Tyrosine Phosphorylation Pathways Research Articles

The effect of temperature shock on enhancing sperm motility: A new insight into molecular and cellular mechanisms through current procedures

Asthenozoospermia, characterized by reduced progressive motility (below 32%), is a leading cause of male infertility. Intrauterine Insemination (IUI) serves as the first-line treatment in sperm-related processes, aiming to increase sperm motility. Although Density Gradient Centrifugation (DGC) and swim-up procedures have become primary treatment options, considered the gold standard for IUI, there is a need to optimize these methods with additional factors to enhance motility, particularly for Asthenozoospermia patients.We hypothesize that a brief cold shock, lasting approximately 15–20 min at 17 °C, followed by incubation and sperm processing, can substantially enhance motility. For the first time, the hypothesis and evaluation of post-exposure incubation following a short-term cold shock, within the temperature range of 17–20 °C, were explored and discussed.This hypothesis appears valid and holds importance for several reasons. Firstly, there is a significant relationship between cold shock and its impact on intracellular components and the molecular organization of the cell membrane. Additionally, this process regulates various physiological cellular processes, including transbilayer distribution, acrosome reaction, and sperm motility. Moreover, lowering the temperature induces a molecular flux, facilitating calcium uptake by sperm. This process significantly impacts the quantity of calcium accessible to the sperm, particularly within the flagellum. Choosing the temperature below 20 °C temperature can lead to the activation of calcium-absorbing protein channels in the shortest possible time. Furthermore, a crucial reaction in IUI is sperm capacitation, which occurs when the protein tyrosine phosphorylation pathway, a key factor, is activated. Alternatively, the cold incubation of sperm reveals an increased level of tyrosine phosphorylation and a higher percentage of induced Acrosome Reaction (AR) compared to sperm incubated at normal temperatures. Hence, when sperm is cooled, it initiates a process called tyrosine phosphorylation, ultimately leading to sperm capacitation. Besides these logical reasons, there is limited empirical and incidental support in two cases that confirmed the hypothesis well.

Myeloid Src-family kinases are critical for neutrophil-mediated autoinflammation in gout and motheaten models.

Autoinflammatory diseases include a number of monogenic systemic inflammatory diseases, as well as acquired autoinflammatory diseases such as gout. Here, we show that the myeloid Src-family kinases Hck, Fgr, and Lyn are critical for experimental models of gout, as well as for genetically determined systemic inflammation in the Ptpn6me-v/me-v (motheaten viable) mouse model. The Hck-/-Fgr-/-Lyn-/- mutation abrogated various monosodium urate (MSU) crystal-induced pro-inflammatory responses of neutrophils, and protected mice from the development of gouty arthritis. The Src-family inhibitor dasatinib abrogated MSU crystal-induced responses of human neutrophils and reduced experimental gouty arthritis in mice. The Hck-/-Fgr-/-Lyn-/- mutation also abrogated spontaneous inflammation and prolonged the survival of the Ptpn6me-v/me-v mice. Spontaneous adhesion and superoxide release of Ptpn6me-v/me-v neutrophils were also abolished by the Hck-/-Fgr-/-Lyn-/- mutation. Excessive activation of tyrosine phosphorylation pathways in myeloid cells may characterize a subset of autoinflammatory diseases.

The non-receptor tyrosine kinase Pyk2 modulates acute locomotor effects of cocaine in D1 receptor-expressing neurons of the nucleus accumbens

The striatum is critical for cocaine-induced locomotor responses. Although the role of D1 receptor-expressing neurons is established, underlying molecular pathways are not fully understood. We studied the role of Pyk2, a non-receptor, calcium-dependent protein-tyrosine kinase. The locomotor coordination and basal activity of Pyk2 knock-out mice were not altered and major striatal protein markers were normal. Cocaine injection increased Pyk2 tyrosine phosphorylation in mouse striatum. Pyk2-deficient mice displayed decreased locomotor response to acute cocaine injection. In contrast, locomotor sensitization and conditioned place preference were normal. Cocaine-activated ERK phosphorylation, a signaling pathway essential for these late responses, was unaltered. Conditional deletion of Pyk2 in the nucleus accumbens or in D1 neurons reproduced decreased locomotor response to cocaine, whereas deletion of Pyk2 in the dorsal striatum or in A2A receptor-expressing neurons did not. In mice lacking Pyk2 in D1-neurons locomotor response to D1 agonist SKF-81297, but not to an anticholinergic drug, was blunted. Our results identify Pyk2 as a regulator of acute locomotor responses to psychostimulants. They highlight the role of tyrosine phosphorylation pathways in striatal neurons and suggest that changes in Pyk2 expression or activation may alter specific responses to drugs of abuse, or possibly other behavioral responses linked to dopamine action.

PH-dependent effects of procaine on equine gamete activation†.

Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) procaine-supplemented capacitating medium (CM) still demonstrated hyperactivated motility, whereas CM without NaCl or Na+ did not. Both procaine and NH4Cl, another weak base, were shown to trigger a cytoplasmic pH increase (BCECF-acetoxymethyl (AM)), which is primarily induced by a pH rise in acidic cell organelles (Lysosensor green dnd-189), accompanied by hypermotility in stallion sperm. As for procaine, 25 mM NH4Cl also induced oocyte cytokinesis. Interestingly, hyperactivated motility was reliably induced by 2.5–10 mM procaine, whereas a significant cytoplasmic cAMP increase and tail-associated protein tyrosine phosphorylation were only observed at 10 mM. Moreover, 25 mM NH4Cl did not support the latter capacitation characteristics. Additionally, cAMP levels were more than 10× higher in boar than stallion sperm incubated under similar capacitating conditions. Finally, stallion sperm preincubated with 10 mM procaine did not fertilize equine oocytes. In conclusion, 10 mM procaine causes a cytoplasmic and acidic sperm cell organelle pH rise that simultaneously induces hyperactivated motility, increased levels of cAMP and tail-associated protein tyrosine phosphorylation in stallion spermatozoa. However, procaine-induced hypermotility is independent of the cAMP/protein tyrosine phosphorylation pathway.

SO4= uptake and catalase role in preconditioning after H2O2-induced oxidative stress in human erythrocytes.

Preconditioning (PC) is an adaptive response to a mild and transient oxidative stress, shown for the first time in myocardial cells and not described in erythrocytes so far. The possible adaptation of human erythrocytes to hydrogen peroxide (H2O2)-induced oxidative stress has been here verified by monitoring one of band 3 protein functions, i.e., Cl-/HCO3- exchange, through rate constant for SO4= uptake measurement. With this aim, erythrocytes were exposed to a mild and transient oxidative stress (30min to either 10 or 100μM H2O2), followed by a stronger oxidant condition (300- or, alternatively, 600-μM H2O2 treatment). SO4= uptake was measured by a turbidimetric method, and the possible role of catalase (CAT, significantly contributing to the anti-oxidant system in erythrocytes) in PC response has been verified by measuring the rate of H2O2 degradation. The preventive exposure of erythrocytes to 10μM H2O2, and then to 300μM H2O2, significantly ameliorated the rate constant for SO4= uptake with respect to 300μM H2O2 alone, showing thus an adaptive response to oxidative stress. Our results show that (i) SO4= uptake measurement is a suitable model to monitor the effects of a mild and transient oxidative stress in human erythrocytes, (ii) band 3 protein anion exchange capability is retained after 10 μM H2O2 treatment, (iii) PC response induced by the 10 μM H2O2 pretreatment is clearly detected, and (iv) PC response, elicited by low-concentrated H2O2, is mediated by CAT enzyme and does not involve band 3 protein tyrosine phosphorylation pathways. Erythrocyte adaptation to a short-term oxidative stress may serve as a basis for future studies about the impact of more prolonged oxidative events, often associated to aging, drug consumption, chronic alcoholism, hyperglycemia, or neurodegenerative diseases.

Large-scale comparative phosphoprotein analysis of maize seedling leaves during greening

MAIN CONCLUSION : Large-scale comparative phosphoprotein analysis in maize seedlings reveals a complicated molecular regulation mechanism at the phosphoproteomic level during de-etiolation. In the present study we report a phosphoproteomic study conducted on Zea mays etiolated leaves harvested at three time points during greening (etiolated seedlings and seedlings exposed to light for 6 or 12 h). We identified a total of 2483 phosphopeptides containing 2389 unambiguous phosphosites from 1339 proteins. The abundance of nearly 692 phosphorylated peptides containing 783 phosphosites was reproducible and profiled with high confidence among treatments. Comparisons with other large-scale phosphoproteomic studies revealed that 473 of the phosphosites are novel to this study. Of the 783 phosphosites identified, 171, 79, and 138 were identified in 0, 6, and 12 h samples, respectively, which suggest that regulation of phosphorylation plays important roles during maize seedling de-etiolation. Our experimental methods included enrichment of phosphoproteins, allowing the identification of a great number of low abundance proteins, such as transcription factors, protein kinases, and photoreceptors. Most of the identified phosphoproteins were involved in gene transcription, post-transcriptional regulation, or signal transduction, and only a few were involved in photosynthesis and carbon metabolism. It is noteworthy that tyrosine phosphorylation and calcium signaling pathways might play important roles during maize seedling de-etiolation. Taken together, we have elucidated a new level of complexity in light-induced reversible protein phosphorylation during maize seedling de-etiolation.

Biphasic role of calcium in mouse sperm capacitation signaling pathways.

Mammalian sperm acquire fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with up-regulation of a cAMP-dependent pathway, changes in intracellular pH, intracellular Ca(2+), and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca(2+) ions have a biphasic role in the regulation of cAMP-dependent signaling. Media without added Ca(2+) salts (nominal zero Ca(2+)) still contain micromolar concentrations of this ion. Sperm incubated in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca(2+). However, chelation of the extracellular Ca(2+) traces by EGTA induced both cAMP-dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca(2+) media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca(2+) ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin. Interestingly, contrary to wild-type mouse sperm, sperm from CatSper1 KO mice underwent PKA activation and an increase in tyrosine phosphorylation upon incubation in nominal zero Ca(2+) media. Therefore, sperm lacking Catsper Ca(2+) channels behave as wild-type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca(2+) involved in the regulation of cAMP-dependent and tyrosine phosphorylation pathways required for sperm capacitation.

Quantitative Phosphoproteomics Analysis Reveals a Key Role of Insulin Growth Factor 1 Receptor (IGF1R) Tyrosine Kinase in Human Sperm Capacitation*[S

One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men.

Ecdysone receptor and ultraspiracle proteins are tyrosine phosphorylated during adult development of silkmoths

20-hydroxy ecdysone (20E) is essential to promote adult development in diapausing silkmoth pupae. Increases in protein tyrosine/serine-phosphorylations observed soon after 20E administration supported the initial hypothesis that activation of receptor tyrosine kinase-ras-MAPK pathway could be responsible for the growth promoting effects of 20E. This report pertains to the high levels of protein tyrosine phosphorylations (PTP) that occurred later during the growth to differentiation transition because of its novelty and relevance to 20E dependence of adult development. Further analyses demonstrated that both ecdysone receptor (EcR) and ultraspiracle (USP), the two dimerizing partners of the functional ecdysone receptor, are tyrosine phosphorylated coincidental with high PTP. Enhanced PTP during growth to differentiation transition and concomitant tyrosine phosphorylation of EcR and USP was shown to occur in another silkmoth species pointing to the necessity of similar protein tyrosine phosphorylation pathways for adult development. Properly timed increases in tissue protein tyrosine kinase (PTK) activity could explain the enhancement of PTP in the wing epidermis of both the silkmoths. Thymidine incorporation measurements showed that cessation of DNA synthesis preceded the increase in PTK activity thus emphasizing a role for PTP in aspects of tissue physiology related to differentiative events rather than cell proliferation. Phosphatase and tyrosine kinase inhibitors (Tyrphostins) had minimal effects on adult wing development in vivo. However, the escape of the adult from the pupal case was blocked by a tyrphostin indicating the importance of PTKs in eclosion.

Receptor-like Tyrosine Phosphatases CD45 and CD148 Have Distinct Functions in Chemoattractant-Mediated Neutrophil Migration and Response to S. aureus

Neutrophils, critical innate immune effectors, use bacterial-derived chemoattractant-induced G protein-coupled receptor (GPCR) signaling for their pursuit of bacteria. Tyrosine phosphorylation pathways and receptor-like tyrosine phosphatases (RPTPs) are rarely considered in chemoattractant-mediated GPCR signaling. Here, we report that two RPTPs, CD45 and CD148, previously shown to share redundant roles in positively regulating Src family kinases (SFKs) in immunoreceptor signaling pathways in B cells and macrophages, are critical in the neutrophil response to S. aureus infection and, surprisingly, in chemoattractant-mediated chemotaxis. Remarkably, deficiency in either of these RPTPs influenced neutrophil GPCR responses in unique ways. Our results reveal that CD45 positively while CD148 positively and negatively regulate GPCR function and proximal signals including Ca(2+), phosphatidylinositol 3'OH kinase (PI3K), and phospho-extracellular regulated kinase (pERK) activity. Moreover, our results suggest that CD45 and CD148 preferentially target different SFK members (Hck and Fgr versus Lyn, respectively) to positively and negatively regulate GPCR pathways.

Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.

Sphingosine kinase 1 overexpression is regulated by signaling through PI3K, AKT2, and mTOR in imatinib-resistant chronic myeloid leukemia cells

As a better understanding of the molecular basis of carcinogenesis has emerged, oncogene-specific cell-signaling pathways have been successfully targeted to treat human malignances. Despite impressive advances in oncogene-directed therapeutics, genetic instability in cancer cells often manifest acquired resistance. This is particularly noted in the use of tyrosine kinase inhibitors therapies and not more evident than for chronic myeloid leukemia. Therefore, it is of great importance to understand the molecular mechanisms affecting cancer cell sensitivity and resistance to tyrosine kinase inhibitors. In this study, we used continuous exposure to stepwise increasing concentrations of imatinib (0.6-1 μM) to select imatinib-resistant K562 cells. Expression of BCR-ABL increased both at RNA and protein levels in imatinib-resistant cell lines. Furthermore, expression levels of sphingosine kinase 1 (SphK1) were increased significantly in resistant cells, channeling sphingoid bases to the SphK1 pathway and activating sphingosine-1-phosphate-dependent tyrosine phosphorylation pathways that include the adaptor protein Crk. The partial inhibition of SphK1 activity by N,N-dimethylsphingosine or expression by small interfering RNA increased sensitivity to imatinib-induced apoptosis in resistant cells and returned BCR-ABL to baseline levels. To determine the resistance mechanism-induced SphK1 upregulation, we used pharmacological inhibitors of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathway and observed robust downmodulation of SphK1 expression and activity when AKT2, but not AKT1 or AKT3, was suppressed. These results demonstrate that SphK1 is upregulated in imatinib-resistant K562 cells by a pathway contingent on a phosphoinositide 3-kinase/AKT2/mammalian target of rapamycin signaling pathway. We propose that SphK1 plays an important role in development of acquired resistance to imatinib in chronic myeloid leukemia cell lines.

Modulation of Platelet Function and Signaling by Flavonoids

Blood platelets play a crucial role in the primary hemostasis and vessel wall repair. However; platelet hyperactivation is implicated in the pathogenesis of cardiovascular diseases such as thrombosis, atherosclerosis and stroke. Epidemiological data have suggested that regular consumption of fruits and vegetables, which are rich in flavonoids, is associated to a reduction in cardiovascular events. The cardioprotective effect of flavonoids is partly due to the inhibition of platelet function. However; the mechanisms underlying the anti-platelet effect of these compounds remain unclear. The aim of this review is to discuss the role of platelets in cardiovascular disease and to provide an overview of the potential anti-platelet effect of flavonoids, focusing on the various platelet signaling pathways modulated by flavonoids, including oxidative stress, protein tyrosine phosphorylation, calcium mobilization and nitric oxide pathway. The understanding of these mechanisms will be helpful in the development of new anti-platelet agents based on flavonoids with fewer or no adverse effects.

Evolutionary Constraints of Phosphorylation in Eukaryotes, Prokaryotes, and Mitochondria

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life.

Lymphocyte dysmetabolism: an immunocytochemical comparative approach in IDDM and control subjects

We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulindependent diabetes mellitns) patients and agematched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly reduced in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

Lymphocyte dysmetabolism: an immunocytochemical comparative approach in IDDM and control subjects

We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulin-dependent diabetes mellitus) patients and age-matched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly increased in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

A New Approach for Quantitative Phosphoproteomic Dissection of Signaling Pathways Applied to T Cell Receptor Activation

Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.

Tyrosine phosphoproteome of hamster spermatozoa: Role of glycerol‐3‐phosphate dehydrogenase 2 in sperm capacitation

Capacitation confers on the spermatozoa the competence to fertilize the oocyte. At the molecular level, a cyclic adenosine monophosphate (cAMP) dependent protein tyrosine phosphorylation pathway operates in capacitated spermatozoa, thus resulting in tyrosine phosphorylation of specific proteins. Identification of these tyrosine-phosphorylated proteins and their function with respect to hyperactivation and acrosome reaction, would unravel the molecular basis of capacitation. With this in view, 21 phosphotyrosine proteins have been identified in capacitated hamster spermatozoa out of which 11 did not identify with any known sperm protein. So, in the present study attempts have been made to ascertain the role of one of these eleven proteins namely glycerol-3-phosphate dehydrogenase 2 (GPD2) in hamster sperm capacitation. GPD2 is phosphorylated only in capacitated hamster spermatozoa and is noncanonically localized in the acrosome and principal piece in human, mouse, rat, and hamster spermatozoa, though in somatic cells it is localized in the mitochondria. This noncanonical localization may imply a role of GPD2 in acrosome reaction and hyperactivation. Further, enzymatic activity of GPD2 during capacitation correlates positively with hyperactivation and acrosome reaction thus demonstrating that GPD2 may be required for sperm capacitation.

B cells drive lymphocyte activation and expansion in mice with the CD45 wedge mutation and Fas deficiency

CD45 and Fas regulate tyrosine phosphorylation and apoptotic signaling pathways, respectively. Mutation of an inhibitory wedge motif in CD45 (E613R) results in hyperresponsive thymocytes and B cells on the C57BL/6 background, but no overt autoimmunity, whereas Fas deletion results in a mild autoimmune disease on the same genetic background. In this study, we show that these two mutations cooperate in mice, causing early lethality, autoantibody production, and substantial lymphoproliferation. In double-mutant mice, this phenotype was dependent on both T and B cells. T cell activation required signaling in response to endogenous or commensal antigens, demonstrated by the introduction of a transgenic T cell receptor. Genetic deletion of B cells also prevented T cell activation. Similarly, T cells were necessary for B cell autoantibody production. However, B cells appeared to be intrinsically activated even in the absence of T cells, suggesting that they may drive the phenotype of these mice. These results reveal a requirement for careful control of B cell signaling and cell death in preventing inappropriate lymphocyte activation and autoimmunity.

Abstract 3552: Pharmacological Inhibition or Genetic Disruption of Protein Tyrosine Phosphatase 1B Attenuates both Myocardial and Endothelial Dysfunction in Mice with Heart Failure

We have shown previously that acute, in vitro inhibition of protein tyrosine phosphatase 1B (PTP1B) improved endothelial function of peripheral resistance arteries in mice with chronic heart failure (CHF), as demonstrated by the restored flow-mediated, NO-dependent vasodila-tation (FMD). This is most likely due to increased tyrosine phosphorylation pathways involved in shear stress-induced activation of eNOS. The present study evaluates the impact of chronic pharmacological inhibition or genetic disruption of PTP1B, on cardiac and endothelial dysfunction in CHF mice. CHF was induced by coronary ligation, either in C57BL/6 mice, or in wild type (WT) or PTP1B-deficient (PTP1B−/−) BALB/c mice. CHF mice (either C57BL/6 or BALB/c WT) were untreated or treated with the PTP1B inhibitor AS279 (60 mg/kg/day) for 2 months. Mice were then anesthetized with isoflurane for echocardiographic evaluation of left ventricular (LV) function and remodeling. After euthanasia, small mesenteric artery segments were isolated and mounted in an arteriograph for the evaluation of FMD. Echocardiographic results are shown in the Table . In parallel, in vitro vascular studies showed that chronic AS279 restored FMD both in C57BL/6 (max FMD: control: 17±2, n=9; CHF untreated: -1±1, n=9; CHF + AS279: 14±2%, n=19; p<0.01 vs. CHF untreated) and BALB/c CHF mice (Control: 29±4, n=16; CHF untreated: 7±1, n=5; CHF + AS279: 24±6%, n=7; p<0.01 vs. CHF untreated). Compared to CHF WT, FMD was also increased in PTP1B−/− CHF mice (WT: 7±1, n=5, PTP1B−/−: 16±6%, n=6, p<0.05). Additionally, in vitro downregulation of PTP1B (by a 3 day incubation with shRNA) also increased FMD in arteries isolated from CHF mice (max FMD: untreated: 6±2; scrambled shRNA: 7±2; shRNA PTP1B: 27±2%, p<0.01). Thus, chronic pharmacological inhibition or genetic disruption of PTP1B both restores endothelial function and improves cardiac dysfunction and remodeling, suggesting that this enzyme may be a new target for the treatment of CHF. Echocardiographic results in mice with 2 months CHF