Induced Protein Phosphorylation Research Articles

Biodegradable Microspheres and Hydrogel Drug Delivery System of Tumor Necrosis Factor (TNF) Inhibitor and Growth Differentiation Factor 5 (GDF5) Reduces Disk Inflammation in the Rabbit Model.

Preclinical study. Develop and test a drug delivery system (DDS) composed of anti-inflammatories and growth factors in the rabbit disk injury model. Biological therapies that inhibit inflammation or enhance cell proliferation can alter intervertebral disk (IVD) homeostasis to favor regeneration. As biological molecules have short half-lives and one molecule may not cover multiple disease pathways, effective treatments may require a combination of growth factors and anti-inflammatory agents delivered in a sustained manner. Biodegradable microspheres were generated separately to encapsulate tumor necrosis factor alpha (TNFα) inhibitors [etanercept (ETN)] or growth differentiation factor 5 (GDF5) and were embedded into a thermoresponsive hydrogel. Release kinetics and activity of ETN and GDF5 were measured in vitro . For in vivo testing, New Zealand White rabbits (n=12) underwent surgery for disk puncture and treatment with blank-DDS, ETN-DDS, or ETN+GDF5-DDS at levels L34, L45, and L56. Radiographic and magnetic resonance images of the spines were obtained. The IVDs were isolated for histologic and gene expression analyses. ETN and GDF5 were encapsulated into poly (L-lactide-co-glycolide) microspheres and had average initial bursts of 2.4±0.1 and 11.2±0.7μg from DDS, respectively. In vitro studies confirmed that ETN-DDS inhibited TNFα-induced cytokine release and GDF5-DDS induced protein phosphorylation. In vivo studies showed that rabbit IVDs treated with ETN+GDF5-DDS had better histologic outcomes, higher levels of extracellular, and lower levels of inflammatory gene expression than IVDs treated with blank-DDS or ETN-DDS. This pilot study demonstrated that DDS can be fabricated to deliver sustained and therapeutic dosages of ETN and GDF5. In addition, ETN+GDF5-DDS may have greater anti-inflammatory and regenerative effects than ETN-DDS alone. Thus, intradiscal injection of controlled release TNF-α inhibitors and growth factors may be a promising treatment to reduce disk inflammation and back pain.

Quantitative Mass Spectrometry Reveals a Proteome‐wide Role for Cyclin A and Cks1 in Multisite, Non‐Proline Directed Phosphorylation by CDK1

Cdk1 is a master kinase of mitotic cell division, phosphorylating thousands of substrates in a highly temporally regulated manner. How Cdk1 phosphorylates the right substrate at the right time remains an open question.Cdk1 activity requires complex formation with a cyclin subunit: cyclin A or cyclin B. Additionally, active Cdk1 is found in complex with a small adaptor protein called Cks1. Cyclin subunits activate Cdk1 and tune Cdk1 substrate choice through interactions with short linear motifs on substrates. While Cks1 is essential in all organisms tested to date, a general function for Cks1 in Cdk1 substrate choice has not been demonstrated.In this study, we investigated the roles of the non‐catalytic subunits of Cdk1 in substrate phosphorylation in human cells. We designed an in vitro assay in which we induced protein phosphorylation in fixed and permeabilized human lymphoblasts using active recombinant Cdk1 either in complex with cyclin A, cyclin B, or cyclin B+Cks1. Global phosphorylation levels were then measured using quantitative mass spectrometry.Our data demonstrate that, contrary to the textbook model of Cdk1 as a proline‐directed kinase, both cyclin A and Cks1 facilitate widespread Cdk1 phosphorylation of sites lacking a +1 proline motif. Our results also suggest a role for Cks1 in multisite phosphorylation. We propose a model whereby cyclin A and Cks1 have indispensable functions in promoting non‐proline directed, multisite phosphorylation of Cdk1 substrates to mediate dynamic, switch‐like changes in protein function required for mitotic entry, proper chromosome segregation and cell division.

Functional Interaction of Spexin and Adiponectin Forming a Local Feedbackin Goldfish Hepatocytes for Feeding Regulation

Spexin (SPX), a neuropeptide with pleiotropic functions, has been confirmed to be a novel satiety factor in fish models. Adiponectin (AdipoQ), the most abundant adipokine in circulation, is involved in lipid and glucose metabolism and its insulin-sensitizing, cardioprotective, and anti-inflammatory actions are also well-documented. However, the interaction between SPX and AdipoQ has not be reported and very little is known regarding the functions of AdipoQ in non-mammalian species. In this study, AdipoQ was cloned in goldfish and found to be widely expressed at tissue level including the liver. Sequence alignment and in silico protein modelling revealed that its a.a. sequence and 3D protein structure were highly comparable to the mammalian counterparts. Recombinant protein of goldfish AdipoQ was expressed in E. coli and IP injection of the protein purified could suppress foraging activity and food intake in goldfish. Food intake in goldfish, interestingly, could elevate plasma levels of SPX and AdipoQ with parallel rises of their transcript expression in the liver. In primary culture of goldfish hepatocytes, SPX treatment was shown to induce protein phosphorylation of MEK1/2 and ERK1/2 with a parallel rise in AdipoQ mRNA level. SPX-induced AdipoQ mRNA expression, however, was sensitive to the blockade of PLC/PKC, Ca2+/CaMK-II and MEK1/2/ERK1/2 but not cAMP/PKA cascades. In reciprocal experiments, AdipoQ treatment could induce protein phosphorylation of AMPK, Akt and P38 MAPK in goldfish hepatocytes. Meanwhile, AdipoQ was also effective in reducing SPX mRNA level and this inhibitory effect was negated by blocking the AMPK/PPAR, PI3K/Akt and P38 MAPK but not the MEK1/2/ERK1/2 or PLC/PKC pathways. Apparently, the PI3K/Akt and P38 MAPK cascades were functionally coupled with AMPK activation. These results imply that (i) AdipoQ, similar to SPX, can be induced by food intake and serve as a satiety signal in goldfish, (ii) AdipoQ expression in goldfish liver can be up-regulated by SPX via the PLC/PKC, Ca2+/CaMK-II and MEK1/2/ERK1/2 pathways, which may enhance the satiation response caused by SPX after food intake, and (iii) AdipoQ can inhibit SPX expression at hepatic level via the AMPK/PPAR, PI3K/Akt and P38 MAPK cascades, which may lead to signal termination of SPX. These findings, as a whole, suggest that AdipoQ production in goldfish liver not only can form a signal amplification step for the satiation response of SPX but also constitute a local feedback to turn off SPX signal at the hepatic level.

LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells.

LDL receptor-related proteins 6 (LRP6) is a type I transmembrane receptor (C-terminus in cytosol), which appears to be essential in numerous biological processes, since it is an essential co-receptor of Wnt ligands for canonical β-catenin dependent signal transduction. It was shown that tissue plasminogen activator (tPA), physically interacting with LRP6, induces protein phosphorylation, which may have large implication in the regulation of neural processes. In this investigation we analyzed whether LRP6 is associated with lipid rafts following tPA triggering in neuroblastoma cells and the role of raft integrity in LRP6 cell signaling. Sucrose gradient separation revealed that phosphorylated LRP6 was mainly, but not exclusively present in lipid rafts; this enrichment became more evident after triggering with tPA. In these microdomains LRP6 is strictly associated with ganglioside GM1, a paradigmatic component of these plasma membrane compartments, as revealed by coimmunoprecipitation experiments. As expected, tPA triggering induced LRP6 phosphorylation, which was independent of LRP1, as revealed by knockdown experiments by siRNA, but strictly dependent on raft integrity. Moreover, tPA induced β-catenin phosphorylation was also significantly prevented by previous pretreatment with methyl-β-cyclodextrin. Our results demonstrate that LRP6 mediated signal transduction pathway triggered by tPA acts through lipid rafts in neuroblastoma cells. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents. Indeed, these data, pointing to the key role of lipid rafts in tPA triggered signaling involving β-catenin, may have pharmacological implications, suggesting that cyclodextrins, and potentially other drugs, such as statins, may represent an useful tool.

Protective effects and mechanisms of Xingnaojing Injection on early global brain ischemic-induced deep coma in rats

To study the protective effect of Xingnaojing Injection on early global brain ischemia-induced deep coma in rats. Methods: The deep coma model was induced by global brain ischemia by using four-vessel occlusion method in male SD rats. According to the body weight, the rats were randomly divided into 8 groups: a model control group, three different dose of Xingnaojing Injection (1.8, 3.6 and 5.4 mL.kg-1) groups, a Xingnaojing Injection (3.6 mL.kg-1) plus PI3K inhibitor group, a naloxone injection (0.04 mL.kg-1) group and a naloxone injection (0.04 mL.kg-1) plus Xingnaojing Injection (3.6 mL.kg-1) group (n=8 per group). In addition, eight animals served as the sham group were performed same operation with the model group excepting no blockage of the blood vessels. After the operation, three different doses of Xingnaojing Injection and/or naloxone injection were given intravenously once a day for three days. Ten μL PI3K inhibitor (LY294002, 10 mmol/L) was injected via anterior cerebral ventricle at once after global brain ischemia. The awakening time after the first drug treatment, the grasping power and the autonomous activity within 10 min after the last drug treatment were recorded. The levels of both dopamine (DA) and glutamate (Glu) in cerebrospinal fluid were detected by ELISA. The pathological changes were observed in brain tissue slices with HE staining and the protein levels of Akt/p-Akt and cAMP-response element binding protein (CREB)/p-CREB in hippocampus were detected by Western blotting. Results: Comparing with the model group, single administration of Xingnaojing Injection could significantly shorten the waking time (P<0.05) and continuous administration of Xingnaojing Injection for 3 d could increase grasping power, distance, frequency and duration of autonomous activities (P<0.05 or P<0.01) in the deep coma rat. Also, Xingnaojing Injection could inhibit these increases in neurotransmitters DA and Glu contents (P<0.05 or P<0.01), and improve pathological changes of hippocampal tissue. Xingnaojing Injection significantly induced protein phosphorylation of both Akt and CREB (P<0.05 or P<0.01); this effect was inhibited by PI3K inhibitor (P<0.05 or P<0.01). Moreover, the protective effects of naloxone on awakening time, grasping power, the autonomous activity and hippocampus damage in global brain ischemia-induced deep coma could be enhanced by joint use of Xingnaojing Injection (P<0.05 or P<0.01). Conclusion: Xingnaojing Injection could significantly improve deep coma induced by global brain ischemia in rat, which is related to inducing PI3K/Akt-dependent protein phosphorylation of CREB, and reducing hippocampal damage. The protective effect of Xingnaojing Injection is synergistically enhanced by naloxone.

The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation.

Holometabolous insects stop feeding at the final larval instar stage and then undergo metamorphosis; however, the mechanism is unclear. In the present study, using the serious lepidopteran agricultural pest Helicoverpa armigera as a model, we revealed that 20-hydroxyecdysone (20E) binds to the dopamine receptor (DopEcR), a G protein-coupled receptor, to stop larval feeding and promote pupation. DopEcR was expressed in various tissues and its level increased during metamorphic molting under 20E regulation. The 20E titer was low during larval feeding stages and high during wandering stages. By contrast, the dopamine (DA) titer was high during larval feeding stages and low during the wandering stages. Injection of 20E or blocking dopamine receptors using the inhibitor flupentixol decreased larval food consumption and body weight. Knockdown of DopEcR repressed larval feeding, growth, and pupation. 20E, via DopEcR, promoted apoptosis; and DA, via DopEcR, induced cell proliferation. 20E opposed DA function by repressing DA-induced cell proliferation and AKT phosphorylation. 20E, via DopEcR, induced gene expression and a rapid increase in intracellular calcium ions and cAMP. 20E induced the interaction of DopEcR with G proteins αs and αq. 20E, via DopEcR, induced protein phosphorylation and binding of the EcRB1-USP1 transcription complex to the ecdysone response element. DopEcR could bind 20E inside the cell membrane or after being isolated from the cell membrane. Mutation of DopEcR decreased 20E binding levels and related cellular responses. 20E competed with DA to bind to DopEcR. The results of the present study suggested that 20E, via binding to DopEcR, arrests larval feeding and promotes pupation.

Phosphorylation-guarded light-harvesting complex II contributes to broad-spectrum blast resistance in rice

Environmental conditions are key factors in the progression of plant disease epidemics. Light affects the outbreak of plant diseases, but the underlying molecular mechanisms are not well understood. Here, we report that the light-harvesting complex II protein, LHCB5, from rice is subject to light-induced phosphorylation during infection by the rice blast fungus Magnaporthe oryzae We demonstrate that single-nucleotide polymorphisms (SNPs) in the LHCB5 promoter control the expression of LHCB5, which in turn correlates with the phosphorylation of LHCB5. LHCB5 phosphorylation enhances broad-spectrum resistance of rice to M. oryzae through the accumulation of reactive oxidative species (ROS) in the chloroplast. We also show that LHCB5 phosphorylation-induced resistance is inheritable. Our results uncover an immunity mechanism mediated by phosphorylation of light-harvesting complex II.

Intracellular Signaling in Key Pathways Is Induced by Treatment with Ultrasound and Microbubbles in a Leukemia Cell Line, but Not in Healthy Peripheral Blood Mononuclear Cells.

Treatment with ultrasound and microbubbles (sonoporation) to enhance therapeutic efficacy in cancer therapy is rapidly expanding, but there is still very little consensus as to why it works. Despite the original assumption that pore formation in the cell membrane is responsible for increased uptake of drugs, the molecular mechanisms behind this phenomenon are largely unknown. We treated cancer cells (MOLM-13) and healthy peripheral blood mononuclear cells (PBMCs) with ultrasound at three acoustic intensities (74, 501, 2079 mW/cm2) ± microbubbles. We subsequently monitored the intracellular response of a number of key signaling pathways using flow cytometry or western blotting 5 min, 30 min and 2 h post-treatment. This was complemented by studies on uptake of a cell impermeable dye (calcein) and investigations of cell viability (cell count, Hoechst staining and colony forming assay). Ultrasound + microbubbles resulted in both early changes (p38 (Arcsinh ratio at high ultrasound + microbubbles: +0.5), ERK1/2 (+0.7), CREB (+1.3), STAT3 (+0.7) and AKT (+0.5)) and late changes (ribosomal protein S6 (Arcsinh ratio at low ultrasound: +0.6) and eIF2α in protein phosphorylation). Observed changes in protein phosphorylation corresponded to changes in sonoporation efficiency and in viability, predominantly in cancer cells. Sonoporation induced protein phosphorylation in healthy cells was pronounced (p38 (+0.03), ERK1/2 (−0.03), CREB (+0.0), STAT3 (−0.1) and AKT (+0.04) and S6 (+0.2)). This supports the hypothesis that sonoporation may enhance therapeutic efficacy of cancer treatment, without causing damage to healthy cells.

Phosphorylation of gp91phox/NOX2 in Human Neutrophils.

The phagocyte NADPH oxidase NOX2 was the first NOX family member to be discovered. It is responsible for the production of reactive oxygen species that are required for bacterial killing and host defense. Activated NOX2 is an enzymatic complex composed of two membrane proteins, p22phox and gp91phox (renamed NOX2), which form the cytochrome b558, and four cytosolic proteins, p47phox, p67phox, p40phox, and the small GTPase Rac2. Except for Rac2, all proteins from the complex become phosphorylated during neutrophil activation, suggesting the importance of this process in NOX2 regulation. The phosphorylation of the cytosolic components, and in particular p47phox, has been extensively studied; however, the phosphorylation of the membrane proteins was less studied, in part due to the lack of good antibodies and accurate membrane solubilization techniques. In this chapter, we describe a method we have used to study NOX2 phosphorylation, which is based on the labeling of the intracellular ATP pool with 32P prior to applying a stimulus inducing protein phosphorylation. We also describe the solubilization of membrane-bound gp91phox/NOX2 and analysis by immunoprecipitation, polyacrylamide gel electrophoresis, electrophoretic transfer, phosphoamino acid analysis, and autoradiography. This protocol can also be used to study the possible phosphorylation of other NOX family members.

S-Nitrosylation of Calcium-Handling Proteins in Cardiac Adrenergic Signaling and Hypertrophy.

The regulation of calcium (Ca(2+)) homeostasis by β-adrenergic receptor (βAR) activation provides the essential underpinnings of sympathetic regulation of myocardial function, as well as a basis for understanding molecular events that result in hypertrophic signaling and heart failure. Sympathetic stimulation of the βAR not only induces protein phosphorylation but also activates nitric oxide-dependent signaling, which modulates cardiac contractility. Nonetheless, the role of nitric oxide in βAR-dependent regulation of Ca(2+) handling has not yet been explicated fully. To elucidate the role of protein S-nitrosylation, a major transducer of nitric oxide bioactivity, on βAR-dependent alterations in cardiomyocyte Ca(2+) handling and hypertrophy. Using transgenic mice to titrate the levels of protein S-nitrosylation, we uncovered major roles for protein S-nitrosylation, in general, and for phospholamban and cardiac troponin C S-nitrosylation, in particular, in βAR-dependent regulation of Ca(2+) homeostasis. Notably, S-nitrosylation of phospholamban consequent upon βAR stimulation is necessary for the inhibitory pentamerization of phospholamban, which activates sarcoplasmic reticulum Ca(2+)-ATPase and increases cytosolic Ca(2+) transients. Coincident S-nitrosylation of cardiac troponin C decreases myocardial sensitivity to Ca(2+). During chronic adrenergic stimulation, global reductions in cellular S-nitrosylation mitigate hypertrophic signaling resulting from Ca(2+) overload. S-Nitrosylation operates in concert with phosphorylation to regulate many cardiac Ca(2+)-handling proteins, including phospholamban and cardiac troponin C, thereby playing an essential and previously unrecognized role in cardiac Ca(2+) homeostasis. Manipulation of the S-nitrosylation level may prove therapeutic in heart failure.

Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine

BackgroundAmphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone derivatives cathine and norephedrine, in comparison with a defined extract of the psychostimulating herb khat (Catha edulis Forsk.). Intracellular protein modifications in single cells were studied using immunostaining and flow cytometry, cell viability was determined by Annexin V-FITC/Propidium Iodide staining, and T-lymphocyte proliferation was measured by 3H-thymidine incorporation.ResultsCathinone, cathine and norephedrine generally reduced post-translational modifications of intracellular signal transducers in T-lymphocytes, B-lymphocytes, natural killer cells and monocytes, most prominently affecting c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), p38 MAPK (p-Thr180/p-Tyr182) and p53 (both total p53 protein and p-Ser15). In contrast, the botanical khat-extract induced protein phosphorylation of STAT1 (p-Tyr701), STAT6 (p-Tyr641), c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), NF-κB (p-Ser529), Akt (p-Ser473), p38 MAPK (p-Thr180/p-Tyr182), p53 (Ser15) as well as total p53 protein. Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes. Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death.ConclusionsSingle-cell signal transduction analyses of leukocytes distinctively discriminated between stimulation with cathinone and the structurally similar derivatives cathine and norephedrine. Cathinone, cathine and norephedrine reduced phosphorylation of c-Cbl, ERK1/2, p38 MAPK and p53(Ser15), and norephedrine induced T-lymphocyte proliferation. Khat-extract induced protein phosphorylation of signal transducers, p38 MAPK and p53, followed by reduced cell proliferation and cell death. This study suggests that protein modification-specific single-cell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts.

Cryopreservation of common carp (Cyprinus carpio L.) sperm induces protein phosphorylation in tyrosine and threonine residues

The effect of cryopreservation on the protein phosphorylation/dephosphorylation pattern of common carp (Cyprinus carpio) sperm is described. Sperm was diluted in dimethyl sulfoxide (DMSO) and ethylene glycol (EG)-based extenders, followed by equilibration, freezing, and thawing. Proteins extracted from fresh and cryopreserved spermatozoa were separated on SDS-PAGE and two-dimensional gel electrophoresis, blotted on polyvinylidene difluoride membrane, and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. For the subsequent protein identification we used matrix-associated laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated that cryopreservation with either DMSO or EG extender significantly altered the phosphorylation state of sperm proteins on tyrosine or threonine residues. A dramatic decrease in tyrosine phosphorylation was detected in the cryopreservation procedures with DMSO extender. Endoplasmin, transketolase, and S-adenosylhomocysteine hydrolase were identified as proteins that play a key role in cellular stress responses and oxidation and/or reduction reactions. Results indicate that the phosphorylation and/or dephosphorylation modifications of sperm proteins that occur during cryopreservation could stimulate a series of biochemical effects interfering with spermatozoa function and leading to a loss of motility and fertilization ability. Our findings indicated that use of EG extender provided superior protein preservation during sperm storage.

The kinesin-like protein TOP promotes Aurora localisation and induces mitochondrial, chloroplast and nuclear division

The cell cycle usually refers to the mitotic cycle, but the cell-division cycle in the plant kingdom consists of not only nuclear but also mitochondrial and chloroplast division cycle. However, an integrated control system that initiates division of the three organelles has not been found. We report that a novel C-terminal kinesin-like protein, three-organelle division-inducing protein (TOP), controls nuclear, mitochondrial and chloroplast divisions in the red alga Cyanidioschyzon merolae. A proteomics study revealed that TOP is a member of a complex of mitochondrial-dividing (MD) and plastid-dividing (PD) machineries (MD/PD machinery complex) just prior to constriction. After TOP localizes at the MD/PD machinery complex, mitochondrial and chloroplast divisions occur and the components of the MD/PD machinery complexes are phosphorylated. Furthermore, we found that TOP downregulation impaired both mitochondrial and chloroplast divisions. MD/PD machinery complexes were formed normally at each division site but they were neither phosphorylated nor constricted in these cells. Immunofluorescence signals of Aurora kinase (AUR) were localized around the MD machinery before constriction, whereas AUR was dispersed in the cytosol by TOP downregulation, suggesting that AUR is required for the constriction. Taken together our results suggest that TOP induces phosphorylation of MD/PD machinery components to accomplish mitochondrial and chloroplast divisions prior to nuclear division, by relocalization of AUR. In addition, given the presence of TOP homologs throughout the eukaryotes, and the involvement of TOP in mitochondrial and chloroplast division may illuminate the original function of C-terminal kinesin-like proteins.

Bucillamine inhibits CD40-mediated Akt activation and antibody production in mouse B‐cell lymphoma

The improvement of rheumatoid factor titers in patients with rheumatoid arthritis is one of the significant clinical effects of bucillamine (Buc). In this study, we investigated the effects of SA981, an active metabolite of Buc, and methotrexate (MTX) on CD40-mediated antibody production using mouse B‐cell lymphoma, BCL1. SA981 significantly attenuated CD40-mediated antibody production in a concentration-dependent manner, but weakly affected cell proliferation. In contrast, MTX did not attenuate CD40-mediated antibody production until it had strongly inhibited cell proliferation at a concentration of 100nM. CD40 signaling induced protein phosphorylation, including Akt phosphorylation, p38 mitogen-activated protein kinase (p38MAPK), and IκBα. SA981 at a concentration of 30μM attenuated CD40-mediated Akt phosphorylation, but not p38MAPK or IκBα phosphorylation. MTX at a concentration of 100nM did not affect CD40-mediated Akt, p38MAPK, or IκBα phosphorylation. Commercially available Akt inhibitor VIII significantly attenuated CD40-mediated IgM production at a concentration of 100nM without significant inhibition of cell proliferation. These results suggest that SA981 inhibits CD40-mediated antibody production in mouse B‐cell lymphoma, at least in part, by attenuation of Akt phosphorylation.

Alemtuzumab induction of intracellular signaling and apoptosis in malignant B lymphocytes

The molecular changes induced by alemtuzumab following binding of CD52 on B tumor cells were investigated. Alemtuzumab alone had no detectable impact on cell signaling but cross-linking of alemtuzumab on the surface of B tumor lines with anti-human Fc antibodies induced a transient Ca2 + flux followed by phosphorylation of several kinases involved in stress and survival pathways, and expression of associated proteins including TNF-α. Cross-linking of alemtuzumab also induced capping and caspase-dependent apoptosis of the tumor lines. When using primary cells from B-CLL patients, alemtuzumab alone was capable of inducing protein phosphorylation and apoptosis through the cross-linking of alemtuzumab by FcγRIIb receptors on B-CLL cells. Apoptosis was prevented by blocking of FcγRIIb receptors with anti-CD32 antibody. Overall, our results indicate that cross-linking of alemtuzumab on B tumor cells can occur naturally through Fc receptor interaction and leads to the activation of specific cellular pathways and induction of apoptosis.

A new in vitro system for activating the cell cycle checkpoint

In response to DNA damage, cells launch elegant networks of genome surveillance mechanisms, called cell cycle checkpoints, to detect and repair damaged DNA to maintain the genome stability. Key components of cell cycle checkpoints are two PI3K-related protein kinases (PIKK), ATR and ATM, which participate in both sensing the DNA damage and transducing the damage signal through phosphorylating two target protein kinases, Chk1 and Chk2, respectively. However, how exactly cell cycle checkpoints are activated, maintained, and terminated are not completely understood. Given the complexity of the cell cycle checkpoint signaling and the cellular environment, systems that can faithfully mimic the cell cycle checkpoint activation in vitro, such as the Xenopus egg extracts, are of extreme value in dissecting the precise molecular mechanisms underlying DNA damage response. Here we describe that the well-established in vitro transcription and translation (IVTNT) system has the capability to induce protein phosphorylation of substrates for ATR or ATM, including Chk1, Rad17, and ATM itself. These phosphorylation events highly mimic those occurring in cells when treated with DNA damaging agents. Our results demonstrate that the IVTNT system could be developed into a novel in vitro system to facilitating the dissecting of mechanisms leading to cell cycle checkpoint activation in vivo.

Cigarette smoke extract induced protein phosphorylation changes in human microvascular endothelial cells in vitro

Phosphorylation is the most widely studied posttranslational modification (PTM) and is an important regulatory mechanism used during cellular responses to external stimuli. The kinases and phosphatases that regulate protein phosphorylation are known to be affected in many human diseases. Cigarette smoking causes cardiovascular disease (CVD). Endothelial cells play a pivotal role in CVD initiation and development; however, there have been limited investigations of the specific signaling cascades and protein phosphorylations activated by cigarette smoke in endothelial cells. The purpose of this research was to better understand the differential protein phosphorylation in endothelial cells stimulated with extracts of cigarette smoke total particulate matter (CS-TPM) in vitro. Human microvascular endothelial cells were exposed in vitro to CS-TPM at concentrations that were shown to cause endothelial cell dysfunction. The phosphorylated proteins were isolated using phosphoprotein-specific chromatography, followed by enzymatic digestion and nano-flow capillary liquid chromatography (ncap-LC) coupled to high resolution mass spectrometry. This study putatively identified 94 proteins in human microvascular endothelial cells that were differentially bound to a phosphoprotein-specific chromatography column following exposure to CS-TPM suggesting differential phosphorylation. Pathway analysis has also been conducted and confirmations of several observations have been made using immunoaffinity-based techniques (e.g., Western blotting).

Differential Expression of Novel Tyrosine Kinase Substrates during Breast Cancer Development

To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ technology, and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.

Environmentally Modulated Phosphoproteome of Photosynthetic Membranes in the Green Alga Chlamydomonas reinhardtii

Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of the green alga Chlamydomonas reinhardtii revealed that the major environmentally dependent changes in phosphorylation are clustered at the interface between the photosystem II (PSII) core and its light-harvesting antennae (LHCII). The photosynthetic membranes that were isolated form the algal cells exposed to four distinct environmental conditions affecting photosynthesis: (i) dark aerobic, corresponding to photosynthetic State 1; (ii) dark under nitrogen atmosphere, corresponding to photosynthetic State 2; (iii) moderate light; and (iv) high light. The surface-exposed phosphorylated peptides were cleaved from the membrane by trypsin, methyl-esterified, enriched by immobilized metal affinity chromatography, and sequenced by nanospray-quadrupole time-of-flight mass spectrometry. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii. Amino-terminal acetylation of seven proteins was concomitantly determined. Sequenced amino termini of six mature LHCII proteins differed from the predicted ones. The State 1-to-State 2 transition induced phosphorylation of the PSII core components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10. Exposure of the algal cells to either moderate or high light caused additional phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to specific hyperphosphorylation of CP29 at seven distinct residues, phosphorylation of another minor LHCII constituent, CP26, at a single threonine, and double phosphorylation of additional subunits of a major LHCII complex including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Environmentally induced protein phosphorylation at the interface of PSII core and the associated antenna proteins, particularly multiple differential phosphorylations of CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions and for LHCII uncoupling from PSII under high light stress to allow thermal energy dissipation.

Intracellular Ca2+ regulates the phosphorylation and the dephosphorylation of ciliary proteins via the NO pathway.

The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO syntase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating.