Meanwhile, pEGFP-C1-CAV1 and CAV1-shRNA plasmids were transfected into hippocampal neurons for CAV-1 overexpression and silence, respectively. The serum containing RSBFL was prepared for the intervention of AD model cells. The expression of CAV-1, GSK-3β, and p-Tau in normal hippocampal neurons and AD model cells in the presence of serum containing RSBFL was evaluated. The model hippocampal neurons with AD induced by Aβ1–42 revealed an obvious CAV-1 inhibition, enhanced GSK-3β activity, and abnormal Tau phosphorylation. In contrast, the treatment with serum containing RSBFL could upregulate CAV-1 in AD hippocampal neurons (P < 0.05) with improved p-GSK-3βSer9 and reduced p-GSK-3βTyr216 (P < 0.01), as well as suppressed abnormal phosphorylation of Tau protein. Therefore, RSBFL has an excellent protective effect on hippocampal neurons through increasing CAV-1 expression, inhibiting GSK-3β activity, and reducing excessive abnormal phosphorylation of Tau protein.In order to explore the effect of root-securing and brain-fortifying Liquid- (RSBFL-) mediated caveolin-1 (CAV-1) on phosphorylation of Tau protein and to uncover underlying mechanisms of RSBFL for the prevention and treatment of Alzheimer's disease (AD), hippocampal neurons isolated from neonatal SD rats and cultured in DMEM-F12 medium were induced by exogenous A The number of NFTs is closely correlated with dementia degree of AD patients [β (GSK-3β), Cdk5, MAPK, and other protein kinases. Thr231 and Ser396 are recognized as the major phosphorylation sites of Tau in AD. Moreover, GSK-3β reveals the highest affinity and the strongest phosphorylation for Tau protein. Currently, a number of studies confirm that the activation of GSK-3β can obviously promote the phosphorylation of Tau [β could be considered as the target to explore the possible mechanisms of AD during the application of traditional Chinese medicine.Alzheimer's disease (AD) is a progressive degenerative disease of central nervous system and has two major characteristics including the formation of neurofibrillary tangles (NFTs) from the aggregates of excessively phosphorylated Tau protein in neurons  and the patients . In addis [β GSK-β, Cdk5, n of Tau . Chinese Codonopsis tangshen, Lycium barbarum, Poria cocos, Ziziphus jujuba Mill. var. spinosa, and Crataegus pinnatifida Bunge has the multiple functions of scavenging free radicals [ Codonopsis tangshen and Lycium barbarum. As a famous formula with application history of more than 20 years for fortifying brain and boosting intelligence of RSBFL, many corresponding investigations have been conducted to validate its functions and treatment efficacy. The polysaccharides from Codonopsis tangshen in this formula can execute antioxidant and antiaging effects in D-gal-induced aging mice [ Lycium barbarum can reduce methylmercury-induced hippocampal neural stem cell injury and promote the differentiation of hippocampal neural stem cells into neurons and the growth of neurons [β1–42 induction were applied to uncover the molecular mechanisms of RSBFL for the prevention and treatment of AD and the improvement of learning and memory capacity through attenuating oxidative stress and alleviating free radical damage.Root-securing and brain-fortifying liquid (RSBFL) composed ofradicals , modulatradicals , and proradicals  for conting mice  and can ing mice . Similar neurons . Our pre neurons . Similar neurons . However5 cells/well in DMEM-F12 medium with 10% FBS. Then, neuronal cells after being adhered to the bottom of plate were cultivated in fresh DMEM-F12 medium with 10% FBS, 2% B27, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and neurobasal-A at 37°C and 5% CO2 in a humidified incubation chamber. The cultivation medium was replaced by fresh medium every 3 days. The cultured hippocampal neurons were identified by immunofluorescence and Western blot.Neonatal specific pathogen-free (SPF) Sprague Dawley (SD) rats of the age of 24 h were provided by the Experimental Animal Center of Three Gorges University in China. According to the methods from previous studies , SD ratsμL of DNA was purified by using Universal DNA Purification Extraction Kit  according to the manufacturer's instructions. The pEGFP-C1 vector and purified CAV-1 gene product were subjected to double digestion by restriction endonucleases EcoR I and Sal I. The digested products were purified and harvested by electrophoresis. The CAV-1 gene was spliced into linear pEGFP-C1 vector to obtain pEGFP-C1-CAV1. The recombinant pEGFP-C1-CAV1 plasmid was subjected to extraction, double digestion by EcoR I and Sal I, identification, and sequence analysis. The validated pEGFP-C1-CAV1 plasmid was transferred into hippocampal neuronal cells for coming experiments.According to gene sequence of CAV-1 from rats in Gene Bank , the primers of CAV-1 for polymerase chain reaction (PCR) amplification were synthesized in Shanghai Sangon Biotech Co., Ltd. . Total RNA was extracted from rat brain through Total RNA Extraction Kit  according to the manufacturer's instructions. The reverse transcription reaction of the extracted RNA was conducted by using reverse transcription kit  to obtain cDNA according to the manufacturer's protocols. Totally 30 According to the previous report , the haiTotally 60 SPF SD rats of the age of 4 months were randomly divided into three groups: normal control, RSBFL, and donepezil groups with 20 rats in each group. RSBFL and donepezil were purchased from the Affiliated Hospital of Hubei University for Nationalities and Eisai China Inc. The rats were subjected to regular gavage with RSBFL and donepezil at the doses of 31 g/kg·bw  and 2.25 g/kg·bw every day, respectively. The rats from normal control group were subjected to identical volume of distilled water every day. After drug administration for 1 week, the sera were collected from abdominal aortic artery of the rats for future use.β1–42  was diluted with distilled water and incubated in 37°C water bath for 3 d to prepare the aggregates of Aβ1–42 to 125 mmol/L as the stock solution. Then the stock solution was added to the cultured hippocampal neurons to a final concentration of 2.5 mmol/L. The viability of hippocampal neurons subjected to Aβ1–42 treatment was evaluated by MTT assay and Aβ1–42-induced p-Tau level in hippocampal neurons was also determined.A2 in a humidified incubation chamber and then divided into 5 groups including normal neuron cultivation medium as the normal control (NC), normal neuron cultivation medium plus 10% serum without any drugs as the blank (B), normal neuron cultivation medium coupled with Aβ1–42 plus 10% RSBFL-containing serum (RSBFL), normal neuron cultivation medium coupled with Aβ1–42 plus 10% donepezil-containing serum (Don), and normal neuron cultivation medium coupled with Aβ1–42 plus 10% serum without any drugs as the AD model (Mod) groups. After intervention by RSBFL- or donepezil-containing serum for 12 h, the cells were harvested for the evaluation of CAV-1, GSK-3β, and Tau protein expression by Western blot.The established model cells with AD were cultivated in DMEM-F12 medium with 10% FBS, 2% B27, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and neurobasal-A at 37°C and 5% COβ (Ser9), p-GSK-3β (Tyr216), and GAPDH , as well as sequential secondary antibody. ECL luminescence was used for imaging, and finally the optical density of the band was analyzed by gel imaging system .The extracted protein from hippocampal neurons in each group was analyzed by Western blot. The protein was quantified by BCA Protein Assay Kit  according to BCA (bicinchoninic acid) method. The proteins were separated in 4–6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred on a PVDF membrane. The membrane was blocked with 5% nonfat milk. The target protein was probed with p-Tau (Thr231) and caveolin-1  and p-Tau (Ser396), p-GSK-3 P < 0.05 and P < 0.01, respectively.Image analysis system Image-Pro plus software was used for the semiquantitative analysis of the results from Western blot and immunofluorescence. All resultant data were expressed as mean ± standard deviation (M ± SD). The data comparison between groups was conducted by using single factor analysis of variance (one-way ANOVA) through SPSS 18.0 software. The statistically significant difference and very statistically significant difference were considered atμL plasmid to 7.5 μL liposome could result in the strongest fluorescence intensity in transfected cells at the largest population  with the best MOI value of 10. Meanwhile, the constructed plasmid revealed excellent expression in targeted hippocampal neurons. The MOI of 100 could lead to the highest GFP expression due to the strongest fluorescence intensity and the largest amount of fluorescent cells , as well as the obviously enhanced expression of p-GSK-3βTyr216, p-TauThr231, and p-TauSer396. In contrast, hippocampal neurons transfected with pEGFP-C1-CAV1 plasmid exhibited the upregulated CAV-1 and p-GSK-3βSer9 (P < 0.01) and downregulated p-GSK-3βTyr216 (P < 0.01), p-TauThr231 (P < 0.05), and p-TauSer396 (P < 0.05) when compared with normal hippocampal neurons.Similarly, as shown in β at the sites of Ser9 and Tyr216, respectively, as well as leading to the increased phosphorylation of Tau at the sites of Thr231 and Ser396. These results indicated that RSBFL could promote the expression of CAV-1, inhibit the activity of GSK-3β, and reduce the phosphorylation of Tau.According to the statistical analysis, CAV-1 gene silence revealed an ideal effect. On the other hand, the serum containing SRBFL could rescue the expression of CAV-1 in the presence of CAV1-shRNA and cause the reduced and enhanced phosphorylation of GSK-3β, and p-Tau in collected hippocampal neurons was evaluated by Western blot. As shown in β1–42 induction . When compared with donepezil and control groups, RSBFL could reinforce the expression of CAV-1 (P < 0.05). On the other hand, AD cell model exhibited the decreased expression of CAV-1 (P < 0.05) and p-GSK-3βSer9 (P < 0.01) and the increased expression of p-GSK-3βTyr216 (P < 0.01), as well as the enhanced phosphorylation of Tau protein at sites of Thr231 and Ser396 (P < 0.01).Similarly, as shown in β at the site of Ser9 and reduce the phosphorylation of GSK-3β at the site of Tyr216, as well as suppressing the phosphorylation of Tau protein at Thr231 and Ser9 sites.Based on the above analysis, the intervention using RSBFL and donepezil can improve the phosphorylation of GSK-3β. Moreover, the mice with Tau knockout can prevent Aβ toxicity due to the low expression level of Tau [β as a critical protein kinase for Tau can phosphorylate APP in cells, thus effectively suppressing the generation of Aβ [Alzheimer's disease is a common neurodegenerative disease, mainly characterized by excessive abnormal phosphorylation of Tau protein, thus leading to the formation of neurofibrillary tangles (NFT) and senile plaques (SP) caused by APP protein metabolic disorders, free radical damage, and nerve cell apoptosis , which fl of Tau , 19. Anoon of Aβ . These sβ25–35 and Aβ1–42 are incubated with rat primary neurons, Aβ25–35 does not reveal the significant change in the morphology of neurons, while Aβ1–42 can result in the morphologic change of neurons. Accordingly, we used sterile triple-distilled water to dilute Aβ1–42 to a final concentration of 2.5 mmol/L. The diluted Aβ1–42 was incubated at 37°C for 72 h to form an aggregation state and was used to induce the damage of hippocampal neurons for 12 h. This cell model can represent the neurotoxic effect during the pathogenesis of AD and the inducing process of abnormal phosphorylation of Tau protein. Thus, this model is a successful AD cell model in vitro. In addition, a report has demonstrated a vesicular structure due to the retraction of plasma membrane in cells, and this nest vesicle is also named as Caveolae. CAV is the marker protein of Caveolae [β-APP and β-secretase can be localized to Caveolae and result in decreased β-amyloid production, thus suggesting the protective role of CAV-1 [β in CAV-1 knockout mice, whose characteristics are similar with the pathological changes of AD; therefore, CAV-1 knockout accelerates neurodegeneration and aging. If mouse primary neurons with CAV-1 knockout are subjected to the transfection of CAV-1 plasmid, the neurons can result in a significant decrease of Aβ level. Trushina et al. [Casal et al.  have fouCaveolae . When CAof CAV-1 . Head etof CAV-1  have alsa et al.  have conβ is considered as the most powerful kinase [β presents as an active state. Therefore, the formation of paired helical fiber (PHF) may be correlated with the active state of GSK-3β, suggesting that excessive abnormal phosphorylation of Tau protein may be associated with GSK-3β activity [β to result in excessive abnormal phosphorylation [β activity can be improved as the increase of intracellular calcium ion concentration, thus sequentially increasing the level of p-Tau. In addition, miR-12-3p with the function of direct targeting to CAV-1 can suppress abnormal hyperphosphorylation of Tau by regulating CAV-1-PI3K/Akt/GSK3β signal pathway in AD [β activity is not clear. Through our systematic investigations, in AD hippocampal neurons, Aβ1–42 could result in the fracture of a large of number of protrusions, serious disintegration of cell bodies, and poor growth state. On the other hand, RSBFL treatment can execute the protective role in hippocampal neurons subjected to Aβ1–42 induction, thus greatly improving cell growth state, delaying cell senescence and apoptosis through inhibiting GSK-3β activity. At the same time, RSBFL treatment can reduce abnormal phosphorylation of Tau protein at Thr231 and Ser9 sites, which further suggests the function of RSBFL to suppress excessive abnormal phosphorylation of Tau and reduce the expression level of p-Tau in AD cells. All of these results indicate that RSBFL may accomplish the protection of hippocampal neurons and exert normal physiological functions through modulating the balance between phosphorylation and dephosphorylation, maintaining the stability of microtubules, and regulating the balance of cytoskeleton. New evidence suggests that endoplasmic reticulum (ER) stress will cause extensive phosphorylation of Tau protein. The excessive abnormal phosphorylation of Tau protein will also cause ER stress, which is a pathological feedback loop [β activity, thereby reducing excessive abnormal phosphorylation of Tau protein and executing the neuroprotective role in AD.Tau reveals the highest content in microtubule-associated proteins. Under the physiological state, Tau can bind to tubulin to promote the polymerization of tubulin proteins into microtubules and inhibit its dissociation. Tau also can further bind with microtubules to promote their assembly for enhancing their stability . Excessil kinase . Previouactivity . Severalrylation . Hartigarylation  have fouay in AD . Howeverack loop . Therefoβ activity, and inhibiting excessive abnormal phosphorylation of Tau protein may be one of the molecular mechanisms of RSBFL for the prevention and treatment of AD. In addition, whether RSBFL can execute the prevention and treatment of AD through suppressing neuronal apoptosis in hippocampal tissues needs to be further explored.In summary, upregulating CAV-1 expression, reducing GSK-3


Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function. The activity of proteins can be finely and reversibly tuned by post-translational modifications. The attachment of phosphate groups to tyrosine residues is one of such modifications. While the existence of extracellular phosphoproteins has been known, the functional significance of extracellular phosphorylation is poorly understood. Here we describe a single extracellular tyrosine whose inducible phosphorylation may represent an archetype for a new class of mechanism mediating protein—protein interaction and regulating protein function. We show that the interaction between EphB2—which occurs upon receptor activation by its ligand ephrin-B—and the N-methyl-D-aspartate receptor (NMDAR) depends on extracellular phosphorylation of EphB2. This interaction regulates the localization of the NMDA receptor to synaptic sites in neurons. In vivo, EphB2 is phosphorylated in response to injury, and the subsequent up-regulation of the interaction between EphB2 and NMDA receptors enhances injury-induced pain behavior and mechanical hypersensitivity in mice. Importantly, our study defines a specific extracellular phosphorylation event as a mechanism driving protein interaction and suggests that extracellular phosphorylation of proteins is an underappreciated mechanism contributing to the development and function of the nervous system and synapse. Modification of protein function by phosphorylation controls many aspects of cellular function and signaling . InteresAt excitatory synapses, glutamate receptors must be recruited and stabilized at synaptic sites. Of particular importance are interactions that maintain the proper localization of N-methyl-D-aspartate receptors (NMDARs), glutamate receptors that are essential for synaptic plasticity and development . The synThe direct extracellular interaction between the EphB receptor tyrosine kinase and the NMDAR appears to play important roles in the localization, function, and signaling of NMDARs . The EphUnderscoring the importance of the EphB—NMDAR interaction, the EphB—NMDAR interaction has been linked to a number of human diseases that are associated with NMDAR dysfunction. The pathological disruption of the ability of the EphB and NMDAR to interact has been linked to NMDAR dysfunction in Alzheimer disease , 20 and Here we show that the EphB2 receptor tyrosine kinase undergoes ephrin-B ligand-induced extracellular phosphorylation of tyrosine residues (Y481 and Y504). Sequence analysis indicates that 1 of these amino acids, Y504, is widely conserved in fibronectin type III (FN3) domains of Eph proteins across phylogeny and is present in all Eph proteins known to interact with the NMDAR. In cortical and spinal cord neurons, ephrin-B—dependent induction of the EphB—NMDAR interaction is mediated by extracellular phosphorylation of Y504 on EphB2. The charge of Y504 is both necessary and sufficient for EphB—NMDAR interaction and regulates the amount of NMDARs found at synaptic sites. Virally mediated spinal cord expression of EphB2 or a phosphomimetic EphB mutant that induces the EphB—NMDAR interaction increases NMDAR levels in the dorsal horn of the spinal cord and results in mechanical hypersensitivity. Intrathecal injections of a membrane-impermeable ectokinase inhibitor that blocks the EphB—NMDAR interaction in cortical and spinal cord neurons reduces long-term hypersensitivity induced by EphB2 wild type (WT) but not pathological pain induced by injection of a phosphomimetic EphB mutant. These findings suggest that extracellular phosphorylation of EphB2 regulates NMDAR synaptic localization and function. Together the data suggest extracellular phosphorylation as a novel, dynamic mechanism that regulates protein—protein interactions at synapses to drive assembly of macromolecular complexes.2, LC-MS/MS identified the known tyrosine phosphopeptides in the juxtamembrane and kinase domains (YNATAIK [Y481] and AGAIYVFQVR [Y504]) from the extracellular portion of EphB2 (http://www.phosphosite.org), further supporting our analysis.EphB receptors interact directly with NMDA-type glutamate receptors through an undefined region of their extracellular domain . The ext domains  of EphB2of EphB2 . Each exY504 and neighboring amino acid residues in the identified peptide are well conserved among different species  , within Our analysis of the F-strand region of cFN3 domains indicates that Y504 is well conserved in proteins that contain homologous domains , 26. TheTo begin to determine whether Y504 might be phosphorylated, we generated a polyclonal phospho-specific antibody to tyrosine 504 of EphB2. Recognition by the phospho-Y504 antibody (p*Y504) was blocked by preabsorption with the immunogenic phospho-Y504 peptide . In addiTo test whether Y504 phosphorylation of EphB2 is enriched in the cortex and spinal cord, we purified synaptosomes from these regions of WT mice. To validate our synaptosome purification, blots were probed for EphB2, PSD-95, and GluN1 . The EphFinally, to begin to address whether phosphorylation of Y504 on EphB2 might change as synapses mature, we asked whether the phosphorylation of Y504 was developmentally regulated. Synaptosomes were purified from P9, P15, and P21 WT mouse brains and probed for Y504 phosphorylation. We found that the levels of p*Y504 were elevated at P9 and P15 and declined with age. Interestingly, the pattern of p*Y504 parallels decreases in the proportion of NMDARs containing the developmentally regulated NMDAR subunit GluN2B  28, 29], 2928, 2To begin to examine the mechanism of EphB2 phosphorylation, we next asked whether EphBs might be phosphorylated on the cell surface after ephrin-B2 stimulation. Cultured cortical neurons were treated with activated ephrin-B2 for 45–60 minutes and then lysates were probed with the p*Y504 antibody. Consistent with the ephrin-B2-dependent activation of EphB2, ephrin-B2 treatment led to a significant increase in Y504 phosphorylation . BlockadEphB2 can be internalized by clathrin-mediated endocytosis after ephrin-B stimulation . To blocp < 0.01, ANOVA followed by Dunn's multiple comparison test).To further test whether the extracellular domain of EphB2 was phosphorylated on the surface of neurons, we conducted live-cell cell surface staining experiments. Because our Y504 phospho-specific antibody is not suitable for immunostaining, these experiments were conducted with the broad-spectrum and well-characterized phosphotyrosine antibody PY99. To induce Y504 phosphorylation, day in vitro (DIV) 7–16 cultured neurons were stimulated with activated soluble ephrin-B2 for 45 minutes, and live-cell staining was conducted with α-phosphotyrosine (PY99) or control antibodies recognizing intracellular proteins. As expected, after treatment with ephrin-B2 or control reagents, live-cell staining with intracellular α-actin antibodies only showed weak background staining . In contp < 0.05, ANOVA followed by Dunn's multiple comparison test). These findings suggest that the majority of the surface phosphotyrosine signal induced by ephrin-B2 treatment can be attributed to phosphorylation at Y504.We next asked whether any phosphotyrosine staining might be due to phosphorylation of Y504. To test this, cultured neurons were transfected at DIV 3 with either EphB2 WT or EphB2 Y504F. After ephrin-B2 treatment, surface staining with PY99 resulted in a significant increase in phosphotyrosine staining in the EphB2 WT-transfected neurons but not Y504F-transfected neurons . Moreover, preincubation with the phosphatase inhibitor vanadate potentiated the ephrin-B—induced increase in surface PY99 staining of ephrin-B2 binding sites . These findings suggest that ephrin-B2 stimulation of EphBs in neurons results in the tyrosine phosphorylation of the EphB ectodomain in the extracellular space.To begin to determine whether EphB2 might be phosphorylated by a kinase outside the cell, we blocked kinase activity selectively in the extracellular space by pretreating neurons with a membrane-impermeable ectokinase inhibitor, K252b . In contp < 0.005, ANOVA followed by Fisher’s exact test). This activity was absent in control ACSF with ATP added or ephrin-B2–treated media without ATP and blocked by heating the ephrin-B2–treated ACSF to 73°C  for 1 hour before ephrin-B treatment, and the effect on the EphB—NMDAR interaction was determined by co-IP. Bath application of ATPγS significantly reduced the ephrin-B—induced EphB—NMDAR interaction without affecting intracellular tyrosine phosphorylation of EphB2 , suggesting that the structure of the extracellular domain remains intact after mutation of this residue. In HEK293T cells, mutations to Y504 modulated the rate of removal of EphB2 from the cell surface; however, these effects were not seen in neurons . Similar effects were observed when antibodies against FLAG (FLAG-EphB2 or FLAG-EphB2 mutants Y504E or Y504F) were used to immunoprecipitate GluN1 (Mutation of the second phosphotyrosine identified by MS (Y481) had no effect on the ability of EphB2 to interact with the NMDAR, suggesting that this amino acid is not required for the EphB—NMDAR interaction . In contte GluN1 . These rp = 0.0056, ANOVA followed by Fisher’s exact test). In contrast, neurons transduced with Y504E mutant EphB2 showed robust GluN1 pull-down even in the absence of ephrin-B treatment , and ephrin-B treatment of neurons transduced with EYFP-tagged EphB2 Y504E did not further increase the EphB—NMDAR interaction . These findings suggest that Y504 phosphorylation is sufficient to induce the EphB2–NMDAR interaction in neurons. Neurons transduced with the Y504F mutant form of EphB2 failed to show GluN1 co-IP with EphB2, either with or without ephrin-B treatment . These data indicate that phosphorylated EphB2 Y504 is both necessary and sufficient for the EphB—NMDAR interaction in neurons and suggest that extracellular phosphorylation of a tyrosine in the cFN3 domain of EphBs may drive protein—protein interactions.To test the effect of EphB2 Y504 mutants in neurons, DIV 2 cultured cortical neurons were transduced with lentiviruses expressing enhanced yellow fluorescent protein (EYFP)-tagged EphB2 WT, EphB2-Y504E, or EphB2-Y504F. Neurons were then challenged with ephrin-B2 treatment at DIV 9 for 45–60 minutes, and the ability of the GluN1 subunit of the NMDAR to co-IP with EphB2 was tested. In the control group, cultured neurons were transduced with EphB2 WT. In these neurons, little EphB—NMDAR interaction was detectable without ephrin-B stimulation, but ephrin-B stimulation induced a significant increase in the co-IP of GluN1 with EYFP-tagged EphB2 WT , while the sequences of EphA3 and EphA4, which do not interact with the NMDAR, differ . Interesp < 0.001, ANOVA followed by Fisher’s exact test). In addition, the amplitude of Y504E-expressing neurons was significantly higher than that of WT-expressing neurons .Early in development, EphBs function to control synapse formation , but latp < 0.01; ANOVA followed by Fisher’s exact test). Cumulative probability histograms of events from neurons transfected with EphB2 Y504E or EphB2 Y504F indicate that the NMDAR-dependent component of mEPSC amplitude is greater in EphB2 Y504E than in EphB2 Y504F—expressing neurons .To determine whether these effects were due to changes to the NMDAR at synaptic sites, spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presence of tetrodotoxin and blockers of GABAergic channels. Consistent with previous work , no signp < 0.001, K–S test). However, Ro-25 treatment had no significant effect on mEPSC amplitude in Y504F-overexspressing neurons  microscopy of FLAG-EphB2 or FLAG-EphB2 mutants (Y504E and Y504F) and endogenous GluN1 combined with 2-color confocal imaging of vGlut1 and cell-filling EGFP expressed in DIV 21–23 cortical neurons . EphBs ap < 0.05, **p < 0.01, ANOVA followed by Fisher’s exact test; Using 2-color STED super-resolution imaging, we determined the proportion of vGlut1-positive spines containing FLAG-EphB2 and GluN1 puncta. Approximately 30% of spines contained FLAG-EphB2 WT puncta that colocalized with 1 or more puncta of GluN1 . We nextDespite normal levels of NMDARs in EphB triple-knockout mice, in these animals NMDARs are redistributed from synaptic sites to extrasynaptic locations, suggesting that EphBs play an important role in regulating NMDAR localization . To testp < 0.05, ANOVA followed by Tukey’s range test; p < 0.0005, ANOVA followed by Tukey’s range test; The superficial nociceptive layers of the dorsal horn are vGlut1-negative and vGlut2-positive , 37. To p < 0.0001, ANOVA followed by Dunnett’s multiple comparison test; Ephrin-B—EphB signaling to the NMDAR is implicated in pain plasticity, leading to pathological pain states , 40. GivBecause EphB1 is expressed more highly than EphB2 in the spinal cord and is known to interact with the NMDAR  23, 42,,42, 43, p < 0.05, Mann—Whitney U test, 8 hemispinal cords pooled per sample, n = 3, Next, we asked whether injury-induced mechanical hypersensitivity might induce the EphB—NMDAR interaction . ConsistNext, to begin to test whether extracellular phosphorylation might be important for the behavioral response to injury, we tested whether blockade of extracellular kinase activity with the membrane-impermeant drug K252b might rescue injury-induced mechanical hypersensitivity . We usedTo begin to test whether the EphB—NMDAR interaction might play a role in pathological pain, we asked whether inducing the EphB—NMDAR interaction with transduction of EYFP-tagged EphB2 WT or Y504E that results in increased levels of NMDAR in the spinal cord  might rep < 0.01, 2-way ANOVA followed by Dunnett’s multiple comparison test; Injections of lentivirus transducing WT EphB2 and EphB2-Y504E resulted in long-lasting mechanical hypersensitivity, even 2 months after virus injection . To testWe next conducted a series of controls. Although we did not observe evidence of action of K252b outside of the spinal cord, and intrathecal infusion of drugs is used clinically to avoid side effects seen with other injection methods , the druRegulation of cell function and animal behavior by phosphorylation of the intracellular portions of proteins has long been appreciated. Here we show that the ligand-induced phosphorylation of the extracellular domains of proteins may have similarly important impacts on synaptic function and animal behavior. Extracellular tyrosine phosphorylation of EphB2 occurs on a specific well-conserved residue within the FN3 repeat domain. We show that the direct extracellular interaction between EphBs and NMDARs regulates recruitment of NMDARs to synaptic sites and that phosphorylation of Y504 on EphB2 is likely to impact the maintenance of injury-induced mechanical hypersensitivity. Taken together, our findings suggest that extracellular phosphorylation of EphB cFN3 is a critical regulator of synaptic NMDAR function and has implications for understanding synaptic plasticity and disease, especially pathological pain.Drosophila wing development, neurite outgrowth, osteoclast migration, and aggregation of amyloid beta [Phosphorylation of protein ectodomains can regulate oid beta –49; howeoid beta  and ATP oid beta , 52. TogIntracellular phosphorylation sites enable protein—protein interactions with specific amino acid motifs. Similarly, the conserved region surrounding the Y504 residue on EphB2 appears to define a new extracellular interaction domain. Based on homology, we could accurately predict that EphA8 would also interact with the NMDAR. In contrast, the corresponding regions of EphA3 and EphA4 that do not co-IP with the NMDAR are less homologous to EphB2 , althougHow might extracellular phosphorylation enable protein—protein interaction? In the intracellular space, changes in the charge of tyrosine residues in specific amino acid motifs control the binding of specific SH2 domain—containing molecules . InteresExtracellular phosphorylation of EphB2 at Y504 stabilizes GluN2B-containing NMDARs on the cell surface and drives the accumulation of GluN2B-containing NMDARs at synaptic sites in young neurons. During development, GluN2B-containing NMDARs predominate at synaptic sites, and the presence of GluN2B-containing NMDARs at synapses can enhance synaptic plasticity . InteresThe kinase regulating the phosphorylation of Y504 on EphB2 remains to be identified. Interestingly, Y504 is not phosphorylated on the kinase-dead version of EphB2 (EphB2 KD) implicating the kinase domain of EphB2. It is not likely, though, that the ephrin-B—induced extracellular phosphorylation of EphB2 Y504 is phosphorylated directly by the EphB2 kinase because (1) p*Y504 requires extracellular ATP, (2) p*Y504 occurs on the cell surface, (3) p*Y504 is blocked by a membrane-impermeable kinase inhibitor K252b, (4) extracellular treatment with phosphatases augments the interaction, (5) an activity that can phosphorylate Y504 is secreted following ephrin-B treatment, and 6) although K252b effectively inhibited p*Y504, it failed to block EphB2 kinase activity. However, while the EphB2 kinase is unlikely directly responsible for phosphorylating Y504, given that p*Y504 requires ephrin-B activation of EphB2, it is likely that the EphB2 kinase participates in the signal transduction pathway necessary for p*Y504. Further work will be needed to identify the genuine Y504 kinase. One interesting set of candidates is the recently identified family of extracellular kinases (such as Fam20C) that function in the secretion pathway . A secon althoughWhile disorders like Alzheimer disease and NMDAR-encephalitis may be linked to pathological reduction of the EphB—NMDAR interaction , 21, indIn addition to functions in the induction of pathological pain, we find that the EphB—NMDAR interaction also appears to play an important role in maintaining long-lasting mechanical hypersensitivity. Virally induced expression of WT EphB2 in dorsal horn neurons results in sustained mechanical hypersensitivity. Importantly, injection of K252b 2 months after induction by an EphB2-expressing virus rapidly alleviates this mechanical hypersensitivity. These findings suggest that inhibition of extracellular phosphorylation might provide a target suitable to alleviate more chronic pain states.In animals injected with virus transducing the EphB2 Y504E point mutant that induces a constitutive, ligand-independent interaction with the NMDARs, injection of K252b did not influence this established mechanical hypersensitivity. This underscores the specific, local effect of extracellular EphB2 phosphorylation. Consistent with clinical practices , our finAll animal procedures were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University, the University of Pennsylvania, the University of Texas at Dallas, and the University of Arizona and were in accordance with International Association for the Study of Pain guidelines . Embryonic day 17 (E17) to E18 Long—Evans rats (Charles River) were used for preparing dissociated cortical neural culture as previously described , 14. Pos2, LC-MS/MS analysis was conducted (Thermo Fisher Scientific LTQ-Orbitrap).For LC-MS/MS analysis, NG108 cells were transfected with FLAG-tagged EphB2 receptor, treated with ephrin-B1, and lysed. Samples immunoprecipitated with α-FLAG antibody were separated by SDS-PAGE, the gels were stained with Coomassie Blue, and the EphB2 band was excised and digested with trypsin in-gel. After enrichment of phosphopeptides using TiOGeneration of FLAG-tagged full-length EphB2, truncated EphB2 (fEphB2 Tr), and kinase-dead  EphB2 were previously described . GeneratHEK293T cells were transfected using the calcium phosphate method. Dissociated cortical neurons were prepared from embryonic day 17 (E17) to E18 rats. Dissociated spinal cord neurons were prepared from E14.5 rats. Cultured neurons were transfected at DIV 3 or 14 using Lipofecatmine 2000. Immunoprecipitations and western blot analysis were performed as previously described with small changes . Cell-suFor phosphotyrosine surface staining, live neurons were incubated for 10 minutes at room temperature in PBS with α-phosphotyrosine, then washed and fixed. For synaptic NMDAR staining, cells were fixed and permeabilized with 0.1% saponin at room temperature. For immunohistochemical analysis, spinal cord tissues were frozen in OCT compound and cut into sections and then fixed and stained. Images were obtained using Leica TCS SP5 confocal scanning microscopy and Leica TCS SP8 STED microscopy. Analysis was done using NIH ImageJ. All data shown as mean ± SEM.2 .Phospho-specific antibodies against α-EphB2 Y504 were generated against a phosphopeptide of the sequence Ac-CKGLKAGAI-pY-VGQVRA-NHSynaptosomes were prepared as previously described .Electrophysiological recordings from DIV 21–23 cultured rat cortical neurons were performed using whole-cell patch methods as previously described . Evoked Behavioral testing and drug administration were performed as described previously .S1 Fig(A-D) Validation of co-immunoprecipitation for mass spectrometry. Lysates of NG108 cells expressing FLAG-tagged EphB2 treated without or with clustered Fc or ephrin-B1 were immunoprecipitated with anti-FLAG or PY99. Western blotting with α-FLAG or α-PY99 showed that transfected FLAG-tagged EphB2 (arrow heads in A and D) as well as other protein (arrow in B) were phosphorylated on tyrosine residues after treatment with clustered ephrin B1- Fc. The total amount of EphB2 was decreased after ephrin-B1 treatment (arrow head in C). (E-F) Alignment of cFN3 in EphB2 with Eph Family Proteins and FN3-containing molecules. (E) Alignment of EphB2 cFN3 domain in vertebrates and Eph in invertebrates using ClustalW2 software. EphB2 Y504 (red) corresponds to a conserved tyrosine residue whereas Y481 (blue) is only conserved in mammals (gray). (F) Alignment of EphB2 cFN3 domain with other FN3- containing proteins with phosphorylated tyrosine residues that are comparable to EphB2 Y504. (G) Untransfected cultured rat cortical neurons (DIV 6–10) were treated with ephrin-B2 (eB2) or control reagents (Fc) for 45–60 minutes. Endogenous EphB2 was immunoprecipitated using α-EphB2 antibodies and blots were probed with α-EphB2 p*Y504 pre-absorbed with non-phospho- or phospho-EphB2 Y504 peptide (top blots) or α-EphB2 extracellular region (bottom blots). Preabsorption of α-EphB2 Y504 with phospho- but not non-phospho-peptide eliminates the EphB2 p*Y504 signals induced by ephrin-B2-treament. Similar results were obtained in three separate experiments. (H) Top blot shows HEK293T lysates probed with a phospho-specific antibody generated against EphB2 Y504 (α-EphB2 p*Y504). Second blot shows same lysates probed for EphB2. Third blot shows lysates probed for EphB2 pY662 (EphB2 kinase activity). Bottom blot shows lysates probed for tubulin loading control. Lanes were loaded with lysates of HEK293T cells transfected with control, (WT) full-length EphB2, or full-length EphB2 Y481F. (I) Full blots of synaptosomes probed for α-p*Y504 in (PDF)Click here for additional data file.S2 FigIn vitro kinase assay revealed that autophosphorylation of intracellular fragments of EphB2 containing tyrosine kinase domain induced by ATP is inhibited by 4 μM PD161570 (PD) in culture medium  or MOPS buffer, but not inhibited by membrane-impermeable kinase inhibitor K252b (10 uM). All lanes are from the same blot with irrelevant intervening lanes removed. (B) Although pretreatment with PD161570 (PD) inhibits both extracellular and intracellular tyrosine phosphorylation of EphB2 induced by ephrin-B2-treatment, presence of this inhibitor following lysis did not affect phosphorylation of EphB2. (C) Sucrose treatment inhibited internalization of ephrin-B2-Fc in cultured cortical neurons. To strip ephrin-B2-Fc binding to membrane surface and only detect the internalized ephrin-B2-Fc, neurons were incubated with 0.2 M acetic acid and 0.5 M NaCl on ice followed by washes with ACSF. Intracellular without or with surface ephrin-B2-Fc was probed for human IgG (ephrinB2-Fc) or actin (control). (D) Quantification of effects of pretreatment with 450 mM sucrose significantly inhibited incorporation of ephrin-B2-Fc into cultured cortical neurons . (E) Regular immunocytochemistry (ICC) and surface staining of living neurons with α-actin revealed weak background staining in a living condition. (F) Ephrin-B2-treated conditioned ACSF in cultured cortical neurons was treated without (-) or with (+) 73–75°C for 20–30 min (+) prior to being used for kinase assay. Upper blots were probed for phospho-tyrosine. Bottom blots were probed for EphB2. (G) Quantification of tyrosine phosphorylation of EphB2-Fc. Heat treatment significantly reduced tyrosine kinase activity present in ephrin-B2-treated conditioned ACSF in cultured cortical neurons .(A) (PDF)Click here for additional data file.S3 Figp < 0.001, ANOVA followed by Fisher’s, n = 5 experiments). (D) Quantification of the ratio of GluN1 pulled down with EphB2 and GluN1 levels in the input .(A) Quantification of the effects of ephrin-B2 treatment after blockade of extracellular kinase activity with K252b on the phosphorylation of Y662 in neurons . No significant difference was found (p > 0.05) between ephrin-B2 treated samples. (B) Untransfected cultured rat cortical neurons (DIV 6–7) were treated with ephrin-B2 (+) or control reagents (-) for 45–60 minutes and either control (C) or ATPγS applied to the extracellular space (1 μM). EphB2 was immunoprecipitated and blots were probed for GluN1 (top blots). Lower blots are input controls showing GluN1 staining, EphB2 pY662, EphB2, and tubulin. (C) Quantification of the ratio of EphB2 pY662 to total EphB2. (****(PDF)Click here for additional data file.S4 Fig(A) Quantification of ephrin-B2-Fc binding to surface FLAG-tagged EphB2 WT and Y504E and Y504F mutants . (B) Regular immunocytochemistry (ICC) and surface staining of living HEK293T cells expressing FLAG-tagged EphB2 WT with α-actin revealed weak background staining in a living condition. (C) Images of HEK293T without transfection, incubated with ephrin-B2 for 45 minutes, stained as in . (D) Ima(PDF)Click here for additional data file.S5 Fig(A) Model of how extracellular phosphorylation at Y504 modulates EphB receptor surface retention and the EphB-NMDAR interaction. (B and C) EphB2 Y504 regulates surface retention of NMDAR in HEK293T cells. Co-expression of EphB2 Y504E with GluN1 and GluN2B significantly increased the fraction of surface GluN1 receptors on the plasma membrane compared to HEK293T cells transfected with GluN1 and GluN2B alone or with EphB2 WT or Y504F . In contrast, co-expression of non-phosphorylatable Y504F mutant EphB2 receptors that fail to interact with NMDARs resulted in a significant decrease of GluN1 receptors on the cell surface compared to WT EphB2 (p = 0.0272). (D and E) Co-expression of NMDAR increases the surface retention of EphB2 Y504E mutant in HEK293T cells . (F and G) Cultured neurons (DIV 7) infected with EphB2 YFP or Y504 mutants at DIV 2 were used to detect the surface level of EphB2 WT or Y504 mutants. In neurons, Y504E mutants are retained on the plasma membrane .(PDF)Click here for additional data file.S6 Fig(A) Effects of EphB2 Y504 mutation on the mEPSC frequency recorded at -65 mV in mature cultured cortical neurons. Whole-cell patch-clamp recordings from DIV 21–23 cultured rat cortical neurons transfected with EGFP and control, EphB2-YFP-WT, Y504E or Y504F. There were no significant differences between control (9 neurons) and EphB2 WT , Y504E  or Y504F  mutants in mean mEPSC frequency. Data are shown as mean ± s.e.m. (B) Example whole-cell patch-clamp recordings from cortical neurons in each condition showing the mEPSC frequency. (C) Quantification of mean mEPSC amplitude. An increase in mEPSC amplitude recorded at -65 mV was observed with overexpression of EphB2 WT  and Y504E  and Y504F  in compared with control neurons (1366 events from 10 neurons). In addition, mEPSC amplitude of neurons expressing Y504F was significantly lower than neurons expressing EphB2 WT or Y504E . Data are shown as mean ± s.e.m. (D) Sample traces of whole cell patch-clamp recording at -65 mV shows that mEPSC amplitude of neurons expressing EphB2 WT and Y504E, but not Y504F are higher than control neurons. (E) Effects of overexpression of EphB2 WT and Y504 mutants without or with APV on the mean amplitude of mEPSC (15–25 msec after the mEPSC peak) in mature cortical rat DIV 21–23 neurons. Neurons were transfected at DIV 14. Overexpression of EphB2 WT or Y504E significantly increased amplitude of the NMDAR dependent component of mEPSC compared to control or Y504F mutants . (F) Cumulative probability histogram of mEPSC amplitude at 15–25 msec after the mEPSC peak for Y504E and Y504F mutants before (dark shading) and after (light shading) NMDAR blockage with 50 μM APV. Inset, Mean traces of mEPSCs after NMDAR blockage with APV. Vertical Scale bar = 10 pA, horizontal scale bar = 10 msec.(PDF)Click here for additional data file.S7 Fig(A) Co-immunoprecipitation (Co-IP) of GluN1 and EphB1 with α-GluN1 antibody from HEK293T. All lanes are transfected with HA-GluN1 and GluN2B. Control lane is transfected with GFP, WT lane is transfected with EphB1 WT, E lane is transfected with EphB1 Y502E, F lane is transfected with EphB1 Y502F. Top blots were probed for FLAG, bottom blots probed for GluN1. Left panels are IP samples pulled down with α-GluN1 antibody, right panels are lysates. Tubulin is used as loading control. (B) Top blot shows HEK293T lysates probed with a phospho-specific antibody α-EphB2 p*Y504. Middle blot shows same lysates probed for EphB1. Bottom blot shows lysates probed for tubulin loading control. Lanes were loaded with lysates of HEK293T cells transfected with either GFP Control, EphB1 WT, or EphB1 Y502F. (C) Adult mice underwent unilateral plantar incision. 24 hours following incision spinal cord was separated into ipsilateral (+) and contra-lateral (-) sides to injury. Endogenous EphB2 or EphB1 were immunoprecipitated with α-EphB2 or α-EphB1 antibodies, respectively from these tissues. Blots were probed with phospho-specific antibody α-p*Y504 and EphB.(PDF)Click here for additional data file.S8 Fig(A) Model of effects of extracellular phosphorylation of EphB cFN3 on the surface retention, accumulation and function of GluN2B-containing NMDARs and pain plasticity. (B) Under physiological conditions, ephrin-B-induced extracellular tyrosine phosphorylation of EphB cFN3 is not required for pain sensitivity through regulating EphB-NMDAR interaction, Ca2+-dependent signaling, and NMDAR-dependent gene expression. (C) Injury increases the level of ephrin-B and/or EphB expression. In turn, this induces NMDAR-dependent pain plasticity potentially leading to pathological pain. (D) Potential approach for therapies to alter pain plasticity: In chronic malignancy-induced or neuropathic pain diseases, EphB-dependent enhancement of NMDAR activity may be prevented by blocking the extracellular tyrosine phosphorylation of EphB cFN3 using ecto-kinase inhibitors such as K252b.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


In yeast, inactivation of TORC1 restricts G1–S transition due to the rapid clearance of G1 cyclins (Cln) and the stabilization of the B-type cyclin (Clb) cyclin-dependent kinase (CDK) inhibitor Sic1. The latter mechanism remains mysterious but requires the phosphorylation of Sic1-Thr173 by Mpk1 and inactivation of the Sic1-pThr173-targeting phosphatase (PP2ACdc55) through greatwall kinase-activated endosulfines. Here we show that the Sic1-pThr173 residue serves as a specific docking site for the CDK phospho-acceptor subunit Cks1 that sequesters, together with a C-terminal Clb5-binding motif in Sic1, Clb5-CDK-Cks1 complexes, thereby preventing them from flagging Sic1 for ubiquitin-dependent proteolysis. Interestingly, this functional switch of Sic1 from a target to an inhibitor of cyclin-CDK-Cks1 also operates in proliferating cells and is coordinated by the greatwall kinase, which responds to both Cln-CDK-dependent cell-cycle and TORC1-mediated nutritional cues.Eukaryotic cell cycle progression through G The phl region . This prodegrons , 10. Botin yeast .1–S transition, this process is tightly coupled to growth- and nutrient-related signals that are mediated by TORC1, the central controller of growth in eukaryotes [1 cyclins (Cln1–3) [173) within the C-terminal part of Sic1 that critically contributes to its stabilization [173 in Sic1 [173 in Sic1 by inhibiting a signaling branch, which restrains the activity of the Sic1-pThr173-targeting PP2ACdc55 protein phosphatase [Cdc55 (or B55-PP2A in higher eukaryotes) [1061 and Thr1075), which cooperatively mediate tandem 14-3-3 protein binding to sequestrate inactive Rim15 in the cytoplasm [Cdc55 inhibitory Igo1/2 proteins. Combined with the coordinated activation of Mpk1, the latter process ensures the phosphorylation of Sic1 at Thr173 under conditions that inhibit TORC1 such as nutrient limitation or rapamycin treatment. How this particular phosphorylation event stabilizes Sic1 remains currently mysterious.Consistent with the important role of Sic1 degradation for Gkaryotes , 13. Acc(Cln1–3) , 15. In lization . TORC1 caryotes) . TORC1 cytoplasm . Downreg173 in Sic1 creates a docking site for the phospho-acceptor subunit Cks1 that tethers Clb5-CDK-Cks1 complexes away from the respective N-terminal docking sites on Sic1 where they could otherwise favor multi-phosphorylation and degradation of Sic1. The spatial sequestration of the pThr173-associated Clb5-CDK-Cks1 complexes also promotes their inhibition via the additional association of Clb5 with the RXL motif within the CDK-inhibitory C-terminus (RXLCDKI) of Sic1. Thus TORC1 inactivation (and consequently Mpk1 and Gwl pathway activation) triggers the phosphorylation of Thr173 in Sic1, thereby converting Sic1 from a target to a potent inhibitor of Clb5-CDK-Cks1 complexes. Interestingly, this switch of Sic1 also occurs in proliferating cells in G1 and is rapidly reversed at START as a result of the increase of Cln-CDK, which, cooperatively with TORC1, inhibits Rim15 by stimulating its confinement to the cytoplasm. Thus, our data pinpoint the Gwl pathway as a central node that interprets both growth and cell cycle cues to coordinate G1–S transition.Here we show that the phosphorylation of Thr173 in Sic1 may compromise its capacity to interact with components of the SCFCdc4 complex [173 may, rather than directly interfering with SCFCdc4 binding, indirectly affect the phosphorylation status of the N-terminal residues in Sic1. To address this possibility, we first compared on phos-tag gels the phosphorylation levels of Sic1 and Sic1T173A from rapamycin-treated cdc4-2ts strains, which were preincubated at the restrictive temperature  to inactivate Cdc4 and consequently block SCFCdc4-mediated degradation of the Sic1 variants. Under these conditions, the slowly migrating, hyperphosphorylated wild-type (WT) Sic1, but not the Sic1T173A variant, rapidly collapsed into several fast-migrating, hypophosphorylated isoforms  prevented the observed sustained hyperphosphorylation of Sic1T173A, indicating that Cln- and/or Clb-CDK complexes are not properly inhibited in Sic1T173A-expressing cells  or S-phase (Clb5) cyclins. As shown in cdc4-2ts and cdc4-2tssic1T173A cells. Expression of the Sic1T173A allele does therefore not alter the dynamic regulation of G1/S cyclin levels in rapamycin-treated cells. Because Sic1T173A-expressing cells exhibited sustained phosphorylation of a preferred Cln-CDK target , we next speculated that the phosphorylation of Thr173 in Sic1 may expand its inhibitory specificity in rapamycin-treated cells to include Cln-CDKs. However, this cannot be the case as Cln2 did not bind Sic1 or Sic1T173A under any conditions tested  docking site in Clb5 , we purified Sic1 and Sic1T173A from rapamycin-treated cells and analyzed the coprecipitating proteins by mass spectrometry. Remarkably, when applying stringent criteria, this experiment pinpointed Cks1 as the only protein that interacted specifically and more robustly with Sic1 when compared with Sic1T173A. To exclude that the presumably lower affinity between Cks1 and Sic1T173A is simply due to lower Cks1 levels in sic1T173A strains, we compared the Cks1 expression levels in WT and sic1T173A cells. Cks1 levels were equally high in exponentially growing cells and rapidly dropped with similar kinetics in rapamycin-treated cells of both strains  that the lysates from rapamycin-treated sic1T173A cells contained much lower amounts of the instable Sic1T173A than the ones of WT cells containing the stable Sic1 protein, and (ii) that our co-IP experiments may, to some extent, probe the association of Cks1 with the N-terminal Cks1-docking sites in Sic1/Sic1T173A. To address both issues at once, we carried out additional Cks1 co-IP experiments using myc13-tagged fragments of Sic1 (Sic1150-285), which lacked the N-terminal phosphodegron and Cks1-docking sites that drive Sic1 degradation. As expected, these Sic1 fragments, whether they contained or not the Thr173-to-Ala mutation, were equally stable under all conditions tested. The respective co-IP experiments with Cks1 confirmed the ones in which full-length variants of Sic1 were expressed (173 and its neighboring residues (Pro-Gly-Thr173-Pro) perfectly match the Cks1-binding consensus motif (F/I/L/P/V/W/Y)XTP [173 a role as bona fide Cks1-docking site.An alternative model we entertained to explain the higher Clb/Cln-CDK activity in Sic1 strains . Directeed cells . These rxpressed . Togethe/W/Y)XTP , our res173-dependent manner, in exponentially growing cells. This is not surprising, as low levels of Sic1-pThr173 molecules can also be detected in these cells [173 in rapamycin-treated cells. This mechanism likely contributes to the swift clearance of free Cks1 molecules and may potentially contribute to a more robust inhibition of Clb5-CDK-Cks1 complexes due to their insertion between the Clb5-tethering RXLCDKI domain and the pThr173 Cks1-docking site in Sic1. Several additional experiments support this model. For instance, mutation of the RXLCDKI motif in Sic1 strongly weakened the association between Clb5 and this Sic1 mutant protein. This resulted in the release of Clb5-CDK from Sic1 inhibition, as Clb5-CDK immune complexes isolated from rapamycin-treated Sic1R262A/L264A-expressing cells (unlike the ones from WT cells) exhibited largely unrestrained Clb5-CDK activity in vitro , both Sic1 variants were unstable in an additive manner when assayed in rapamycin-treated cells  when TORC1 is low. Our co-IP analyses using extracts from rapamycin-treated cells endorsed this hypothesis, as Cks1 interacted well with Clb5 and Sic1 in WT cells, while Cks1 only bound Clb5 but not Sic13A cells . Under tWT cells . Finally173 in Sic1 via Mpk1 and the Gwl pathway (Rim15-Igo1/2-PP2ACdc55), we next quantified the relative levels of Sic1 that were precipitated with Cks1 in exponentially growing and rapamycin-treated WT, mpk1Δ and cdc55Δ cells. As expected, inhibition of TORC1 strongly stimulated the relative affinity between Sic1 and Cks1  and the mutation of Thr173 in Sic1 per se, whether combined or not with cdc55Δ, measurably weakened the affinity between Sic1 and Cks1 in exponentially growing cells  raises more rapidly and subsequently oscillates at a significantly higher level in sic1T173A than in WT cells when released from an α-factor arrest . Combinr arrest , these d173 species (when cells were released from the α-factor arrest) temporally coincided with a significant decrease in the level of the active version of the PP2ACdc55-inhibitory endosulfine Igo1  [in vivo [1–S transition phase and be released from this inhibition when CDK is low . Consistent with this idea, inactivation of Cdc28as with 1NM-PP1 stimulated the activity of Rim15, which was not due to indirect effects on TORC1, because the phosphorylation of Thr737 in Sch9, a proxy for TORC1 activity, remained largely constant throughout the experiment  . Because[in vivo , this inperiment . Notablyng cells , which ang cells .1061 and Thr1075) within adjacent classical 14-3-3-binding domains that mediate cooperative association with dimeric 14-3-3 proteins in the cytoplasm [1061) and the phosphate-responsive cyclin-CDK Pho80-Pho85 module (Rim15-Thr1075) [1075 residue together with Pho85. Supporting this hypothesis, we found Rim15 to specifically interact with Cln2, but not with Clb5 or the control protein Lst7, when probed in exponentially growing cells  , 23, 24.Thr1075) , 34, we ng cells . Moreovein vitro , and ina in vivo . Therefoe manner . Taken t1–S [2–M cell cycle transition [2–M instead of G1–S. Alternatively, the observed divergent roles of the Gwl pathways in budding yeast and metazoans could have evolved from an ancestral version of this pathway that functioned in both G1–S and G2–M, which raises the possibility that higher eukaryotes may have retained some aspects of the Gwl-mediated G1–S control mechanism identified in this study. In this context, it is compelling that mammalian cells express the cyclin-CDK2 inhibitor p27Kip1, which is structurally similar to Sic1 and also functions in G0/1–S cell cycle transition control [Kip1 is flagged for degradation by the combined action of cyclin-CDK, Cks1 and the SCFSkp2 ubiquitin ligase complex, while it is stably maintained in G0 cells only when phosphorylated at specific amino-acid residues [Kip1 and Cks1 in suppressing and promoting cancer progression, respectively [1–S transition control.The budding yeast and metazoan Gwl pathways are organized in a strikingly similar way. Indeed, as shown here and in previous studies , they ar1–S , 37, 38,ansition , 39–41.  control . Moreoveresidues . Consideectively , 44, theSaccharomyces Genome Database). To remain consistent with the previously published literature, we refer to Sic1-Thr173 throughout our study although the respective residue corresponds to Sic1-Thr174 in JK9-3D. Saccharomyces cerevisiae yeast cells were pregrown overnight at 30 °C in standard rich medium (YPD) with 2% glucose or in synthethic defined medium  and 2% glucose) for maintenance of plasmids. Prior to the experiments, cells were diluted to an OD600 of 0.15 and grown until they reached an OD600 of 0.6. Rapamycin , 1NM-PP1 , and α-factor  were used at final concentrations of 200 ng ml−1, 0.5 μM, and 5 μg ml−1, respectively.Strains and plasmids are listed in M Phos-tag . The sources and dilutions of antibodies used are listed in Protein extraction was performed by mild alkali treatment of cells followed by boiling in standard electrophoresis buffer . Sodium M TRIS (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.5% NP40 and 1× protease and phosphatase inhibitor cocktails ) with glass beads (0.5 mm diameter) using a Precellys cell disruptor  and subsequent clarification by centrifugation . HA- and myc-tagged proteins were immunoprecipitated with an anti-HA and anti-myc magnetic matrix , respectively. GST-tagged proteins were pulled down with glutathione-sepharose beads .For co-IP analyses, lysates were prepared by disruption of frozen cells in lysis buffer (50 m3 or Clb5-HA3 immunocomplexes were incubated at 30 °C for 20 min (or for the times indicated) in kinase buffer  for radioactive kinase assays). The reactions were stopped with sample buffer and boiled for 5 min. For the CDK inhibition assays, the immunoprecipitated kinase complexes were incubated for 1 h on ice with GST-Sic1, GST-Sic1T173A or GST-Sic1R262A L264A (that were subjected to in vitro phosphorylation by Mpk1 before use) and then assayed in CDK assays using GST-Sic11-100 as substrate. The levels of Thr5 in Sic1 were detected with phosphospecific anti-Sic1-pThr5 antibodies.Protein kinases were immunoprecipitated as above. For CDK assays, Cln2-HAImages were captured with an inverted Spinning Disk Confocal Microscope  that was equipped with an Evolve 512  EM-CCD camera and a 100× 1.3 NA oil immersion Nikon CFI series objective .


Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity.Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite  Plasmodium falciparum is an apicomplexan parasite that causes the most virulent form of human malaria. All of the pathology of the disease is caused by the asexual blood-stage parasite, which develops and multiplies within red blood cells (RBC). Intracellular development through the trophozoite and schizont phases results in a multinucleate cell and then cell division produces individual haploid merozoites. This process is accompanied by the elaboration of apical organelles such as micronemes and rhoptries, and an inner membrane complex (IMC) that forms part of the parasite’s surface pellicle. Following release (or egress) from the schizont, the extracellular merozoites invade new RBC and the cycle continues. The invasion of RBC is a crucial step in the parasite life-cycle and, because the parasite's exposed surface antigens are especially susceptible to immune attack, rapid host cell entry is important. Interactions between parasite and host cell proteins are very important in this process.The full life-cycle of this parasite is complex and includes other extracellular stages that are motile and that invade host cells and tissue. The motile sporozoite and ookinete stages move by a process called gliding motility, which is powered by a multi-molecular complex called the glideosome. In the current model, the glideosome, containing a class XIV myosin, myosin A (MyoA); a myosin light chain called the MyoA tail domain-interacting protein (MTIP); and glideosome-associated proteins (GAPs) such as GAP50, GAP45, and GAP40 , is locain vitro with aldolase  ATP was added to 10μl 1mM ATP, and 2μl of this mixture added to each sample to initiate the kinase reaction. Following incubation at 30°C for 3 hours, samples were mixed with SDS-PAGE loading buffer (1:1 vol/vol), and heated to 90°C for 5 minutes. Each sample was loaded in two halves on a 10% Bis-Tris NuPAGE gel using MOPS running buffer. The gel was fixed, dried and exposed to Kodak Biomax MR film to visualize the radiolabelled proteins, or the proteins were visualised by Coomassie Brilliant Blue staining to ensure that equivalent amounts of protein were loaded.Phosphorylation assays were performed using 10 μM recombinant CDPK1 , PKA (PrZipTip® Pipette Tips (Millipore) containing either C18 or C4 according to the size of the protein to be desalted. For CID analysis, the eluted proteins in 70% (vol/vol) acetonitrile (ACN) and 0.1% (vol/vol) acetic acid were injected into the mass spectrometer using a 10 μl min-1 flow rate. Phosphorylation sites were determined using stand-alone nanospray on an LTQ Orbitrap Velos (Thermo Fisher Scientific). Nanospray needles  were operated at 1.6 kV and collision induced dissociation MS/MS was performed in the ion trap and recorded in the Orbitrap. Spectra were acquired over a collision energy range of 20-30eV at a resolution of 100k. The averaged MS/MS spectrum was then deconvoluted using Xtract and interpreted using Prosight (https://prosightptm.northwestern.edu/).Non-phosphorylated, phosphorylated and biotinylated polypeptides were analysed by electrospray (ES)-mass spectrometry and collision-induced dissociation (CID) fragmentation pathway analysis. The samples (100 pmol) were first desalted using P10 -1 of endopeptidase, overnight at 32°C. Digestion in solution was performed in a 20 μl final reaction volume with a molar ratio of 1:100 enzyme:protein in 5 mM ammonium bicarbonate, with overnight digestion at 32°C. One to ten microlitres of peptide solution acidified to a final concentration of 0.1% TFA was loaded at 5 μl min-1 onto a 2 mm x 100 μm Acclaim Pepmap C18 trap column attached to an Ultimate 3000 nanoRSLC HPLC (Thermo Scientific), then peptides were eluted at 0.22 μl min-1 through a 50 cm x 75 μm Acclaim Pepmap C18 column. Starting with Buffer A , a gradient from 9% to 25% buffer B  over 37 minutes, and 25% to 40% buffer B over 18 minutes was used; followed by a short gradient to 100% B. Eluant was introduced into a LTQ Orbitrap Velos Pro (Thermo Scientific) using a Proxeon NanoES source (Thermo Scientific) and a 30 μm ID stainless steel emitter operated at 2 kV. The Orbitrap was operated in “Data Dependent Acquisition” mode with a survey scan at a resolution of 60,000 from m/z 300–1500, followed by MS/MS in the LTQ of the top 6–12 ions. Dynamic exclusion was used with a time window of 40 s. The Orbitrap charge capacity was set to a maximum of 106 ions in 10 ms, whilst the LTQ was set to 104 ions in 100 ms. Raw files were processed using Proteome Discoverer (PD) 1.3 (Thermo Scientific) with Mascot 2.4  as the search engine against the appropriate protein fasta database. A decoy database of reversed sequences was used to filter the results at a false detection rate of 1%. Label free quantitation was achieved using the precursor-ion quantitation module of PD.For protein analysis, peptides were generated by in-gel or in-solution digestion using sequencing-grade modified endopeptidases, trypsin, Asp-N or chymotrypsin (Promega). The in-gel digestion was performed after first resolving proteins by SDS-PAGE and subsequent staining using the Colloidal Blue staining kit (Invitrogen), according to the manufacturer’s instructions. The proteins bands were excised and both SDS and stain removed with 100% (vol/vol) ACN, and 5 mM ammonium bicarbonate containing 50% (vol/vol) ACN, for 30 min at room temperature. The proteins were then reduced using 20 mM DTT for 1 hour and alkylated with 5 mM iodoacetamide for 20 min at room temperature. The gel slices were first dehydrated in 100% (vol/vol) ACN and then the proteins digested in 20 μl final volume in 5 mM ammonium bicarbonate using 2 ng μlmrw) Secondary structure content was estimated using methods described elsewhere [Far-UV CD spectra were recorded on a Jasco J-715 spectropolarimeter fitted with a cell holder thermostatted by a PTC-348 Peltier unit. All CD measurements were made in 20 mM Tris (pH 8) at 20°C using fused silica cuvettes with 1 mm path length . The spectra were typically recorded with 0.1 nm resolution and baseline corrected by subtraction of the appropriate buffer spectrum. CD intensities are presented as the CD absorption coefficient calculated on a mean residue weight basis  at 25°C in 96-well microplates, as described previously . All assFor competition experiments, for example of MTRAP in the interaction between AMA1 or RH4 and aldolase, the Octet Red system was also used. Biotinylated AMA1 or RH4 peptide was loaded onto the streptavidin coated sensor and following washing incubated with 20 or 70 μM aldolase with (or without) increasing concentrations of unlabelled competitor MTRAP CTD peptide. Following incubation the data were collected and analysed as described above. The experiments were performed in duplicate.P. falciparum MTRAP with a GFP tag situated distal to the transmembrane (TM) sequence and proximal to the CTD was placed into the pHH4 vector [mtrap locus in the parasite by homologous recombination (see CCCGGGTCGAAAAATGCTAGAG GTTATAGAGG (forward) and GGCCTAGGTTTTTCTTTACGTAAGAAATAG (reverse) to incorporate XmaI and AvrII restriction enzyme sites (underlined). The sequence coding for MTRAP CTD was synthesized in a recodonised form: TATTTTCTGCGCAAAGAAAAAACCGAAAAAGTGGTGCAGGAAGAAACCAAAGAAGAAAATTTTGAAGTTATGTTTAATGATGATGCCCTGAAAGGCAAAGATAATAAAGCCATGGATGAAGAAGAATTTTGGGCACTGGAATAA (GeneArt®). The GFP sequence was obtained from the pHH4 plasmid and fused to MTRAP CTD sequence by PCR amplification using the primers: CCCCTAGGAGTAAAGGAGAAGAACTTTTCAC, TTTTTTCTTTGCGTTTG TATAGTTCATCCATGCCATGTGTAATCCC, and GGCCGCGGTTATTCCAGTGCCC to introduce AvrII and SacII restriction enzyme sites. These DNA fragments were then incorporated upstream of the P. berghei dhfr 3’ UTR in the pHH4 plasmid and Sanger sequencing (Beckman Genomics) was used to confirm the correct sequence assembly.Sequence coding for 4 vector  for insetion see . The regP. falciparum 3D7 ring stage parasites were transfected with 100 μg of plasmid DNA and cultured continuously under a WR99210 (2.5 nM) drug selection cycle for 4 cycles over 12 weeks. After each cycle parasite cultures were examined for genomic integration by PCR and for GFP expression by fluorescence microscopy. PCR was performed with the following primers: F1 (GTGGGAGAGACATTTAAGAAGTAATA) and R1 (CTCGGTGGGGCTTTAAATAATG) targeting sequences upstream and downstream of endogenous mtrap, F1 and R2 , and F1 and R3 . Following detection of integration the parasites were cloned by limiting dilution. A schematic representation of the parasite integration, diagnostic PCR and western blot is presented in To incorporate the modified gene, P. falciparum asexual blood stages were cultured in human RBCs, and analysed by fluorescence microscopy and western blotting, using preparations of schizonts and merozoites purified as described previously [eviously . GFP-tageviously .S1 Fighttps://prosightptm.northwestern.edu/) or Proteome Discoverer (PD) 1.3 (Thermo Scientific).Phosphorylation sites were interpreted from the mass data using either Prosight ( A. MTRAP phosphorylation (MTRAP_1 to MTRAP_6) interpreted using Prosight. There are only two potential phosphorylation sites and each of these threonines can be phosphorylated. B. AMA1 phosphorylation (AMA1_1 to AMA_5) interpreted using PD1.3. Phosphorylation was by CDPK1 (AMA1_1 to AMA1_3) and PKA (AMA1_4 and AMA1_5).The data are consistent with phosphorylation of Ser610 and not of the other 6 serine/threonine residues in the sequence. C. RH1 phosphorylation by CDPK1 interpreted using Prosight. The method did not resolve whether Ser2950 or Thr2951 was phosphorylated. D. RH4 phosphorylation by CK2 (RH4_1 to RH4_7) interpreted using PD1.3. The data are consistent with phosphorylation of either Ser1667 or Ser1674 with a very small amount of phosphorylation of both residues. E. EBA140 phosphorylation by CDPK1 (EBA140_1 to EBA140_2) interpreted using Prosight. No phosphorylated residues were identified unambiguously. F. EBA175 phosphorylation by CDPK1 (EBA177_1 to EBA175_2) interpreted using Prosight. Phosphorylation of both Thr1483 and Ser1489 was detected. G. EBA181phosphorylation by CDPK1 (EBA181_1 to EBA181_21) interpreted using PD1.3. Phosphorylation of both Ser1523 and Ser1528 was detected.(PDF)Click here for additional data file.S2 FigA. Phosphorylation of recombinant MTRAP peptide by protein kinase A. The MTRAP peptide  was biotinylated  and then phosphorylated using PKA (lower panel). The peaks corresponding to the protein and its phosphorylated forms are indicated. B. Phosphorylation of synthetic biotinylated AMA1 peptide by CDPK1 (upper panel) and protein kinase A (lower panel). The non-phosphorylated peptide  and a single phosphorylated form (6968.4) were detected. C. Phosphorylation of synthetic biotinylated RH1 peptide by CDPK1 (upper panel) and protein kinase A (lower panel). Only the non-phosphorylated peptide  was detected after incubation with PKA and an additional single (6619.9) phosphorylated form was detected following incubation with CDPK1. D. Phosphorylation of synthetic biotinylated RH4 peptide by CDPK1 (upper panel), protein kinase A (middle panel) and casein kinase 2 (lower panel). Only the non-phosphorylated peptide  was detected after incubation with CDPK1 or PKA and a single (8386.2) phosphorylated form was detected following incubation with CK2. E. Phosphorylation of synthetic biotinylated EBA140 peptide by CDPK1 (upper panel) and protein kinase A (lower panel). Only the non-phosphorylated peptide  was detected after incubation with PKA. F. Phosphorylation of synthetic biotinylated EBA175 peptide by CDPK1 (upper panel) and protein kinase A (lower panel). Only the non-phosphorylated peptide  was detected after incubation with PKA and a single (7243.2) phosphorylated form was detected following incubation with CDPK1. G. Phosphorylation of synthetic biotinylated EBA181 peptide by CDPK1 (upper panel) and protein kinase A (lower panel). Only the non-phosphorylated peptide  was detected after incubation with PKA and single (7094.9) and dual (7173.9) phosphorylated forms were detected following incubation with CDPK1.(PDF)Click here for additional data file.