 (Cdc34ΔC(S97A)6XHis) were grown in 15 ml of LB+ampicillin for 16 hours at 37°C. Five ml of the starter culture was used to inoculate 500 ml LB+ampicillin. The 500 ml culture was grown with vigorous shaking at 37°C for 2 hours or until the OD600 reached 1.2 at which time IPTG was added to a final concentration of 500 mM. The cells were allowed to grow for an additional 3 hours and were then collected by centrifugation. The bacterial pellet was resuspended in 5 ml of Lysis Buffer  adjusted to pH 8 using NaOH). Five hundred µl of 10 mg/ml lysozyme was added and the cell suspension was sonicated for five cycles of ten seconds with a twenty-five percent duty cycle. The lysate was repeatedly drawn through a 23G1 needle to shear the DNA and then spun quickly in a clinical centrifuge. The supernatant was collected and mixed with 1.5 ml of Nickel Sepharose 6 Fast Flow resin  that had been washed twice with Lysis Buffer. The nickel sepharose slurry was incubated on a rocker at 4°C for three hours. The beads were then washed twice with 1 ml of Wash Buffer  for 1 hour at 4°C. Bound proteins were eluted by adding 500 µl of Elution Buffer  and mixing at 4°C for 15 minutes. Two additional rounds of elution, accomplished by adding 750 µl of Elution Buffer to the beads and mixing at 4°C for 15 minutes, were necessary to recover the maximal amount of protein. The purified protein was then dialyzed overnight at 4°C in 1 L of Dialysis Buffer 1  and for an additional hour in Dialysis Buffer 2 .Cdc34ΔCMATahis3-200, leu2-3,112, ura3-52, pep4::URA3], harboring a unique plasmid expressing a single GST-kinase fusion gene under control of the PCUP1 promoter An array of strain EJ758 [in vivo, each strain was grown overnight in 5 ml SD-Ura then diluted to 4×105 cells/ml in 100 ml SD-Ura and grown for 8 hours at 30°C. After 8 hours of growth, copper sulfate was added to a final concentration of 500 µM to induce GST-Kinase expression. Induction lasted for 3 hours at which time the cells were pelleted in a Beckman Centrifuge at 4000×g, 4°C for 5 minutes and immediately frozen with liquid nitrogen. The level of Cdc34-S97 phosphorylation in each strain was measured qualitatively by western blot analysis.For analysis of Cdc34-S97 phosphorylation GSTKinase from yeast, a patch of cells was inoculated into 50 ml SD-Ura to an OD600 ∼0.2 and grown for the remainder of the day, shaking at 30°C. At the end of the day, the 50 ml culture was diluted into 500 ml so that it reached an OD600 of 0.8 the following morning. The following morning, copper sulfate was added to a final concentration of 500 µM to induce transcription of the GSTKinase. The cultures were left to shake at 30°C for 2 hours. After 2 hr, the culture was split into 250 ml fractions and each was harvested by centrifugation in a Beckman Centrifuge at 4000×g. One tube was used for protein purification and the other was stored at −70°C for future purification. The cell pellet was resuspended in 1 ml EXTRACTION buffer (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 4 mM MgCl2, 5 mM DTT, 10% (v/v) glycerol, 1 M NaCl) with one protease inhibitor cocktail tablet  added per 10 ml buffer. Suspension was transferred to a microfuge tube and glass beads were added to the top of the tube. Cells were lysed at 4°C for 200 sec in a Biospec mini bead beater, using 10 cycles of shearing for 20 sec, followed by 1 minute cooling in an ice-water bath. The cell extract was removed from beads by puncturing the bottom of the tube with a flamed 25G needle and the extract was gently forced out with low-pressure air or light centrifugation. The remaining protein was washed off the beads with 0.5 ml EXTRACTION buffer. Two µl of 1.0 M PMSF was added to two ml of protein solution and insoluble cellular debris was removed by gentle centrifugation. The supernatant was transferred to a fresh microfuge tube and protein concentration was determined by the Bradford method To express and purify the 2, 1 mM DTT, 10% Glycerol) to bring the final salt concentration to 0.5 M NaCl. This salt concentration was chosen because it prevents most nonspecific protein-ligand or protein-protein interactions, while still preserving required functional interactions. 100 µl of pre-equilibrated glutathione-Sepharose resin were added per 2 ml of extract . The tube was mixed gently at 4°C for 3 hours. To remove non-specific proteins, the mixture was centrifuged for 20–30 sec at low speed in a microfuge and the supernatant was decanted. The beads were washed twice with 1 ml of WASH BUFFER by mixing for 15 minutes, centrifuged and wash buffer was decanted. Bound proteins were eluted by adding 2 ml ELUTION BUFFER to the resin, followed by 1 hour of mixing, and then low speed centrifugation. The elution was dialyzed overnight against DIALYSIS BUFFER I  glycerol) for 2 hours at 4°C and then overnight against DIALYSIS BUFFER II  glycerol) and stored at −20°C. Normal GSTKinase yield from a 250-ml culture is 0.5 ml of protein at ∼250 µg/ml.The crude protein extract was diluted with an equal volume of No Salt Wash Buffer . The phosphopeptide DGRLCI(pS)ILHQ was synthesized and conjugated to Keyhole Limpet Hemocyanin (KLH). On day one, 500 µg of KLH-phosphopeptide conjugate was emulsified with Freund's complete adjuvant and subsequently used to immunize two New Zealand white rabbits. On days 14, 28 and 42 the rabbits were injected with an additional 250 µg of KLH-phosphopeptide conjugate emulsified with Freund's incomplete adjuvant. On days 35 and 56, approximately 25 ml of serum was collected from each rabbit. Production bleeds of 50 ml were taken from each rabbit on day 70 at which time the animals were euthanized.6XHis was electrophoresed on a 10% SDS-PAGE gel. The protein was transferred to PVDF membrane for 2.5 hours at 30 volts at 4°C in Transfer Buffer . The membrane was blocked with 5% milk in 1× KPBS-T for 1 hour at room temperature. After blocking, the membrane was exposed to 2 ml of crude anti-pS97 antisera which had been diluted with 13 ml of sterile 1× KPBS-T+0.02% sodium azide. The membrane was incubated overnight at 4°C with the antisera. The unbound fraction was collected and 11.5 of the 13 ml were purified with protein A beads as previously described 280 measurement estimates the α-pS97 antibody concentration to be 1.7 mg/ml. Sodium azide was added to a final concentration of 0.02% prior to storage at 4°C.To ensure that the antibody specifically recognized Cdc34 phosphorylated on serine amino acid residue 97, 360 µg of purified, bacterially expressed Cdc34ΔCOnce grown to the desired density, cells were collected by centrifugation for 5 minutes at 4000×g in a Beckman centrifuge at 4°C. The cell pellet was immediately frozen in liquid nitrogen and stored at −80°C until lysis. To extract the protein, the cell pellet was washed with water and then resuspended in 300 µl of Breaking Buffer  per 10 ml of breaking buffer). 500 µm acid-washed, glass beads  were added to the cell suspension and the cells were broken by repeated rounds of glass bead beating until a protein concentration of ∼5 µg/µl was achieved. Typically, this required three rounds of bead beating at one minute per round. Protein concentration was determined by the Bradford method The Horvath-Riezman protocol 2HPO4, 1.5 mM KH2PO4, 0.2% Tween-20, pH to 7.2) and blocked with 5% milk in KPBS-T. Antibodies were used at the following dilutions, affinity purified α-Cdc34  and and α-TAP (1∶2000) . Primary antibody was detected with an HRP conjugated goat α-rabbit secondary antibody at a 1∶10,000 dilution .5× Laemmli Buffer  was added to the protein extract (∼50 µg of protein) made via the glass bead method. Protein from either extraction method was loaded in equal amounts onto a 10% or 12% SDS-PAGE gel. Separated proteins were transferred to a PVDF membrane using Transfer Buffer . The membranes were washed with KPBS-T ΔC6XHis (1.3 ηmoles) were incubated with 10 µl (1 U/µl) of bovine Protein Kinase A  in PKA phosphorylation buffer  plus 10 µl of 5× ATP solution  in a total reaction volume of 100 µl. When PKA Inhibitor  was used, 1 µl of 1 mg/ml solution was added to the reaction. The reactions were incubated at 30°C for 20 minutes and stopped by addition of 25 µl of 5× Laemmli Buffer. Samples were heated at 95°C for 3 minutes and 30 µl of each reaction was loaded onto a 12% SDS-PAGE gel. The gel was run and then dried and exposed to film for 4 hours. To quantitate the relative amounts of 32P incorporation, bands were cut from the dried gel and analyzed by a liquid scintillation counter.Bacterially expressed Cdc34ΔC6XHis (165 picomoles) was incubated with 10 µl (1 unit/µl) of bovine Protein Kinase A  or 5 µl of GSTTpk3 or GSTSch9 purified from yeast in PKA phosphorylation buffer  plus 10 µl of 5× ATP solution  in a total reaction volume of 50 µl. When PKA Inhibitor  was used, 1 µl (1 mg/ml) was added to the reaction. The reactions were incubated at 30°C for 10 minutes and the samples were placed on ice. 10 µl of each was spotted onto a piece of PVDF membrane which had been pre-soaked in methanol. The spot was allowed to dry for 30 minutes. The membrane was re-wet with methanol and then processed as described in the western blotting section above.Bacterially expressed Cdc34ΔC


Almost a 20% of the residues of tau protein are phosphorylatable amino acids: serine, threonine, and tyrosine. In this paper we comment on the consequences for tau of being a phosphoprotein. We will focus on serine/threonine phosphorylation. It will be discussed that, depending on the modified residue in tau molecule, phosphorylation could be protective, in processes like hibernation, or toxic like in development of those diseases known as tauopathies, which are characterized by an hyperphosphorylation and aggregation of tau. Tau protein was discovered as one of the brain microtubule-associated proteins bound to in vitro assembled tubulin . Tau proTau is a neuronal microtubule-associated protein, and some of its functions are related to that association that may result in microtubule stabilization or the regulation of axonal transport, but tau protein is a “sticky” protein that could bind to other proteins, apart from tubulin (the main component of microtubules), which could also facilitate its subcellular localization at the cytoplasm close to the membrane or in the nucleus. On the other hand, in the neuron, tau is mainly present in axons, owing to a sorting mechanism in which its level of phosphorylation may play a role , but tauSome of tau functions could be regulated by phosphorylation. In many cases, tau phosphorylation can cause its detachment from microtubules , and thoTau is a phosphoprotein that could be modified by different protein kinases at tyrosine, threonine, or serine residues. In the longest CNS tau molecule, there are 80 serine/threonine residues that are mainly present at the proline-rich and C-terminal regions .Tau kinases have been divided in proline- (PDPK) and nonproline- (NPDPK) directed protein kinases, being GSK3 the PDPK that could modify more sites in tau molecule . These sβ, one of its isoforms, is abundant in the central nervous system and can modify several neuronal proteins like tau [GSK3 is a protein kinase originally identified due to its role in glycogen metabolism regulation. GSK3like tau .Tau hyperphosphorylation takes place, at the CNS, in several diseases (tauopathies), being the most predominant of these disorders, Alzheimer disease. In the tauopathies, tau protein is not only phosphorylated but also could be found in aggregated form. In Alzheimer's disease (AD) tau protein can be assembled into the paired helical filaments that form the aberrant structure known as neurofibrillary tangles . Also, iIn Alzheimer's disease postmortem brain material, tau phosphorylation has been found to increase at several sites modified by GSK3. In this way, the analysis of how an increase in GSK3 activity could modify tau protein has been deeply studied. It is known the involvement of factors like wnt, insulin, or insulin-like growth factor in the regulation of GSK3 . It is aβ, and a consequence of that is a reduced phosphorylation of amyloid precursor protein (APP), another important protein in AD. In the absence of phosphorylation, the turnover of APP is increased [α, can regulate APP processing and the production of amyloid beta peptide [One of the consequences of tau phosphorylation by GSK3 is a decrease of its interaction with microtubules , which cncreased . Also, Pncreased . About G peptide . Going back to tau phosphorylation, it has been observed that the interaction of tau with cell membranes decreases upon its phosphorylation . In thisSince tau phosphorylation may affect tau functions, it was suggested that phosphotau could be toxic. However, this point has been widely discussed and is commented if phosphorylated tau is toxic, protective, or none of the above .Drosophila as a model it was indicated that phosphotau is not toxic [Working on ot toxic , 30. Alsot toxic  or that ot toxic , or to iot toxic , 34. Morot toxic . A possiot toxic . In anotot toxic , being tot toxic , 39.A protective role for tau phosphorylation could be suggested when that modification could take place at threonine 181  that couOn the other hand, tau toxicity resulting from tau phosphorylation could affect to axonal transport, mitochondrial respiration, or cytoskeletal changes , 46. AlsA way to study the role of tau phosphorylation on cell toxicity is the use of pseudophosphorylated tau variants where a serine or threonine residue is replaced by glutamatic acid. Thus, it has been found that pseudophosphorylation at the site recognized by antibody AT8, but not at the site recognized by abPHF-1, prevents the interaction of tau with neuronal membranes , 51.Pseudophosphorylation of tau in ten different sites of a single molecule  results in a toxic action and the induction of apoptosis .Combined pseudophosphorylation at threonine 212, threonine 231, and serine 262 causes neurodegeneration, mainly through the presence of pseudophosphorylation at threonine 212 . A possiAll those results suggest that, depending on the modified site, a protective or toxic function may take place and that it could be due to different conformational changes of tau molecule. Although tau is an unstructured molecule, it has been suggested that, in some conditions, it could have a paperclip conformation. In this conformation, the C-terminal end of tau interacts with the microtubule-binding domain, whereas the N-terminal end could bind to the C-terminal region, adopting a paperclip folding . PseudopAt the level of degeneration of a single neuron, occurring in Alzheimer's disease, it was suggested that first takes place phosphorylation of tau molecule at some specific sites and afterwards there is a further phosphorylation and aggregation of tau protein. Thus, early tau phosphorylation can be used as an initial marker for neurodegeneration .The phosphorylation of tau, by GSK3, at specific sites can be analyzed by the use of antibodies that specifically recognize some of those sites. In this way, analysis of phosphorylation at serine 202 or at serines 396–404 could be achieved by using the antibodies AT8  or PHF-1Tau phosphorylation (recognized by AT8 antibody) at regions like CA3 of the hippocampus has been correlated with regression of synaptic components and memory deficits . Indeed,A main difference between neuron atrophy and neuron death is that in the first case the neuron remains alive and could be possible to reactivate it. In fact, we have recently observed that in patients with AD the accumulation of phosphotau in a pretangle state does not induce changes in the dendrites of pyramidal neurons, whereas the presence of tau aggregates forming intraneuronal neurofibrillary tangles gives rise to a progressive dendritic atrophy and the loss of dendritic spines (synaptic disconnection) . Thus, the presence of phosphotau in a pretangle state in neurons of the human brain does not necessarily result in severe and irreversible effects. Thus, it is of great interest to search for possible physiological conditions in which neurons with a reduced metabolism and altered synaptic connectivity that may result in the lack of cognitive functions could be reactivated to regular functionally neurons. The group of Arendt et al.  answers Mesocricetus auratus (another hibernating rodent) has been sequenced and characterized . The reason of that characterization has been to perform a first step to know if tau protein in hibernating animals, like M. auratus, could be taken as a single marker of the process or it may play a role in it. Thus, tau differences between this hibernating animal and nonhibernating organisms like the human being should be carried out. Our results have indicated the presence of several variations at the N-terminal half between M. auratus and human tau. Some of these variations have been also found in nonhibernating rodents. However, there are three variations, in that region, which are only present in the two hibernating rodents, M. auratus and S. citellus. In addition, there are another differences between M. auratus and human tau like the presence of 10 phosphorylatable residues present in human but not in M. auratus tau . Many of these phosphorylatable residues could be possible substrates for ck1, a protein kinase that has been related with circadian rhythm in nonhibernating organism [Thus, if topor stage, in hibernation, is similar to the pretangle stages in AD and it could be reversed, we could ask if that reversion could also take place in AD. This question has pros and cons. One of the cons is that the primary structure of tau from hibernating animals could be different than that of a human being. Thus, tau from hibernating mammals have been sequenced , but no organism , 73. AltIt could be a possible relationship between hibernation/wake in hibernating mammals and sleep/wake in humans. Indeed, AD patients exhibit sleep/wake disorders . HoweverA more related behavior to that of hibernation could be anesthesia. Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia , a proceFurthermore, we should look for better analysis to detect a decreased metabolic rate in damaged CA3 neurons in AD, and we should look for an answer about how to increase that metabolic rate in AD neurons. A probable common link among some of these disorders is the appearance of oxidative damage that results in neurodegeneration , 80.β  may cause mitochondrial defects and ATP depletion.A possible mechanism has been suggested to explain an energy depletion in AD. It involves that oxidative stress may lead to the formation of reactive carbonyl compounds  that appIn summary, since neuron atrophy in neurodegenerative diseases, like AD, has been correlated with tau phosphorylation by GSK3, it should be tested in the future if by decreasing that phosphorylation that atrophy could be reversed or not.


Kif26b expression is restricted to the metanephric mesenchyme, and its transcription is regulated by a zinc finger transcriptional regulator Sall1. However, the mechanism(s) by which Kif26b protein is regulated remain unknown. Here, we demonstrate phosphorylation and subsequent polyubiquitination of Kif26b in the developing kidney. We find that Kif26b interacts with an E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) in developing kidney. Phosphorylation of Kif26b at Thr-1859 and Ser-1962 by the cyclin-dependent kinases (CDKs) enhances the interaction of Kif26b with Nedd4. Nedd4 polyubiquitinates Kif26b and thereby promotes degradation of Kif26b via the ubiquitin-proteasome pathway. Furthermore, Kif26b lacks ATPase activity but does associate with microtubules. Nocodazole treatment not only disrupts the localization of Kif26b to microtubules but also promotes phosphorylation and polyubiquitination of Kif26b. These results suggest that the function of Kif26b is microtubule-based and that Kif26b degradation in the metanephric mesenchyme via the ubiquitin-proteasome pathway may be important for proper kidney development.Kif26b, a member of the kinesin superfamily proteins (KIFs), is essential for kidney development.  The kidney is composed of minimum units called nephrons, which maintain an appropriate homeostatic balance of water and salt levels and remove nitrogenous metabolic waste products. The tubular epithelial network of the nephron originates from two different tissues, the ureteric bud and the metanephric mesenchyme, that form the collecting duct system and the renal tubules, respectively Saccharomyces cerevisiae, is thought not to be motile, especially along microtubules, due to the deviance in both a catalytic pocket for ATP hydrolysis and the microtubule-binding sites The kinesin superfamily proteins (KIFs) are known to be important molecular motors that are involved in the microtubule- and ATP-dependent transport of various cargos, including membranous organelles, protein complexes, and mRNAs, to specific destinations Kif26b-knockout mice exhibit kidney agenesis or hypoplasia Kif26b-null embryos, the ureteric buds elongate and migrate in proximity to, but do not invade, the metanephric mesenchyme. This defect is attributable to impaired maintenance of Gdnf, which is regulated by α8β1 integrin signaling Kif26b-null mesenchyme does not condense around the ureteric buds, indicating that the localization of the integrin α8 subunit is disrupted. Furthermore, transcription of Kif26b is regulated by Sall1, a zinc finger transcription factor that has been reported to be crucial for kidney development Kif26b contains tandem Sall1-binding consensus sequences, and binding of Sall1 to these sites enhances Kif26b-transcription. In the developing kidney, Kif26b is expressed in the undifferentiated metanephric mesenchyme but is rapidly downregulated after renal vesicle formation. While our study revealed the regulatory mechanism of Kif26b transcription, the regulation of Kif26b protein during kidney development remains to be elucidated.We recently reported that In the present study, we identify Nedd4, a HECT E3 ubiquitin ligase, as a Kif26b-interacting partner. Nedd4 polyubiquitinates Kif26b and thus targets it for degradation via the ubiquitin-proteasome pathway. We also show that phosphorylation of Kif26b by CDKs is important for its interaction with Nedd4. Interestingly, disruption of microtubules by nocodazole triggers phosphorylation and polyubiquitination of Kif26b. These findings suggest that the degradation of Kif26b is important for kidney development.Roscovitine, a selective CDK inhibitor, was purchased from Biomol . The proteasome inhibitor MG132, the MEK1/2 inhibitor U0126, and nocodazole were from Peptide Institute , Wako , and Sigma-Aldrich , respectively. Anti-Kif26b rabbit polyclonal antibody was previously described The full-length Kif26b construct and the Kif26b-ΔC and Kif26b-C deletion constructs were previously described HEK293, COS-7, and HeLa (American Type Culture Collection) cells were maintained in DMEM supplemented with 10% fetal bovine serum. Cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For organ culture, kidneys dissected from embryonic day (E) 14.5 embryos were cultured as previously described Duplex siRNAs for human Nedd4 were purchased from Invitrogen. The siRNA nucleotide sequences were as follows: Nedd4-siRNA#1, sense (5′-UAUUGGUGACAACUAUUUCUGAUCC-3′) and antisense (5′-GGAUCAGAAAUAGUUGUCACCAAUA-3′), Nedd4-siRNA#2, sense (5′-UUCAAUUGCCAUCUGAAGUUUAUCC-3′) and antisense (5′-GGAUAAACUUCAGAUGGCAAUUGAA-3′), and negative control, sense (5′-AAAUGGCGUCGCGUUCCUUUAGAGG-3′) and antisense (5′-CCUCUAAAGGAACGCGACGCCAUUU-3′).g at 4°C, and the supernatants were incubated with the appropriate antibodies and protein G-Sepharose beads  for 2 h at 4°C. Immunoprecipitates were washed four times with lysis buffer, separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. Immunoblotting analyses were performed as previously described Cells were harvested with lysis buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% glycerol, 5 mM EDTA, 0.5% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and phosphatase inhibitor cocktail . For analysis of ubiquitinated proteins, SDS was added to a concentration of 0.1%. Lysates were centrifuged at 17,500 × in situ hybridization and were performed using an automated Discovery System  according to the manufacturer’s protocols. A cDNA fragment of Nedd4 (1648–2664 bp) spanning the open reading frame was used as a riboprobe. All experimental procedures and protocols for animal studies were approved by the Committee on Animal Research of Kumamoto University (B23-179).E14.5 mouse embryos were fixed in 10% formalin and processed for paraffin-embedded sectioning. Immunohistochemistry and Immunofluorescence analysis was performed as previously described 2, and 1% Triton X-100) containing complete protease inhibitor (Roche) and centrifuged at 100,000 × g for 30 min at 4°C. The lysates were supplemented with 0.2 mg/ml of a taxol-stabilized microtubule solution , 10 mM EGTA, 5 mM MgClin vitro kinase assay was performed as previously described with a minor modification Escherichia coli (BL21) with an N-terminal glutathione S-transferase (GST) tag was purified with Glutathione Sepharose 4B . CDK1/cyclin B, CDK2/cyclin A, CDK5/p25 and CDK6/cyclin D3 were purchased from Millipore. Kinase activity was assayed in 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM EGTA, 0.5 mM PMSF, and 0.5 mM DTT for 30 min at 30°C in the presence of 0.1 mM ATP.The In vitro ubiquitination assays for Kif26b proteins were performed as previously described In our previous report, we searched for proteins that interact with Kif26b in the developing kidney and found several, including nonmuscle myosin heavy chain type IIB, that were pulled down by a purified GST-tagged C-terminal tail fragment of Kif26b We next investigated which region of Kif26b was involved in the interaction with Nedd4. We expressed Myc-tagged Nedd4 and FLAG-tagged full-length or partial Kif26b constructs  in HEK29As shown above, the WW domains of Nedd4 interacted with the C-terminal tail region of Kif26b. The WW domains have been grouped into four classes according to their ligand preferences We first addressed whether the C-terminal tail region of Kif26b could be phosphorylated. We searched the C-terminal tail region of Kif26b for protein kinase consensus sequences and found that this region harbored two potential CDK phosphorylation sites . Analysein vitro kinase assay using purified active CDK proteins. We found that CDK2 and CDK5 prominently phosphorylated GST-Kif26b C . This method has previously been reported to remove associated proteins from the immunoprecipitated protein of interest in vitro ubiquitination assays. FLAG-Kif26b was expressed in HEK293 cells and purified by immunoprecipitation with anti-FLAG beads as an ubiquitination substrate. Then, GST, GST-Nedd4 WT, or GST-Nedd4 CS purified from E. coli and ubiquitin, together with other E1 and E2 proteins, were added to initiate the ubiquitination reaction. The positive smeared signal was observed and was dependent on the catalytic activity of Nedd4  in HEK293 cells. At 48 h post-transfection, cells were treated for 8 h with the proteasome inhibitor MG132 and Kif26b was immunoprecipitated with anti-FLAG beads. We observed a smeared signal in the anti-FLAG bead precipitates of the lysates of cells expressing FLAG-Kif26b and Myc-Ub . This smof Nedd4 . We alsoof Nedd4 . Lys48-lPhosphorylation of Kif26b regulated its interaction with Nedd4 . TherefoIn order to verify that endogenous Nedd4 also affected polyubiquitination of Kif26b, we treated HeLa/FLAG-Kif26b cells with siRNAs against Nedd4 and performed immunoprecipitation and immunoblotting analyses. Treatment with two siRNAs against Nedd4 reduced the amount of polyubiquitinated FLAG-Kif26b compared with a control siRNA treatment , implyinWe next addressed how upstream signaling induced the phosphorylation and polyubiquitination of Kif26b. Kif26b is expressed in the metanephric mesenchyme but is absent in the renal epithelia (described below). This suggests the possibility that epithelialization of the metanephric mesenchyme leads to degradation of Kif26b. Dramatic rearrangements to the microtubule-based cytoskeleton have been reported to occur during the formation of polarized epithelial cells We first addressed whether Kif26b associated with microtubules. Kif26b, like Kif26a, is classified as a member of the Kinesin-11 family We next examined whether the phosphorylation of Kif26b was dependent on the microtubule status. When we treated HeLa/FLAG-Kif26b cells with nocodazole, we observed a significant shift of the FLAG-Kif26b signal toward the higher molecular weight area on SDS-PAGE in a time-course dependent manner , and preWe next addressed whether nocodazole treatment affected polyubiquitination of Kif26b. HeLa or HEK293/FLAG-Kif26b cells were pretreated for 6 h with DMSO or nocodazole and then treated for another 8 h with MG132. Immunoprecipitation and immunoblotting analyses showed that nocodazole treatment increased the polyubiquitination of Kif26b . Furtherin vivo complex formation between Kif26b and Nedd4 in the developing kidney is regulated by CDKs. Immunohistochemistry analyses of E14.5 kidneys showed that Kif26b was localized in the peripheral regions of the developing kidney, in which the metanephric mesenchyme containing nephron progenitors exist In situ hybridization for Nedd4 showed an expression pattern similar to that shown by immunostaining  and mediates its ubiquitination, promoting its endocytosis from the plasma membrane and degradation + uptake in kidney epithelial cells, and defects in this regulatory pathway contribute to the pathogenesis of certain forms of hypertension such as Liddle syndrome, a hereditary hypertension that shows increased activity and retention of ENaC at the plasma membrane Ubiquitination is a key regulatory process that determines the fate of a protein and induces various cellular events including protein degradation, endocytosis, and transmembrane protein sorting and trafficking Kif26b is attenuated in the epithelialized structures remains unclear. Once the metanephric mesenchyme forms renal vesicles upon the ureteric bud in response to activation of canonical Wnt signals, Kif26b transcription is rapidly downregulated, while Sall1 expression is sustained in this and subsequent stages of epithelial differentiation Kif26b cannot be explained solely by decreased expression of Sall1. Several studies have demonstrated that Sall1 acts as a transcriptional repressor. Sall1 associates with histone deacetylases, deacetylase corepressor complex, and several components of chromatin remodeling complexes Kif26b-transcription following the initiation of epithelialization induced by canonical Wnt signaling. It will be interesting to investigate the mechanism of transcriptional attenuation of Kif26b.The proteasomal degradation of Kif26b could contribute to its elimination from differentiated structures such as renal vesicles and comma-shaped bodies. However, the mechanism by which transcription of in vivo. It would be necessary to generate a mouse strain that retains the phosphorylation-resistant Kif26b to see whether mis-regulation of Kif26b affects kidney formation. In addition, the physiological role of Nedd4 should be examined in vivo. We performed knockdown analysis of Nedd4 in kidney explants from E12.5 embryos using in vivo morpholino oligonucleotides (MO), which has been shown to be effective in the embryonic kidney While we found phosphorylation and polyubiquitination of endogenous Kif26b, the physiological significance of these phenomena in kidney development awaits further studies In this study, we have identified the molecular mechanism by which Kif26b is degraded. Kif26b appears to be tightly regulated at both the transcriptional and posttranslational levels. Spatiotemporal regulation of Kif26b would be important for mesenchymal cells, which is indispensable for proper kidney formation.Figure S1Identification of Nedd4 as a Kif26b-interacting protein. The lysates from newborn kidneys were subjected to pull-down assay with recombinant GST-tagged C-terminal region of Kif26b. The precipitates were separated by SDS-PAGE, followed by silver staining. The arrowheads indicate proteins that were identified by mass spectrometry .(TIF)Click here for additional data file.Figure S2Kif26b is phosphorylated by CDKs.A. HEK293 cells were transfected with FLAG-Kif26b expressing plasmid. At 48 hrs after transfection, cells were treated with DMSO, Roscovitine (20 µM) or U0126 (20 µM) for 6 hrs. The lysates were subjected to immunoprecipitation with anti-FLAG beads. The precipitants were analyzed by immunoblotting with the indicated antibodies. B. GST, GST-Kif26b-C, GST-Kif26b-C T1859A, GST-Kif26b-C S1962A, or GST-Kif26b-C T1859A/S1962A was incubated with or without recombinant His-tagged CDK5, and a kinase assay was performed. Proteins were separated by SDS-PAGE and detected by Coomassie Brilliant Blue (CBB) staining or immunoblotting with the indicated antibodies. C. HEK293 cells were transfected with the indicated plasmids. At 48 hrs after transfection, cell lysates were subjected to immunoprecipitation with anti-FLAG beads, followed by immunoblotting with the indicated antibodies.(TIF)Click here for additional data file.Figure S3Kif26b is polyubiquitinated via Lys48 on ubiquitin.A. HEK293 cells were transfected with FLAG-Kif26b. At 48 hrs after transfection, cells were treated with MG132 (20 µM) for 8 hrs and lysed with lysis buffer in the presence or absence of 2% SDS. The lysate containing SDS was boiled for 10 min at 95°C, and then diluted 15 times with lysis buffer. Immunoprecipitation was performed with anti-FLAG M2 beads and the precipitants were subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies. B. HEK293 cells were transfected with Myc-tagged WT, K48R, K63R and 0K ubiquitin constructs along with FLAG-Kif26b. After 48 hrs, cells were treated with MG132 (20 µM) for 8 hrs, followed by immunoprecipitation with anti-FLAG M2 beads. The precipitates were subjected to SDS-PAGE and immunoblotting with the indicated antibodies.(TIF)Click here for additional data file.Figure S4Nedd4 is expressed in developing kidney.In situ hybridization showed Nedd4 was expressed in all components of developing kidney such as the metanephric mesenchymes, comma-shaped bodies and the ureteric epithelia. No signal was observed with the sense control probe. Scale bar, 100 µm.(TIF)Click here for additional data file.Figure S5Interaction of Kif26b with CDK5 and expression of Kif26b in the developing kidney.A. Kif26b was immunoprecipitated from E14.5 kidney lysates with anti-Kif26b antibody, and precipitates were analyzed by immunoblotting with the indicated antibodies. B. Kidneys from E17.5 CDK5 mutant embryos were lysed with sample buffer and then separated by SDS-PAGE followed by immunoblotting with the indicated antibodies.(TIF)Click here for additional data file.


Arctium lappa) and to elucidate the inhibitory mechanism in melanogenesis, we analyzed melanin content and tyrosinase activity on B16BL6 murine melanoma and melan-A cell cultures. Water extracts of Fructus Arctii were shown to inhibit tyrosinase activity in vitro and melanin content in α-melanocyte stimulating hormone-stimulated cells to similar levels as the well-known kojic acid and arbutin, respectively. The active compound arctigenin of Fructus Arctii displayed little or no cytotoxicity at all concentrations examined and decreased the relative melanin content and tyrosinase activity in a dose-dependent manner. Melanogenic inhibitory activity was also identified in vivo with zebrafish embryo. To determine the mechanism of inhibition, the effects of arctigenin on tyrosinase gene expression and tyrosinase promoter activity were examined. Also in addition, in the signaling cascade, arctigenin dose dependently decreased the cAMP level and promoted the phosphorylation of extracellular signal-regulated kinase. This result suggests that arctigenin downregulates cAMP and the tyrosinase enzyme through its gene promoter and subsequently upregulates extracellular signal-regulated kinase activity by increasing phosphorylation in the melanogenesis signaling pathway, which leads to a lower melanin content.To identify the active compound arctigenin in Fructus Arctii (dried seed of medicinal plant  Researches on natural products, including traditional medicine and herbs, have been increasing recently due to an increase in demand for complementary and alternative medicines with less side effect and increased safety . In dermα-melanocyte stimulating hormone (α-MSH), which increase tyrosinase expression and melanin content by binding to melanocortin 1 receptor (MC1R). Activated MC1R and chemical compound forskolin and isobutylmethylxanthine (IBMX) also stimulate melanogenesis by activating cAMP in the melanocyte-specific pathway, and cAMP can in turn increase melanogenic signaling enzymes through protein kinase A (PKA), which induces microphthalmia-associated transcription factor (MITF) expression  × 100.Murine melanoma cell line B16BL6 and non-neoplastic cell line melan-A were maintained in Dulbecco's Modified Eagle Medium  and RPMI 1640, respectively, supplemented with 10% (v/v) fetal bovine serum and 1x penicillin-streptomycin mixture  in a humidified 5% COications . In brieμL of DOPA (20 mM), KA (60 μg/mL), FAE, and 100 μL of tyrosinase (250 U/mL) were mixed together in 96-well plate and incubated at 37°C for 10 min. The absorbance was then measured using a microplate reader at 475 nm. DOPA, KA, and tyrosinase were dissolved in 0.1 M potassium phosphate buffer (pH 6.6) at the concentrations indicated above. Concentrations and volumes of each reagent were determined based on preliminary optimization experiments performed in our laboratory (data not shown). For control, cells were treated with DOPA, potassium phosphate buffer and tyrosinase, with no tyrosinase inhibitor. All the experiments were carried in triplicate.Tyrosinase inhibition was determined spectrophotometrically as described previously, with minor modifications . To exam4 cells/dish) were seeded on 6-well plate and treated with arbutin (positive control) or ATG. Melanogenesis stimulator α-MSH (100 nM) was used to induce melanin synthesis in both cell lines. After 72 h, cells were harvested, washed with DPBS, and collected. Cell pellets were then completely lysed with 1 N NaOH at 60°C for 1 h, and the absorbance at 405 nm of 200 μL of the lysates was measured using microplate reader. The protein concentrations and melanin contents for each treatment were determined using the BCA protein assay kit  and were compared with the negative control (without arbutin and inhibitor).The melanin content was measured as described previously . B16BL6 5 cells/dish and incubated for 24 h. After serum starvation for 24 h, the cells were further incubated for 72 h with or without ATG. Cells were harvested, washed in DPBS, and lysed with lysis buffer  and subjected to a freeze-thaw cycle (−80°C 30 min/25°C 25 min/37°C 5 min). After centrifugation , protein concentrations were determined using the BCA protein assay kit and the concentrations were adjusted to 200 μg/mL. Cell lysate supernatants (each 50 μL) were transferred to 96-well plate and mixed with 100 μL of DOPA (20 mM) and 50 μL of DPBS. After incubation at 37°C overnight, the absorbance at 475 nm was measured using a microplate reader. Cellular tyrosinase activities were determined as the ratio of tyrosinase content over total protein content (ng/μg).Tyrosinase activity was measured as described previously, with minor modifications . In brieα-MSH treatment. Total RNA was isolated using TRI reagent and 1 μg of each samples was reverse-transcribed using AccuPower RT PreMix  according to the manufacturer's instructions, and aliquots of cDNA were subjected to real-time quantitative polymerase chain reaction (RT-qPCR) with AccuPower 2X Greenstar qPCR Master Mix . A forward primer sequence of 5′-GGCCAGCTTTCAGGCAGAGGT-3′ and reverse primer sequence of 5′-TGGTGCTTCATGGGCAAAATC-3′ were used for tyrosinase exon 1. Thermal cycling of PCR amplification was carried out as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and elongation at 72°C for 30 s using Eco Real-Time PCR System . For β-actin, forward primer 5′-TGTCCACCTTCCAGCAGATGT-3′ and reverse primer 5′-AGCTCAGTAACAGTCCGCCTAGA-3′ were used in the same thermal cycling conditions as described for the tyrosinase PCR experiments.B16BL6 cells were treated with ATG for 1 h and further incubated for 24 h with GGTACCTTCAACCCTTTTCTATGTCC-3′ (−2267 to −2242) and reverse 5′-CTCGAGCACAGACTTCTTTTCCAGCAAC-3′ (−9 to +19). Genomic DNA information was obtained from http://genome.ucsc.edu/. The underlined sequences of each primer were not present in the genomic DNA and were artificially attached to confer restriction enzyme recognition sites for DNA cloning. The PCR product  was then introduced into the T-Blunt vector system. The inserted PCR product was digested with restriction enzyme Xho I and Kpn I and then subsequently cloned into the same enzyme site of the pGL3-basic vector, which contained a luciferase coding sequence in its upstream region. Cloned DNA was identified by sequencing and enzyme digestion followed by sizing on the agarose gel electrophoresis.Upstream region, including the tyrosinase promoter, was amplified and isolated from genomic DNA by PCR using the following primers: forward 5′-μg of CRE-Luc vector and 0.05 μg of Rlu, using 2 μg of lipofectamine 2000 reagent in a total reaction volume of 200 μL. After 24 h, the transfection medium was changed and the cells were treated with ATG for 1 h and further incubated with α-MSH for 24 h at 37°C. Cells were then washed with DPBS and lysed with Triton X-100  containing ethylenediaminetetraacetic acid  and DTT (2 mM). Luciferase activity was determined using a Dual-Luciferase Assay system. All reagents were prepared as described in the manufacturer's instruction, and the data are represented as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc).For the analysis of CRE (cAMP response element) promoter activity, murine melanoma B16BL6 cells in 24-well dishes were transfected with 0.2 α-MSH, and lysed with Triton X-100 . The proteins in the cell lysate were separated by 10% sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The blotted membrane was incubated for 1 h with 5% nonfat milk in Tris-buffered saline (pH 7.6) containing 0.05% Tween 20. Subsequently, the membrane was incubated overnight with the anti-goat tyrosinase antibody (1 : 1000), and further incubated for 1 h with the anti-goat antibody conjugated with horseradish peroxidase . For ERK, the membrane was incubated overnight with ERK and p-ERK antibody (1 : 1000), and then incubated for 1 h with the anti-rabbit antibody conjugated with horseradish peroxidase . The β-actin antibody  was used for normalization. The signals were visualized using the enhanced chemiluminescence reagent .Cells were treated with ATG or arbutin for 1 h, further incubated for 24 h at 37°C with μM. As control, DMSO or PTU (well-known melanogenic inhibitor) treated embryos were analyzed together with the embryos of ATG treated. Live embryos at 40 hpf (hour post-fertilization) were classified and taken pictures.Inhibitory effect of ATG on melanin pigmentation was observed in vivo using zebrafish embryo following the previous report with slight modification . ZebrafiArctium lappa (common name Burdock) has been used as a therapeutic herb in Asia, Europe, and North and South America for hundreds of years. It has also long been cultivated and used as a dietary and nutritional food, especially in Asia [ in Asia . Here, wμM  prior to the emergence of pigmentation in 15 hpf developing zebrafish embryos showed the moderate reduction of pigment deposition  embryos did not undergo normal development but were instead stalled at between 18 and 20 somite stage . Lower dose of ATG (1 μM) failed to suppress the body pigmentation . Analysis of tyrosinase activity has been widely used as a measure of melanin synthesis in cell culture systems [α-MSH-mediated tyrosinase activity. Consistent with the results observed in the melanin content assay, ATG treatment significantly suppressed tyrosinase activity. Other natural herbs and medicines have been shown to inhibit tyrosinase activity and melanin contents. For example, Zhong et al. screened 90 traditional Chinese herb extracts to identify extracts displaying depigmentation activity [Galla Chinensis and ethanol extract of Radix Clematidis showed the highest activity in the cell culture assay. Although they did not identify the active compound in each extracts further investigations are needed for the development of useful dermatological agents. Lin et al. isolated biochanin A from the methanol extract of Trifolium pretense and found that it inhibited melanogenesis and tyrosinase activity [Eupatorium triplinerve Vahl [To further confirm that ATG is an inhibitory active compound, the effect of ATG on melanin content and tyrosinase activity was analyzed in B16BL6 melanoma cells and nonneoplastic melan-A cells. Cellular melanin contents and tyrosinase activities were significantly reduced by ATG treatment in a dose-dependent manner in both B16BL6 and melan-A cells, with similar inhibition pattern between two cell lines Figures . As show systems . For thiactivity . Among tactivity . They alrve Vahl .α-MSH-induced tyrosinase mRNA expression and tyrosinase protein expression was also greatly repressed to levels greater than the arbutin control . Tyrosinase gene expression and melanin synthesis are then stimulated by this signaling pathway, and α-MSH stimulates cAMP upregulation. This then induces melanogenesis, which is closely linked to the level of tyrosinase [Moreover, it is well known that ogenesis . It was ogenesis –51. cAMPogenesis . In mammogenesis . cAMP-elrosinase , 55. To Rehmannia glutinosa was shown to inhibit melanogenesis by ERK activation and tyrosinase downregulation [μm) treatment time and the effect was remarkably after 30 min. This result is in agreement with previous reports on the ERK pathway in melanogenesis [In a previous study, acteoside from the leaves of gulation . Accordiogenesis . ATG exeμM was shown to have toxicity in zebrafish embryos with developmental retardation. The results of this study imply that ATG may be used as a natural resource for the treatment of melanogenesis, which is highly important given the current increase in demand for complementary and alternative medicines.In conclusion, we first investigated the inhibitory effect of ATG on tyrosinase activity and melanin biosynthesis and elucidated the signaling mechanism. We found ATG has a significant inhibitory effect on them without producing any significant cytotoxicity. Those results indicate that ATG inhibits melanogenesis in murine melanoma B16BL6 cells by repressing cAMP and tyrosinase gene/protein expression and increasing ERK phosphorylation in the signaling pathway of melanogenesis. Likewise, inhibition of melanogenesis was also confirmed in nonneoplastic cells melan-A and in vivo zebrafish embryos. However, ATG of high concentration 100


The purpose of this study was to compare the interfragmentary compression force across a simulated scaphoid fracture by two commonly used compression screw systems; the Acutrak 2 Standard and the 3.0 mm Synthes headless compression screw.Sixteen  cadaver scaphoids were randomly assigned to receive either the Acutrak 2 or Synthes screw with the contralateral scaphoid designated to receive the opposite. Guide wires were inserted under fluoroscopic control. Following transverse osteotomy, the distal and proximal fragments were placed on either side of a custom load cell, to measure interfragmentary compression. Screws were placed under fluoroscopic control using the manufacturer's recommended surgical technique. Compressive forces were measured during screw insertion. Recording continued for an additional 60s in order to measure any loss of compression after installation was complete. The peak and final interfragmentary compression were recorded and paired t-tests performed.The mean peak compression generated by the Acutrak 2 Standard was greater than that produced by the Synthes compression screw . The mean final interfragmentary compression generated by the Acutrak 2 screw (68.6 ± 36.4 N) was significantly greater (p = 0.04) than the Synthes screw (37.2 ± 26.8 N). Specimens typically reached a steady state of compression by 120-150s after final tightening.Peak interfragmentary compression observed during screw installation was similar for both screw systems. However, the mean interfragmentary compression generated by the Acutrak 2 Standard was significantly greater. Our study demonstrates that the Synthes headless compression screw experienced a greater loss of interfragmentary compressive force from the time of installation to the final steady state compression level. The higher post-installation compression of the Acutrak 2 Standard may be attributable to the greater number of threads throughout the entire length of the screw. The clinical significance of these results, are, at this point uncertain. We do demonstrate that a fully threaded design offers a more reliable compression that may translate to more predictable bony union. The scaphoid is commonly injured, and is one of the most frequently fractured bones of the wrist . TreatmeSurgical fixation of the scaphoid is the accepted standard of care for the treatment of nonunions, delayed unions and displaced fractures ,4-6. Recin-vitro study, the Acutrak Standard screw  provided more compression than the Bold screw  and Acutrak Mini screw  [®, Westchester, PA, USA) provided reliable compression in a cadaveric model [®, Hillsbro, OR, USA). We hypothesized that the Acutrak 2 Standard screw would provide more reliable compression when compared to the Synthes headless compression screw in a cadaveric model.A variety of internal fixation systems are commercially available and have been studied for the treatment of scaphoid fractures. In an OR, USA) . As wellic model . The purEight paired  fresh frozen cadaveric scaphoids with a mean age of 75 (range 47-87) years were tested. Each scaphoid was carefully harvested, stripped of soft tissue, examined with fluoroscopy to ensure the absence of abnormalities and frozen at -20°C. Eight scaphoids were randomly assigned to receive the Synthes 3.0 mm headless compression screw, while the 8 remaining contralateral scaphoids received the Acutrak 2 screw.The Synthes screw is composed of titanium and is a headless design. It consists of a threadless central shaft with threads of differential pitch at either end promoting fracture compression. The proximal threads are dual to increase bone purchase and the screw is available in long (40% of screw length threaded) and short (20% of screw length threaded) configurations. Available sizes range from 10-40 mm for long screws and 16-40 mm for short screws . A longitudinal mark from the distal to proximal pole was then made prior to osteotomy to ensure post-osteotomy rotational alignment. The required screw length was measured, with the width of the load cell taken into account. The scaphoid was then predrilled/reamed under fluoroscopic guidance as per the manufacturer's instructions. With the guide wire removed, the scaphoid was secured to the cutting table with two Babcock clamps. A microsagittal saw was used to create an osteotomy perpendicular to the long axis of the scaphoid simulating a transverse waist fracture. The fracture was reduced with the load cell interposed between the fragments Figure . RotatioThe force of compression was continuously measured during screw insertion and continued for 180s after a steady state had been reached. Steady state was typically reached within 60-90s after peak compression was obtained, and was reached within 150s for all screws tested. The same procedure was repeated in the contralateral scaphoid using the comparison screw. Statistical analysis consisted of paired t-tests to compare the peak and final steady state compression for the two screw systems.The Acutrak 2 screw had higher measured peak and final interfragmentary compression than the Synthes screw, but this difference was only statistically significant in final compression. The mean peak compression Figure  of the AOur study has demonstrated that the Acutrak 2 screw system provides greater mean interfragmentary compression when compared to the Synthes headless compression screw in a static cadaveric scaphoid model. By using contralateral scaphoids to test each screw system, we were able to control for potential differences in bone quality between cadaveric subjects. In addition, the operating surgeon was blinded to the amount of compression generated. The surgeon was instructed to advance each screw based on tactile feedback and fluoroscopic imaging alone, replicating the intraoperative environment. While we found no statistical difference in the peak compression between the two systems, there was a statistically significant difference in the mean final interfragmentary compression .et al [et al [et al [in vitro. Currently there is no consensus on the optimum interfragmentary compression needed to promote reliable bony union and limited conclusions can be made when comparing the absolute compressive forces generated across similar studies.Similar cadaveric studies have previously been reported. A study by Lo et al  found thl [et al  utilizedl [et al  have beel [et al  measuredOne possible reason that the Acutrak 2 screw provided greater final compression than the Synthes system may relate to the thread pattern. The Acutrak 2 is a fully threaded design that generates a large amount of thread-to-bone contact area. In comparison, the Synthes screw has fewer threads, which results in less thread-to-bone contact area, and thus greater stresses on the cancellous bone. The comparatively greater loss of compression experienced by the Synthes screw, may be evidence of the gradual failing of trabeculae due the higher stresses. Maximizing the number of effective threads may help to preserve post-insertion compression. This may mean the selection of screw length is less critical in the case of the Acutrak screws, as the fully threaded design may be less sensitive to minor errors in measured screw length. It should also be noted that the increased purchase afforded by the greater thread surface area, may make removal more difficult if required.A limitation of this study is the age of the cadaveric specimens used. Although we did not formally assess bone quality, given the mean age of the specimens (75 years), we can infer that the bone quality was lower than what would be expected for the average patient with a scaphoid fracture. To address this issue, the matched pair design of this study helped to control for bone quality between the two screw systems.While our results suggest that the Acutrak 2 compression screw maintained greater compression than the Synthes screw, the clinical significance of this finding remains unclear. The literature has yet to demonstrate the amount of interfragmentary compression necessary to promote optimal fracture union. One can infer that reliable compression across the fracture site is required for predictable healing, and yet some degree of micromotion may also be advantageous. Further biological testing is needed to determine the correlation between compression, micromotion and bone biology. Additionally, the Synthes screw is a smaller screw and may cause less articular damage during and after insertion, thus leading to less post-operative pain and morbidity. Whether this is clinically significant is currently unknown and is an area of further investigation.in-vivo differences in fracture healing.Our study has demonstrated that the Synthes and Acutrak 2 screws provide similar mean peak compression in a scaphoid fracture model, but the Acutrak 2 screw generates greater mean final interfragmentary compression. Before any clinical recommendations can be made, a clinical trial is necessary to determine if the identified differences in generated compression correspond to The authors declare that they have no competing interests.The lead author on this paper is RG. She is an experienced hand surgeon who performed the surgical techniques of the study and conceived the initial idea for the study and was responsible for revision of manuscript. The second author on the paper, JA, collected and analyzed the data and drafted the original manuscript. He is also the contact author. KF is an experienced hand surgeon who conceived and performed the study. He was also responsible for revision of the manuscript. DS, LF, and JJ were responsible for manuscript review, study conception and assisting with biomechanical issues associated with our study. All authors have read and approved the final manuscript prior to resubmission.


Results demonstrate that the β-carboline compounds (1) potently reduce the expression of all three phosphorylated forms of tau protein, and (2) inhibit the DYRK1A catalyzed direct phosphorylation of tau protein on serine 396. By assaying several β-carboline compounds, we define certain chemical groups that modulate the affinity of this class of compounds for inhibition of tau phosphorylation.Harmine, a β-carboline alkaloid, is a high affinity inhibitor of the dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) protein. The DYRK1A gene is located within the Down Syndrome Critical Region (DSCR) on chromosome 21. We and others have implicated DYRK1A in the phosphorylation of tau protein on multiple sites associated with tau pathology in Down Syndrome and in Alzheimer's disease (AD). Pharmacological inhibition of this kinase may provide an opportunity to intervene therapeutically to alter the onset or progression of tau pathology in AD. Here we test the ability of harmine, and numerous additional β-carboline compounds, to inhibit the DYRK1A dependent phosphorylation of tau protein on serine 396, serine 262/serine 356 (12E8 epitope), and threonine 231 in cell culture assays and  The dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) gene is located within the Down syndrome critical region on chromosome 21. Overexpression of DYRK1A has been proposed to be a significant contributor to the underlying neurodevelopmental abnormalities associated with Down syndrome. Transgenic animals overexpressing DYRK1A show marked cognitive deficits and impairment in hippocampal dependent memory tasks 2 and 5-HT1AThe family of β-carboline alkaloids, characterized by a core indole structure and a pyridine ring, are naturally occurring compounds in some plant species that affect multiple central nervous system targets. These include the 5-hydroxytryptamine receptor substypes 5-HTIn this study, we extend previous published findings from our lab and others that show DYRK1A is involved in phosphorylation of tau protein on sites that are hyperphosphorylated during the course of tau pathology in AD and show that certain β-carboline alkaloids can significantly reduce the levels of phosphorylated tau protein. Specifically, we identify DYRK1A dependent tau phosphorylation on threonine 231 and serine 396. We further show that harmine and other β-carboline compounds inhibit DYRK1A dependent tau phosphorylation with varying affinities that are dependent upon several structural features of the molecules. These results suggest that this class of compounds warrant further investigation as candidate tau-based therapeutics to alter the onset or progression of tau dysfunction and pathology in Alzheimer's disease and other tauopathies.2. Cell lysates were prepared using the Complete Lysis-M, EDTA-free kit (Roche Applied Science) and total protein concentration was quantified using the BCA protein assay (Pierce). Westerns for the multiple forms of tau were performed as described below.4R0N tau overexpressing H4 neuroglioma cells 2. Cell lysates were then prepared using the Complete Lysis-M, EDTA-free kit (Roche Applied Science) supplemented with phosphatase inhibitor cocktails 1 and 2 (Sigma). Lysates were quantified using the BCA protein assay (Pierce). Protein from lysates  was separated on SDS-PAGE gels and transferred to nitrocellulose membrane. Membranes were blocked in 5% blocking solution for one hour at room temperature (RT). Blocking buffer solution used for detection of non phosphorylated protein contained 5% Non-fat dry milk in 1×-TBS-T . For detection of phosphorylated protein, blocking buffer solution contained 5% Bovine Serum Albumin in 1× TBS-T. Membranes were probed with primary antibody (various dilutions depending on the epitope – see below) in blocking buffer overnight at 4°C on a rocker. Membranes were subsequently washed with 1× TBS-T and probed with secondary antibody in blocking buffer for forty-five minutes using a 1∶25,000 dilution of HRP-GAM or HRP-GAR (Jackson Immunoresearch), depending on the species (mouse or rabbit) in which the primary antibodies were raised. Following incubation with secondary antibody, membranes were washed in 1× TBS-T and developed with Super Signal West Femto Maximum Sensitivity Substrate Kit (Promega) and digitally imaged. Protein band signal saturation was assessed before any further analysis of multiple forms of tau. Alpha Innotech Fluoro Chem SF imaging software verifies degrees of saturation when signal intensity is beyond the dynamic range . Protein band signal intensities used for quantification were within the instrument's dynamic range.For all cell-based experiments, including siRNA treatments and compound treatments, cells were treated for 96 hours at 37°C, with 5% COTo test multiple primary antibodies, membranes were stripped for 15 minutes at RT using ReBlot Plus Mild Antibody Stripping Solution (Millipore). Membranes were then washed for 5 minutes in 1× TBS-T and blocked for one hour in 5% blocking solution at RT. For verification of protein loading, membranes were reprobed overnight at 4°C with an anti-Tubulin primary antibody . Primary antibodies used for detection included anti-tau , 12E8 tau , pT231 tau , pS396 tau , and anti-DYRK1A .50 value for viability was identified, effects on multiple forms of tau were investigated after treating with the β-carbolines indicated in Cells undergoing any treatment, including β-carboline derivative dosing and siRNA treatment, were maintained in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 2 mM L-Glutamine (Invitrogen). Viability assays were performed using a 96-well pate format. Metabolic activity was measured 12 hours after the addition of 10% alamar Blue (Invitrogen) directly to attached cells in full medium. This assay was based on the ability of metabolically active cells to convert alamar Blue reagent into a fluorescent signal proportional to innate metabolic activity. Once the ideal ICin vitro phosphorylation assays.Evaluation of DYRK1A kinase activity was determine by incubating 0.08 ug of recombinant human DYRK1A protein (Invitrogen) with 0.15 ug of 4R2N recombinant human Tau  in 1× kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2 - Cell Signal) and 1 mM ATP in a final volume of 20 ul for 30 minutes @ 30C. For testing the effects of the β-carboline derivatives, recombinant human DYRK1A was pretreated with compounds for 10 minutes prior to the addition of kinase buffer, ATP, and recombinant human tau. The reaction was inactivated upon addition of 1× Novex LDS sample buffer and Novex sample reducing reagent, 50 mM DTT, (Invitrogen) followed immediately by heating for 10 minutes @ 95C. Phosphorylated Tau was resolved using 7% Tris Acetate gels and detected by western analysis. Westerns were probed for Phospho-Tau S396 (abcam) @ 1∶5,000 dilution and a secondary of Goat anti – Rabbit HRP (Jackson Immuno Labs) @ 1∶50,000 in 5% BSA. Membranes were stripped as above and reprobed with rabbit anti Human Total Tau (Dako) @ 1∶15,000 dilution and a secondary of Goat anti – Rabbit HRP (Jackson Immuno labs) @ 1∶100,000 dilution in 5% milk. For quantitation purposes, both bands of pS396 phosphorylated tau were used in the We previously reported that silencing of DYRK1A expression causes reduced tau phosphorylation at the 12E8 epitope (phosphorylated serines 262 and 356) 50 of ∼80 nM in an in vitro kinase assay using synthetic peptide substrate in vitro IC50 value for harmine against DYRK1A, we selected doses of 80 nM, 800 nM and 8 µM for the tau phosphorylation assays. Harmine reduced the expression of each phospho-tau species tested, including 12E8 (pS262/pS356), pT231, and pS396  Based on results for harmine, we tested additional β-carboline derivatives, including harmol, harmane, harmaline, norharmane, 9-ethylharmine, and two novel proprietary compounds MPP-021 and MPP-313 . We first performed toxicity assays for each compound in our H4 cell line . We thenin vitro phosphorylation assay with recombinant DYRK1A and tau proteins, we confirmed that DYRK1A could directly phosphorylate tau protein  and harmine (700 nM). Harmol was the third ranked compound in both the toxicity and cell-based tau assay. Reasons for this slight disconnect are unclear, but could be related to differential cellular metabolism of the free hydroxyl group on carbon 7 of harmol.We next tested each β-carboline derivative compound in this ro assay . Severaly assays , with on50 nearly 2-fold, suggesting that additional modifications on this nitrogen might increase the affinity of harmine for DYRK1A more substantially. Harmane, norharmane, and harmaline were more than an order of magnitude lower affinity than harmine, consistent with the relatively muted effects of these compounds in our cell-based tau assay  and 100 µM (640% control) (The MPP-313 compound reduced phosphorylation of the lower molecular weight form of phosphorylated tau protein with an ICcontrol) . This waPrevious literature indicates that DYRK1A can phosphorylate tau protein on T212, S202, S404, and the 12E8 epitope (S262/S356) DYRK1A can directly phosphorylate tau protein on serine 396 . Due to 50 for tau effects in our cell-based tau assay. However, because harmine significantly affected tau levels prior to observed toxicity, we conclude that the toxicity seen at higher concentrations beyond 8 µM likely results from excessive reductions to tau protein levels. This interpretation is consistent with our prior published findings that reductions to overall tau levels via siRNA knockdown of the tau transcript, and concomitant decreases to levels of phosphorylated tau, lead to significant cellular toxicity Based on our RNA interference data implicating DYRK1A in the phosphorylation of tau protein at multiple sites , we testAlthough harmine significantly reduced total tau levels, reductions to both 12E8 and pT231 phosphorylated tau remained highly significant . HoweverBased on positive results for harmine, we tested several harmine derivatives, including fully aromatic β-carboline compounds  and a dihydro-derivative . Modifications to certain structural components of the β-carboline ring structure significantly affected the ability of these compounds to inhibit tau phosphorylation. A comparison of results for harmaline and harmine indicate that a fully aromatic ring structure provides higher affinity for tau inhibition and toxicity see . CertainAnother important observation from these compound derivative studies was that correlations between tau reductions and cellular toxicity that were first observed with harmine, were also found with all of the β-carbolines tested. While we were initially hopeful that certain derivatives could separate the tau effects from the toxic effects, no compounds separated these effects. This may well result from a causative relationship between excessive tau reductions and toxicity, consistent with our prior results targeting tau expression with siRNA Pharmacologic inhibition of tau phosphorylation at certain key sites that regulate the functional activity of tau or that promote the aggregation of tau in to neurofibrillary tangles may provide a promising approach for the treatment of AD and other tauopathies. We show that the β-carboline alkaloids inhibit DYRK1A kinase activity and reduce the levels of multiple phosphorylated forms of tau protein that are important in the pathological progression of AD. Further refinement of this class of compounds on functional groups that are important determinants of their affinity for DYRK1A could lead to high affinity inhibitors of tau phosphorylation.Figure S1Quantification of the inhibition of tau phosphorylation by multiple β-carbolines. Quantification of the absolute tau phosphorylation data from the H4 cells is shown for each compound tested. Data have not been normalized to account for changes to total tau levels. Significance at p<0.05, as assessed by Student's T-test, is indicated by asterices above the error bars on the graphs. Error bars (standard deviation) from three independent replicates are shown.(TIF)Click here for additional data file.Table S1β-carboline compounds tested in this study. Shown in columns from left to right are the compound names, chemical structures, concentration resulting in 50% viability in the H4 neuroglioma cell line used in all of the cell-based tau assays.(TIF)Click here for additional data file.