Changes In Phosphorylation State Research Articles

Phosphoproteome and proteome analyses reveal low-phosphate mediated plasticity of root developmental and metabolic regulation in maize (Zea mays L.)

Phosphate (Pi) deficiency has become a significant challenge to worldwide agriculture due to the depletion of accessible rock phosphate that is the major source of cheap Pi fertilizers. Previous research has identified a number of diverse adaptive responses to Pi starvation in the roots of higher plants. In this study, we found that accelerated axile root elongation of Pi-deprived maize plants resulted from enhanced cell proliferation. Comparative phosphoproteome and proteome profiles of maize axile roots were conducted in four stages in response to Pi deficiency by multiplex staining of high-resolution two dimensional gel separated proteins. Pro-Q DPS stained gels revealed that 6% of phosphoprotein spots displayed changes in phosphorylation state following low-Pi treatment. These proteins were involved in a large number of metabolic and cellular pathways including carbon metabolism and signal transduction. Changes in protein abundance of a number of enzymes indicated that low-Pi induced a number of carbon flux modifications in metabolic processes including sucrose breakdown and other downstream sugar metabolic pathways. A few key metabolic enzymes, including sucrose synthase (EC 2.4.1.13) and malate dehydrogenase (EC 1.1.1.37), and several signaling components involved in protein kinase or phosphatase cascades, auxin signaling and 14-3-3 proteins displayed low-Pi responsive changes in phosphorylation state or protein abundance. A variety of key enzymes and signaling components identified as potential targets for phosphorylation provide novel clues for comprehensive understanding of Pi regulation in plants. Protein phosphorylation, coordinating with changes in protein abundance, is required for maize root metabolic regulation and developmental acclimation to Pi starvation.

Tau phosphorylation and tau mislocalization mediate soluble Aβ oligomer‐induced AMPA glutamate receptor signaling deficits

In our previous studies, phosphorylation-dependent tau mislocalization to dendritic spines resulted in early cognitive and synaptic deficits. It is well known that amyloid beta (Aβ) oligomers cause synaptic dysfunction by inducing calcineurin-dependent AMPA receptor (AMPAR) internalization. However, it is unknown whether Aβ-induced synaptic deficits depend upon tau phosphorylation. It is also unknown whether changes in tau can cause calcineurin-dependent loss of AMPARs in synapses. Here, we show that tau mislocalizes to dendritic spines in cultured hippocampal neurons from APPSwe Alzheimer's disease (AD)-transgenic mice and in cultured rat hippocampal neurons treated with soluble Aβ oligomers. Interestingly, Aβ treatment also impairs synaptic function by decreasing the amplitude of miniature excitatory postsynaptic currents (mEPSCs). The above tau mislocalization and Aβ-induced synaptic impairment are both diminished by the expression of AP tau, indicating that these events require tau phosphorylation. The phosphatase activity of calcineurin is important for AMPAR internalization via dephosphorylation of GluA1 residue S845. The effects of Aβ oligomers on mEPSCs are blocked by the calcineurin inhibitor FK506. Aβ-induced loss of AMPARs is diminished in neurons from knock-in mice expressing S845A mutant GluA1 AMPA glutamate receptor subunits. This finding suggests that changes in phosphorylation state at S845 are involved in this pathogenic cascade. Furthermore, FK506 rescues deficits in surface AMPAR clustering on dendritic spines in neurons cultured from transgenic mice expressing P301L tau proteins. Together, our results support the role of tau and calcineurin as two intermediate signaling molecules between Aβ initiation and eventual synaptic dysfunction early in AD pathogenesis.

KaiC intersubunit communication facilitates robustness of circadian rhythms in cyanobacteria

The cyanobacterial circadian clock is the only model clock to have been reconstituted in vitro. KaiC, the central clock component, is a homohexameric ATPase with autokinase and autophosphatase activities. Changes in phosphorylation state have been proposed to switch KaiC’s activity between autokinase and autophosphatase. Here we analyse the molecular mechanism underlying the regulation of KaiC’s activity, in the context of its hexameric structure. We reconstitute KaiC hexamers containing different variant protomers, and measure their autophosphatase and autokinase activities. We identify two types of regulatory mechanisms with distinct functions. First, local interactions between adjacent phosphorylation sites regulate KaiC’s activities, coupling the ATPase and nucleotide-binding states at subunit interfaces of the CII domain. Second, the phosphorylation states of the protomers affect the overall activity of KaiC hexamers via intersubunit communication. Our findings indicate that intra-hexameric interactions play an important role in sustaining robust circadian rhythmicity.

Early PK-Analysis Predicts Molecular Response In Patients With Early Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Treated With Frontline Nilotinib

IntroductionTyrosine kinase inhibitor (TKI) trough plasma level testing and pharmacodynamic evaluation (i.e. quantification of phosphorylated BCR-ABL downstream targets) may help to optimize patient-tailored dosing in CML-CP. For 2nd generation TKIs the clinical value of TKI plasma level and protein phosphorylation quantification remains unclear. The ENEST1st study (NCT01061177) is focused on examining the role of first-line nilotinib therapy in CML-CP. This ENEST1st substudy addresses the value of plasma-level testing and protein single cell phosphorylation pharmacodynamics as predictors of response and long-term outcomes. MethodsNilotinib levels were centrally quantified by means of mass spectrometry (HPLC-MS/MS) in 54 patients at 3 hours after first drug intake at day (d) 1 (Cmax) and at d7, d28 as well as at months (mo) 3, 6, 12 prior to morning drug intake (Cmin=trough level). Single cell quantification of intracellular protein phosphorylation was performed by flow cytometry (phosphoflow) in the myelocyte cell population after full blood fixation/cell permeabilization/erythrocyte lysis and analyzed 3 hours after first drug intake at d1 and on d7 and d28. Primary endpoint of the study was MR4 (BCR-ABL <0.01% IS) at 18 months. ResultsOur initial analysis focuses on correlation of drug levels with molecular response by correlating PK data to early molecular response, i.e. ≤10% BCR-ABL (IS) reduction at 3 mo as well as 0.1% IS (MMR), MR4 and MR5. At 3, 6, 12 and 18 mo, 24%, 56%, 74% and 77 % of the here investigated patients achieved MMR; 2%, 22%, 34% and 44% achieved MR4 and MR5 was detected in 0%, 5%, 15% and 14% of patients at the respective time points. Only 1 patient at 3mo had >IS 10% BCR-ABL. On d1 at 3 hours after intake of the first tablet the median peak plasma levels reached 408 ng/ml increasing to a median trough level of 834 ng/ml at d7, 888 ng/ml at d28, 919 ng/ml at 3mo, 1122 ng/ml at 6m and 1003 ng/ml at 12mo. Initial Cmax-levels at d1 significantly correlated with steady state levels thereafter (e.g. with trough levels at 12mo: r=0.56, p=0.004). Three mo drug levels were significantly negatively associated with lower BCR-ABL mRNA burden at 12m and 24m (r=-0.44, p=0.011 for m12 and r=-0.35, p=0.014 for m24). When median steady state drug levels were compared in different response categories (yes/no: MR4, MR5, MMR, >IS 10% at 3mo), significantly higher nilotinib trough levels at day 90 and mean trough level (day 28-180) were seen in patients achieving the primary endpoint of the study (MR4 at 18mo). Similarly, 3mo and mean through levels were also significantly higher in patients achieving MR5 at 12 or 18mo. As only one patient did not reach <10% at 3mo, we cannot evaluate if early PK data are able to predict this endpoint. The probability of achieving MR5 at 12mo and 24mo was significantly higher in the top third of the day 90 PK level (cumulative incidence of MR5 in the respective PK groups at 12mo: 5.6% vs. 5.6% vs. 44.4% and at 24mo: 5.6% vs. 22% vs. 44.4%). Cumulative incidence of MR4 was faster in the higher PK group (6mo: 6% vs. 18% vs. 42%; 12mo: 22% vs. 29% vs. 57% and at 24mo: 46% vs. 67% vs. 58%), which however did not reach statistical significance. Phosphoflow of myelocytes revealed that compared to baseline, phosphorylation of almost all investigated proteins decreases over time, including significant reduction of phospho-Gab2(Y452), Abl(Y245), STAT5(Y694), Erk1/2(T202/Y204), and p38(T108/Y182) at d28. Correlation of plasma levels with changes in phosphorylation status revealed a negative correlation of increasing drug levels with decreasing phospho-p38 and phospho-STAT5 status. A more detailed signaling analysis of cellular subsets related to molecular response criteria are in progress. ConclusionsSingle cell myelocyte phosphoprotein detection allowed monitoring of the BCR-ABL signalling network. Inter-individual plasma levels show a relatively wide variability and Cmax levels upon first drug exposure correlate with later trough levels pointing out to a potential individual pharmacogenetic background regulating drug availability. PK-based optimizing of nilotinib-dosing may help to further improve response depth (i.e. reaching MR4 or MR5), which is assumed to be a prerequisite for treatment discontinuation. Disclosures:Wolf:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mustjoki:Novartis: Honoraria, Research Funding. Greil:Novartis: Honoraria, Research Funding. Hjorth-Hansen:Pfizer, BMS: Honoraria, Travel expenses Other. Verhoef:Novartis: Research Funding. Mark:Novartis: Employment. Haenig:Novartis: Employment. Jurjonas:Novartis: Employment. Giles:Novartis: Consultancy, Research Funding. Hochhaus:Novartis: Research Funding. Porkka:Novartis: Honoraria, Research Funding. Ossenkoppele:Novartis: Consultancy, Research Funding; BMS: Consultancy. Gjertsen:Novartis: Honoraria, Research Funding.

Phospho-Regulation Pathways During Egg Activation in Drosophila melanogaster

Egg activation is the series of events that transition a mature oocyte to an egg capable of supporting embryogenesis. Increasing evidence points toward phosphorylation as a critical regulator of these events. We used Drosophila melanogaster to investigate the relationship between known egg activation genes and phosphorylation changes that occur upon egg activation. Using the phosphorylation states of four proteins-Giant Nuclei, Young Arrest, Spindly, and Vap-33-1-as molecular markers, we showed that the egg activation genes sarah, CanB2, and cortex are required for the phospho-regulation of multiple proteins. We show that an additional egg activation gene, prage, regulates the phosphorylation state of a subset of these proteins. Finally, we show that Sarah and calcineurin are required for the Anaphase Promoting Complex/Cyclosome (APC/C)-dependent degradation of Cortex following egg activation. From these data, we present a model in which Sarah, through the activation of calcineurin, positively regulates the APC/C at the time of egg activation, which leads to a change in phosphorylation state of numerous downstream proteins.

Stimulation of rat liver branched-chain alpha-keto acid dehydrogenase activity by low doses of bezafibrate

Multienzyme branched-chain alpha-ketoacid dehydrogenase complex (BCKDH) catalyzes the regulatory step of oxidative catabolism of indispensable branched-chain amino acids (BCAA). The activity of the BCKDH complex is regulated by a reversible phosphorylation, end-product inhibition and by changes in the gene expression of BCKDH component enzymes. It has been shown previously that a high dose of bezafibrate (an agent added to rat chow at final concentration of 0.5%) changes mRNA levels of BCKDH-related enzymes and increases dephosphorylation of the complex leading to stimulation of liver BCKDH activity and the enhanced BCAA catabolism. The aim of the present study was to determine an in vivo effect of low, clinically relevant doses of bezafibrate on BCKDH activity in rat liver. Bezafibrate was administrated for 14 days by gastric gavage to Wistar male rats (fed low-protein chow; 8% protein) at one of the following daily doses of 5, 10 and 20mg/kgb.wt. The control group was given the vehicle (0.3% methylcellulose) only. The actual BCKDH and total BCKDH activities were assayed spectrophotometrically before and after incubation with a broad-specificity phosphatase, respectively. The mRNA levels of the selected genes (BCKDH catalytic subunits and regulatory enzymes) were quantified by means of semi-quantitative RT-PCR. Current catalytic activity of BCKDH (described as BCKDH activity state – the proportion of the BCKDH complex in its active dephosphorylated form) increased by 2.1±0.2, 2.3±0.2 and 2.7±0.2 fold (p<0.01). Changes in BCKDH activity did not correspond with changes in mRNA levels of the complex catalytic subunits. Moreover, mRNA levels of regulatory enzymes remained unaltered. Initially bezafibrate caused a transient insignificant reduction in body weight, but it had no effect on the final body weight. The highest dose of bezafibrate induced hepatomegaly. In conclusion, these data indicate that under conditions of dietary protein restriction low, clinically relevant doses of bezafibrate have a similar adverse effect on rat liver BCKDH activity and BCAA degradation rate as the high experimental dose. Up-regulation of liver BCKDH activity by low doses of bezafibrate appears to result mainly from changes in phosphorylation status of the complex (increased dephosphorylation) and is not associated with elevations in mRNA levels of BCKDH enzymatic components.

Bak apoptotic function is not directly regulated by phosphorylation

During apoptosis, Bak and Bax permeabilize the mitochondrial outer membrane by undergoing major conformational change and oligomerization. This activation process in Bak is reported to require dephosphorylation of tyrosine-108 close to an activation trigger site. To investigate how dephosphorylation of Bak contributes to its activation and conformational change, one-dimensional isoelectric focusing (1D-IEF) and mutagenesis was used to monitor Bak phosphorylation. On 1D-IEF, Bak extracted from a range of cell types migrated as a single band near the predicted isoelectric point of 5.6 both before and after phosphatase treatment, indicating that Bak is not significantly phosphorylated at any residue. In contrast, three engineered ‘phosphotagged' Bak variants showed a second band at lower pI, indicating phosphorylation. Apoptosis induced by several stimuli failed to alter Bak pI, indicating little change in phosphorylation status. In addition, alanine substitution of tyrosine-108 and other putative phosphorylation sites failed to enhance Bak activation or pro-apoptotic function. In summary, Bak is not significantly phosphorylated at any residue, and Bak activation during apoptosis does not require dephosphorylation.

Quantitation, networking, and function of protein phosphorylation in plant cell

Protein phosphorylation is one of the most important post-translational modifications (PTMs) as it participates in regulating various cellular processes and biological functions. It is therefore crucial to identify phosphorylated proteins to construct a phosphor-relay network, and eventually to understand the underlying molecular regulatory mechanism in response to both internal and external stimuli. The changes in phosphorylation status at these novel phosphosites can be accurately measured using a 15N-stable isotopic labeling in Arabidopsis (SILIA) quantitative proteomic approach in a high-throughput manner. One of the unique characteristics of the SILIA quantitative phosphoproteomic approach is the preservation of native PTM status on protein during the entire peptide preparation procedure. Evolved from SILIA is another quantitative PTM proteomic approach, AQUIP (absolute quantitation of isoforms of post-translationally modified proteins), which was developed by combining the advantages of targeted proteomics with SILIA. Bioinformatics-based phosphorylation site prediction coupled with an MS-based in vitro kinase assay is an additional way to extend the capability of phosphosite identification from the total cellular protein. The combined use of SILIA and AQUIP provides a novel strategy for molecular systems biological study and for investigation of in vivo biological functions of these phosphoprotein isoforms and combinatorial codes of PTMs.

Phosphoproteome Dynamics Upon Changes in Plant Water Status Reveal Early Events Associated With Rapid Growth Adjustment in Maize Leaves

Plant growth adjustment during water deficit is a crucial adaptive response. The rapid fine-tuned control achieved at the post-translational level is believed to be of considerable importance for regulating early changes in plant growth reprogramming. Aiming at a better understanding of early responses to contrasting plant water statuses, we carried out a survey of the protein phosphorylation events in the growing zone of maize leaves upon a range of water regimes. In this study, the impact of mild and severe water deficits were evaluated in comparison with constant optimal watering and with recovery periods lasting 5, 10, 20, 30, 45, and 60 min. Using four biological replicates per treatment and a robust quantitative phosphoproteomic methodology based on stable-isotope labeling, we identified 3664 unique phosphorylation sites on 2496 proteins. The abundance of nearly 1250 phosphorylated peptides was reproducibly quantified and profiled with high confidence among treatments. A total of 138 phosphopeptides displayed highly significant changes according to water regimes and enabled to identify specific patterns of response to changing plant water statuses. Further quantification of protein amounts emphasized that most phosphorylation changes did not reflect protein abundance variation. During water deficit and recovery, extensive changes in phosphorylation status occurred in critical regulators directly or indirectly involved in plant growth and development. These included proteins influencing epigenetic control, gene expression, cell cycle-dependent processes and phytohormone-mediated responses. Some of the changes depended on stress intensity whereas others depended on rehydration duration, including rapid recoveries that occurred as early as 5 or 10 mins after rewatering. By combining a physiological approach and a quantitative phosphoproteomic analysis, this work provides new insights into the in vivo early phosphorylation events triggered by rapid changes in plant water status, and their possible involvement in plant growth-related processes.

Protein phosphorylation changes reveal new candidates in the regulation of egg activation and early embryogenesis in D. melanogaster

Egg activation is the series of events that must occur for a mature oocyte to become capable of supporting embryogenesis. These events include changes to the egg's outer coverings, the resumption and completion of meiosis, the translation of new proteins, and the degradation of specific maternal mRNAs. While we know some of the molecules that direct the initial events of egg activation, it remains unclear how multiple pathways are coordinated to change the cellular state from mature oocyte to activated egg. Using a proteomic approach we have identified new candidates for the regulation and progression of egg activation. Reasoning that phosphorylation can simultaneously and rapidly modulate the activity of many proteins, we identified proteins that are post-translationally modified during the transition from oocyte to activated egg in Drosophila melanogaster. We find that at least 311 proteins change in phosphorylation state between mature oocytes and activated eggs. These proteins fall into various functional classes related to the events of egg activation including calcium binding, proteolysis, and protein translation. Our set of candidates includes genes already associated with egg activation, as well as many genes not previously studied during this developmental period. RNAi knockdown of a subset of these genes revealed a new gene, mrityu, necessary for embryonic development past the first mitosis. Thus, by identifying phospho-modulated proteins we have produced a focused candidate set for future genetic studies to test their roles in egg activation and the initiation of embryogenesis.

Palytoxin and Other Microalgal Toxins Belonging to Different Chemical Classes Induce Cytotoxic Effects Involving a Common Set of Stress Response Proteins

Abstract The complexity of effects exerted by palytoxin (PlTX) in biological systems is apparent from responses observed in different models. We have approached this complexity by proteomic analyses, to study responses induced by PlTX at a system level. The analysis of a sub-proteome of a human cell line has shown that PlTX causes changes in phosphorylation states of Hsp27 and the oxidation of DJ-1, supporting the conclusion that cell stress responses, including oxidative damage, participate to the cytotoxic effect of PlTX. A descriptive meta-analysis of proteomic data about toxic responses induced by other classes of microalgal toxins, such as okadaic acid and microcystins, reveals that several protein components participating to cell stress responses, particularly oxidative stress, are affected when the systems are exposed to effective doses of those toxins. Detected changes comprise either the total levels of individual proteins, or the relative proportions of isoforms distinguishable according to thei...

A Comprehensive Differential Proteomic Study of Nitrate Deprivation in Arabidopsis Reveals Complex Regulatory Networks of Plant Nitrogen Responses

Nitrogen (N) is an important nutrient and signal for plant growth and development. However, to date, our knowledge of how plants sense and transduce the N signals is very limited. To better understand the molecular mechanisms of plant N responses, we took two-dimensional gel-based proteomic and phosphoproteomic approaches to profile the proteins with abundance and phosphorylation state changes during nitrate deprivation and recovery in the model plant Arabidopsis thaliana. After 7-day-old seedlings were N-deprived for up to 48 h followed by 24 h recovery, a total of 170 and 38 proteins were identified with significant changes in abundance and phosphorylation state, respectively. Bioinformatic analyses implicate these proteins in diverse cellular processes including N and protein metabolisms, photosynthesis, cytoskeleton, redox homeostasis, and signal transduction. Functional studies of the selected nitrate-responsive proteins indicate that the proteasome regulatory subunit RPT5a and the cytoskeleton protein Tubulin alpha-6 (TUA6) play important roles in plant nitrate responses by regulating plant N use efficiency (NUE) and low nitrate-induced anthocyanin biosynthesis, respectively. In conclusion, our study provides novel insights into plant responses to nitrate at the proteome level, which are expected to be highly useful for dissecting the N response pathways in higher plants and for improving plant NUE.

Chronic isolation stress compromises JNK/c-Jun signaling in rat brain

The c-Jun NH2-terminal kinases (JNKs) are important stress-responsive kinases. They regulate cellular activities by sequential phosphorylation and activation through a mitogen-activated protein kinase cascade, whereas JNKs activation is altered in response to various stressors. In the present study, we used immunoblotting to assess the effect of 21day of social isolation as the chronic stressor, either sole and in combination with 2h of acute immobilization or cold (4°C) stress on circulating corticosterone level and phosphorylation status of p46 (phospho-p46/total p46) and p54 (phospho-p54/total p54) JNK isoforms in the cytosolic and nuclear fraction of the prefrontal cortex and hippocampus of male Wistar rats. Also, the phosphorylation status of JNK nuclear down-stream target c-Jun (p-c-Jun/c-Jun) on Ser63 was examined. Both acute stressors with elevated CORT levels led to increased phosphorylation status of cytosolic p54 JNK isoforms but not p46 JNK isoforms only in the hippocampus and no change in phosphorylation status of c-jun in both brain regions. Chronic isolation with unaltered CORT level and reduced responsiveness to novel acute stressors, led to unchanged or reduced phosphorylation status of p46 and p54 JNK isoforms in both fractions and both brain regions, whereas the decrease of c-Jun phosphorylation status was found only in the prefrontal cortex. Our results suggest that compromised JNKs activation following chronic isolation may lead to interruption of JNK signaling, which could be related with neuropsychiatric disorders such as depression or long-lasting neuronal remodeling.

P5-13-04: Changes in Phosphorylation Status at VEGFR2 and Basal Tumor Hypoxic Volume Assessed by Misonidazol (MISO) Positron Emission Tomography (PET/CT) as Potential Biomarkers for Predicting Response to Bevacizumab in Breast Cancer.

Abstract Background: To evaluate the expression of novel putative biomarkers as predictors of benefit from bevacizumab in stage II-III, previously untreated breast cancers (BC) patients (pts) in the context of a phase II, single-arm, multicenter and prospective clinical trial. To address this aim, we examined baseline and induced changes after a single bevacizumab administration as potential early predictors of response. Methods: Pts received a single infusion of bevacizumab (15 mg/kg) (C1) 3 weeks prior to the beginning of neoadjuvant chemotherapy consisting in 4 cycles of docetaxel (60 mg/mq), doxorubicin (50 mg/mq) and bevacizumab (15 mg/kg) every 21 days (C2-C5) following by surgery. Early assessment of tumor changes was performed by paired tumor-biopsies and MISO PET/CT before and 14–21 days after bevacizumab administration (C1). Biomarker expression was assessed by immunohistochemistry (IHC) (Ki67, CD31, CD31/Ki67, VEGFR2, pVEGFR2 [Y951]) on formalin-fixed, paraffin-embedded tissue before and after bevacizumab infusion (C1). MISO SUV and tumor volume depicted by PET were calculated. Pathological response on surgical specimens was assessed according to Miller/Payne classification. Pts with tumor reduction &amp;gt;90% were considered as best responders (G4-G5) whether tumor reduction &amp;lt;90% were considered as no responders. Association between pathological response, IHC and MISO biomarkers was analyzed using Mann-Whitney test. ROC curve was performed to test sensibility and specificity of the biomarker found significantly associated with response and its value as independent predictor was tested in the multivariate analysis using logistic regression. Results: This analysis was performed on the training set including 73 patients (49 yr, range 29–70). Twenty (27%) patients obtained best response (G4-G5) whether 50 (68%) were considered as no responder (G1-G2-G3). Response was associated with negative estrogen receptors expression (p=0.02) and high Ki67 basal and after C1 expression (p=0.009 and p=0.01). Six (54%) of triple negative tumors were responders (p=0.05). Interestingly, change in pVEGFR2 [Y951] staining induced by bevacizumab administration and basaline MISO tumor volume was found significantly associated with response (p=0.03 and 0.057). Decrease in the phosphorilation status of VEGFR2 (Y951) &amp;gt;70% yielded a receiver operating characteristic (ROC) curve area of 0.681 (95% CI: 0.536 — 0.825) with 84% sensitivity and 95% specificity. The positive and negative predictive values for this marker were 60% and 64%, respectively. The change in phosphorilation status of VEGFR2p remains a significant predictor biomarker of response in multivariate analysis (OR=0.9, IC%95 0.96−0.99, p=0.04) after adjusting for clinical-pathological characteristics. Conclusion: These findings underline the potential value of early decrease in phosphorilation status of VEGFR2 after bevacizumab infusion as predictive biomarker of response to anti-angiogenic therapy in breast cancer. Moreover, tumor hypoxic volume obtained by MISO might be associated with response. A validation set is warranted to confirm these findings. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-04.

Mass Spectrometry-Based Analysis of Proteomic Changes in the Root Tips of Flooded Soybean Seedlings

Flooding injury is a major problem in soybean cultivation. A proteomics approach was used to clarify the occurrence of changes in protein expression level and phosphorylation in soybeans under flooding stress. Two-day-old seedlings were flooded for 1 day, proteins were extracted from root tips of the seedlings and digested with trypsin, and their expression levels and phosphorylation states were compared to those of untreated controls using mass spectrometry-based proteomics techniques. Phosphoproteins were enriched using a phosphoprotein purification column prior to digestion and mass spectrometry. The expression of proteins involved in energy production increased as a result of flooding, while expression of proteins involved in protein folding and cell structure maintenance decreased. Flooding induced changes of phosphorylation status of proteins involved in energy generation, protein synthesis and cell structure maintenance. The response to flooding stress may be regulated by both modulation of protein expression and phosphorylation state. Energy-demanding and production-related metabolic pathways may be particularly subject to regulation by changes in protein phosphorylation during flooding.

Cardiac myosin binding protein C phosphorylation in cardiac disease

Perturbations in sarcomeric function may in part underlie systolic and diastolic dysfunction of the failing heart. Sarcomeric dysfunction has been ascribed to changes in phosphorylation status of sarcomeric proteins caused by an altered balance between intracellular kinases and phosphatases during the development of cardiac disease. In the present review we discuss changes in phosphorylation of the thick filament protein myosin binding protein C (cMyBP-C) reported in failing myocardium, with emphasis on phosphorylation changes observed in familial hypertrophic cardiomyopathy caused by mutations in MYBPC3. Moreover, we will discuss assays which allow to distinguish between functional consequences of mutant sarcomeric proteins and (mal)adaptive changes in sarcomeric protein phosphorylation.

Modulation by the BK accessory β4 subunit of phosphorylation-dependent changes in excitability of dentate gyrus granule neurons

Large-conductance voltage- and calcium-activated potassium (BK) channels are large-conductance calcium- and voltage-activated potassium channels critical for neuronal excitability. Some neurons express so called fast-gated, type I BK channels. Other neurons express BK channels assembled with the accessory β4 subunit conferring slow gating of type II BK channels. However, it is not clear how protein phosphorylation modulates these two distinct BK channel types. Using β4-knockout mice, we compared fast- or slow-gated BK channels in response to changes in phosphorylation status of hippocampus dentate gyrus granule neurons. We utilized the selective PP2A/PP4 phosphatase inhibitor Fostriecin to study changes in action potential shape and firing properties of the neurons. In β4-knockout neurons, Fostriecin increases BK current, speeds up BK channel activation and reduces action potential amplitudes. Fostriecin increases spiking during early components of an action potential train. In contrast, inhibition of BK channels through β4 in wild-type neurons or by the BK channel inhibitor Paxilline opposes Fostriecin effects. Voltage clamp recordings of neurons reveal that Fostriecin increases both calcium and BK currents. However, Fostriecin does not activate BK α channels in transfected HEK293 cells lacking calcium channels. In summary, these results suggest that fast-gating, type I BK channels lacking β4 can increase neuronal excitability in response to reduced phosphatase activity and activation of calcium channels. By opposing BK channel activation, the β4 subunit plays an important role in moderating firing frequency regardless of changes in phosphorylation status.

Activity-dependent Phosphorylation of Neuronal Kv2.1 Potassium Channels by CDK5

Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible, such that rephosphorylation occurs after removal of excitatory stimuli. Here, we show that cyclin-dependent kinase 5 (CDK5), a Pro-directed Ser/Thr protein kinase, directly phosphorylates Kv2.1, and determines the constitutive level of Kv2.1 phosphorylation, the rapid increase in Kv2.1 phosphorylation upon acute blockade of neuronal activity, and the recovery of Kv2.1 phosphorylation after stimulus-induced dephosphorylation. We also demonstrate that although the phosphorylation state of Kv2.1 is also shaped by the activity of the PP1 protein phosphatase, the regulation of Kv2.1 phosphorylation by CDK5 is not mediated through the previously described regulation of PP1 activity by CDK5. Together, these studies support a novel role for CDK5 in regulating Kv2.1 channels through direct phosphorylation.

English

Aconitine, a strong poisonous type of alkaloid, has a pharmaceutical effect in stimulating the membranes of cardiomyocyte. However, other effects of aconitine on the Connexin43 (Cx43) and PKC&alpha; expression&nbsp;on cardiomyocyte are unknown. In this study, we investigated whether aconitine also mediates the phosphorylation status of Cx43 and PKC&alpha;&nbsp;in cultured ventricular myocytes of neonatal rats. The band intensity of phosphorylated Cx43 and nonphosphorylated Cx43 in cultured and aconitine-treated cardiomyocytes were determined by Western blot analysis. The changes in phosphorylation status occurring in PKC&alpha; in cultures were revealed by quantitative immunofluorescent microscopy. A decreased band intensity (0.37&plusmn;0.04) of phosphorylated Cx43 (P-Cx43) and a concomitant increased band intensity (3.56 &plusmn; 0.65)&nbsp;of nonphosphorylated Cx43 (NP-Cx43) were found, compared to the controls (1.00&nbsp;for P-Cx43 and NP-Cx43). It also revealed that, after aconitine treatment, the amount of phosphorylated PKC&alpha; (P-PKC&alpha;) decreased significantly. Similar changes were revealed in phosphorylation status occurring in PKC&alpha; in the cultures under the same treatment conditions. These observations suggest that aconitine not only induces dephosphorylation of Cx43, but also alters expression of P-PKC&alpha; in cultured cardiomyocytes. &nbsp; Key words:&nbsp;Aconitine, cardiomyocyte, connexin 43 (Cx43), protein kinase C-&alpha; (PKC&alpha;), protein phosphorylation.

Regulation of p53 and cell proliferation by resveratrol and its derivatives in breast cancer cells: An in silico and biochemical approach targeting integrin αvβ3

Resveratrol is a grape polyphenol with cancer preventative activities in tissue culture and animal model studies. Potential of resveratrol as a broad-based chemopreventive agent have been questioned by its limited bioavailability. The bioefficacy of resveratrol was compared with its derivatives, triacetyl-resveratrol (trans-3,5,4'-triacetylstilbene) and trimethoxy-resveratrol (trans-3,5,4'-trimethoxystilbene) in both estrogen receptor-α (ERα)-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cells. Binding to integrin αvβ3 and control of cell proliferation and p53 were chosen as targets for comparative analysis using an in silico and biochemical approach. Resveratrol and triacetyl-resveratrol interacted avidly and specifically with integrin αvβ3 through binding at the site targeted by the high affinity cyclic Arg-Gly-Asp (RGD) peptide. In contrast, binding of trimethoxy-resveratrol to this site was substantially less robust. Moreover, the different stilbenes also elicited diverse cellular and signaling responses in MCF-7 and MDA-MB-231 cells, as evidenced by analysis of colony formation, cell proliferation, cell cycle phase transition, the extent of phosphorylation of p53 at Ser15 and p53-inducible proteins, p21 and p53R2, respectively. Further, stilbene-elicited signaling cascade leading to p53 activation was examined in MCF-7 cells and results showed that resveratrol and triacetyl-resveratrol induced both ERK and p38 phosphorylation, whereas only marginal changes in state of phosphorylation in these two kinases were observed in trimethoxy-resveratrol-treated cells. Taken together, these results support that resveratrol and triacetyl-resveratrol regulate proliferation and gene expression in breast cancer cells by utilizing largely similar signaling molecules and pathways and cellular events, which appear quite distinct from those targeted by trimethoxy-resveratrol.