Increase In Tyrosine Phosphorylation Of Proteins Research Articles

Cholesterol Efflux-mediated Signal Transduction in Mammalian Sperm: β-CYCLODEXTRINS INITIATE TRANSMEMBRANE SIGNALING LEADING TO AN INCREASE IN PROTEIN TYROSINE PHOSPHORYLATION AND CAPACITATION

Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk. The activation of this signaling pathway, as well as capacitation, requires bovine serum albumin (BSA) in the incubation medium. BSA is hypothesized to modulate capacitation through its ability to remove cholesterol from the sperm plasma membrane. Here we demonstrate that the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin and OH-propyl-beta-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these beta-cyclodextrins were also demonstrated to increase protein tyrosine phosphorylation in the absence of BSA in both mouse and bull sperm, and the patterns of phosphorylation were similar to those induced by media containing BSA. The potency of the different beta-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the beta-cyclodextrins with cholesterol-SO4- to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The beta-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO3 and protein kinase A-dependent. The beta-cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the zona pellucida to induce the acrosome reaction and by successful fertilization in vitro. In summary, beta-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further support the coupling of cholesterol efflux to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems, thus defining a new mode of cellular signal transduction initiated by cholesterol release.

Corticotropin-releasing factor induces phosphorylation of phospholipase C-γ at tyrosine residues via its receptor 2β in human epidermoid A-431 cells

This laboratory previously reported that corticotropin-releasing factor (CRF) increased intracellular free calcium concentrations, cellular cAMP, inositol 1,4,5-trisphosphate, protein kinase C activity, and protein phosphorylation in human A-431 cells. The increase was blocked by CRF receptor antagonist. In this study, we identified the type of CRF receptors present and investigated whether CRF induced tyrosine phosphorylation of phospholipase C-γ via CRF receptors. Using novel primers in reverse transcriptase-polymerase chain reaction, we determined the CRF receptor type to be that of 2β. The levels of the CRF receptor type 2β were not altered in cells treated with activators of protein kinase C, Ca 2+ ionophore, or cells overexpressing heat shock protein 70 kDa. Cells treated with CRF displayed increases in protein tyrosine phosphorylation approximately at 150 kDa as detected by immunoblotting using an antibody against phosphotyrosine. Immunoprecipitation with antibodies directed against phospholipase C-β3, -γ1, or -γ2 isoforms (which have molecular weights around 150 kDa) followed by Western blotting using an anti-phosphotyrosine antibody showed that only phospholipase C-γ1 and -γ2 were phosphorylated. The increase in phospholipase C-γ phosphorylation was concentration-dependent with an EC 50 of 4.2±0.1 pM. The maximal phosphorylation by CRF at 1 nM occurred by 5 min. The CRF-induced phosphorylation was inhibited by the protein tyrosine kinase inhibitors genistein and herbimycin A, suggesting that CRF activates protein tyrosine kinases. Treatment of cells with CRF receptor antagonist, but not pertussis toxin, prior to treatment with CRF inhibited the CRF-induced phosphorylation, suggesting it is mediated by the CRF receptor type 2β that is not coupled to pertussis toxin-sensitive G-proteins. Treatment with 1,2-bis(2-iminophenoxy)ethane- N, N, N′, N′-tetraacetic acid attenuated the phospholipase C-γ phosphorylation. In summary, CRF induces phospholipase C-γ phosphorylation at tyrosine residues, which depends on Ca 2+ and is mediated by activation of protein tyrosine kinases via the CRF receptor type 2β.

Mannitol at clinical concentrations activates multiple signaling pathways and induces apoptosis in endothelial cells.

Hyperosmotic mannitol therapy is widely used in the clinical setting for acute and subacute reduction in brain edema, to decrease muscle damage in compartment syndrome, and to improve renal perfusion. Though beneficial rheological effects commonly are attributed to mannitol, its direct effects on endothelial cells are poorly understood. We studied the effect of hypertonic and hypotonic stress on bovine aortic endothelial (BAE) cells, using mannitol, urea, and sodium chloride and medium dilution in vitro. Exposure to incremental osmolar concentrations of 300 mOsm of each osmotic agent increased apoptosis in BAE cells (mannitol congruent withNaCl>urea). Induced programmed cell death was detected by DAPI staining of intact cell nuclei, and by TUNEL and DNA fragmentation ladder assays. Mannitol-induced apoptosis exhibited dose dependence (42% of cells at 300 mOsm [P<0.0001] compared with 1.2% of control cells) and was also observed in bovine smooth muscle cells. Mannitol-induced apoptosis was attenuated approximately 50% in the presence of cycloheximide or actinomycin D. Hypertonic mannitol and NaCl, but not urea, increased tyrosine phosphorylation of the focal adhesion contact-associated proteins paxillin and FAK. Hypotonic medium, which did not lead to apoptosis, increased protein tyrosine phosphorylation of FAK but not of paxillin. Addition of mannitol or NaCl also produced sustained increases in c-Jun NH2-terminal kinase (JNK) activity. In addition, hypertonic mannitol increased intracellular free [Ca2+] in a dose-dependent manner. Chelation of intracellular Ca2+ with quin2-AM (10 micromol/L) inhibited mannitol-induced apoptosis approximately 50%, as to a lesser extent did inhibition of tyrosine kinase activity with herbimycin (1 micromol/L). We have shown that hypertonic mannitol exposure induces endothelial cell apoptosis, accompanied by activation of tyrosine and stress kinases, phosphorylation of FAK and paxillin, and elevation of intracellular free [Ca2+]. The apoptosis is attenuated by inhibition of transcription or translation, by inhibition of tyrosine kinases, or by intracellular Ca2+ buffering. These data suggest that clinical use of the osmotic diuretic mannitol may exert direct deleterious effects on vascular endothelium.

Novel mechanism by which hemoglobin induces constriction of cerebral arteries

Since oxyhemoglobin (OxyHb) is implicated in the pathogenesis of cerebral vasospasm, we have investigated the role of protein tyrosine phosphorylation in OxyHb-mediated signalling in canine cerebral arteries and cultured canine cerebrovascular smooth muscle cells. OxyHb produced a contraction of basilar artery preparations, which was reversed by genistein, an inhibitor of tyrosine kinases, and PD098059, an inhibitor of mitogen-activated protein kinase. In cerebrovascular smooth muscle cells, OxyHb induced tyrosine phosphorylation of 42, 46, 54–60 and 80–100 kDa proteins with a time-course which paralleled the contractile action of OxyHb, suggesting that these events might be functionally linked. The 42 and 60 kDa proteins were immunologically related to the mitogen-activated protein kinase, extracellular signal regulated protein kinase (ERK2), and to p60 c-Src (c-Src), respectively. The increase in protein tyrosine phosphorylation was attenuated by genistein, and the phosphorylation of the 42 kDa protein (ERK2) was inhibited by PD098059. These results suggest that OxyHb-mediated signalling utilizes a protein tyrosine kinase-based mechanism.

Paradoxical effect of reagents for sulfhydryl and disulfide groups on human sperm capacitation and superoxide production

Spermatozoa must undergo capacitation prior to fertilization. In humans, this process appears regulated by oxidoreduction reactions. We investigated the possibility that these reactions involved the sulfhydryl-disulfide pair, which offers a reversible regulation of cellular processes. The effects of reagents targeted for sulfhydryl and disulfide groups on human sperm capacitation, superoxide (O 2 •−) generation and protein tyrosine phosphorylation were evaluated. The sulfhydryl targeted agents, phenylarsine oxide (PAO), diamide, dithiopyridine (DTP), N-ethylmaleimide (NEM), maleimidylpropionyl biocytin (MPB), p-chloromercuribenzoic acid (PCMB), and bromobimane analogs (mBBr and qBBr) triggered sperm capacitation to levels comparable to those observed with a biological inducer, fetal cord serum ultrafiltrate (FCSu). Capacitation induced by NEM, MPB, PCMB, and PAO was prevented by superoxide dismutase (SOD) and associated with an increased sperm production of O 2 •−. However, SOD did not affect the increase in protein tyrosine phosphorylation of spermatozoa treated with NEM, PAO, or MPB. Disulfide reductants, dithiothreitol (DTT), thioredoxin (TRX), glutathione (GSH), tris-(2-carboxyethyl) phosphine (TCEP), and tris-(2-cyanoethyl) phosphine (TCP) partially to totally inhibited FCSu-induced sperm capacitation and O 2 •− production. TCEP, DTT, and TRX decreased the capacitation-associated tyrosine phosphorylation of sperm proteins. The strong time-dependent increase of sperm membrane sulfhydryl groups exposed to the extracellular space occurring during the first hour of capacitation could indicate an important rearrangement of sulfhydryl carrying proteins during the initiation of capacitation. Therefore, protein sulfhydryl-disulfide status may be important for the regulation of human sperm capacitation and the mechanisms involved may be complex and multifactorial.

Reactive oxygen species signaling through regulation of protein tyrosine phosphorylation in endothelial cells.

Tyrosine phosphorylation of proteins, controlled by tyrosine kinases and protein tyrosine phosphatases, plays a key role in cellular growth and differentiating. A wide variety of hormones, growth factors, and cytokines modulate cellular tyrosine phosphorylation to transmit signals across the plasma membrane to the nucleus. Recent studies suggest that reactive oxygen species (ROS) also induce cellular protein tyrosine phosphorylation through receptor or nonreceptor tyrosine kinases. To determine whether protein tyrosine phosphorylation by ROS regulates endothelial cell (EC) metabolism and function, we exposed vascular ECs to H2O2 or H2O2 plus vanadate. This resulted in a time- and dose-dependent increase in protein tyrosine phosphorylation of several proteins (M(r) 21-200 kDa), as determined by immunoprecipitation and Western blot analysis with antiphosphotyrosine antibody. Immunoprecipitation with specific antibodies identified increased tyrosine phosphorylation of mitogen-activated protein kinases (42-44 kDa), paxillin (68 kDa), and FAK (125 kDa) by ROS. An immediate signaling response to increased protein tyrosine phosphorylation by ROS was activation of phospholipases such as A2, C, and D. Suramin pretreatment inhibited ROS stimulation of phospholipase D (PLD), suggesting a role for growth factor receptors in this activation. Further, PLD activation by ROS was attenuated by N-acetylcysteine, indicating that intracellular thiol status is critical to ROS-mediated signal transduction. These results provide evidence that ROS modulate EC signal transduction via a protein tyrosine phosphorylation-dependent mechanism.

β-Amyloid Fibrils Activate Parallel Mitogen-Activated Protein Kinase Pathways in Microglia and THP1 Monocytes

The senile plaques of Alzheimer's disease are foci of local inflammatory responses, as evidenced by the presence of acute phase proteins and oxidative damage. Fibrillar forms of beta-amyloid (Abeta), which are the primary constituents of senile plaques, have been shown to activate tyrosine kinase-dependent signal transduction cascades, resulting in inflammatory responses in microglia. However, the downstream signaling pathways mediating Abeta-induced inflammatory events are not well characterized. We report that exposure of primary rat microglia and human THP1 monocytes to fibrillar Abeta results in the tyrosine kinase-dependent activation of two parallel signal transduction cascades involving members of the mitogen-activated protein kinase (MAPK) superfamily. Abeta stimulated the rapid, transient activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 in microglia and ERK2 in THP1 monocytes. A second superfamily member, p38 MAPK, was also activated with similar kinetics. Scavenger receptor and receptor for advanced glycated end products (RAGE) ligands failed to activate ERK and p38 MAPK in the absence of significant increases in protein tyrosine phosphorylation, demonstrating that scavenger receptors and RAGE are not linked to these pathways. Importantly, the stress-activated protein kinases (SAPKs) were not significantly activated in response to Abeta. Downstream effectors of the MAPK signal transduction cascades include MAPKAP kinases, such as RSK1 and RSK2, as well as transcription factors. Exposure of microglia and THP1 monocytes to Abeta resulted in the activation of RSK1 and RSK2 and phosphorylation of cAMP response element-binding protein at Ser133, providing a mechanism for Abeta-induced changes in gene expression.

Alterations in Human B Cell Calcium Homeostasis by Polycyclic Aromatic Hydrocarbons: Possible Associations with Cytochrome P450 Metabolism and Increased Protein Tyrosine Phosphorylation

Previous studies performed in this laboratory have shown that certain benzo(a)pyrene (BaP) metabolites, such as benzo(a)pyrene-7,8-dihydrodiol (BaP-7,8-diol) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), were more effective in elevating intracellular Ca2+in normal human peripheral blood mononuclear cell (HPBMC) T and B cells than was BaP. Additionally, it has been shown that the suppression of human T cell mitogenesis produced by polycyclic aromatic hydrocarbons (PAHs) and certain BaP metabolites is reversed by treatment with α-naphthoflavone (ANF), a cytochrome P450 1A and 1B inhibitor. ANF also diminishes the elevation in intracellular calcium (Ca2+) produced by BaP in HPBMC. In the present studies, we further defined the relationships between intracellular Ca2+elevation produced by BaP and two immunotoxic P450-derived metabolites, BaP-7,8-diol and BPDE in the Daudi human B cell line. At 1, 4, and 18 h, both BaP-7,8-diol and BPDE produced a significant rise in intracellular Ca2+. This effect, however, was not observed with BaP or benzo(e)pyrene (BeP), a nonimmunotoxic PAH. To evaluate the potential role of cytochrome P450 metabolism in PAH-induced Ca2+elevation, Daudi cells were pretreated with ANF for 4 h, followed by treatment with BaP metabolites for 18 h. ANF completely reversed the rise in Ca2+produced by BaP-7,8-diol, but had no effect on the Ca2+elevation produced by BPDE. These results suggest that BPDE may be the ultimate P450 metabolite responsible for Ca2+elevation in human B cells. BaP-7,8-diol and BPDE were found to increase tyrosine phosphorylation in Daudi whole cell lysates and to increase tyrosine phosphorylation of two important Src-related protein tyrosine kinases (PTKs), Lyn and Syk. Inhibition of tyrosine phosphorylation by herbimycin A was found to largely prevent the increase in intracellular Ca2+produced by BaP-7,8-diol and BPDE, suggesting that Ca2+elevation is coupled to increased tyrosine phosphorylation in Daudi. BPDE was found to produce a statistically significant increase in tyrosine phosphorylation of Lyn and Syk within 10 min of exposure. Collectively, these data demonstrate that certain P450-derived metabolites of BaP may be responsible for PTK activation and an increase intracellular Ca2+, which may alter antigen receptor signaling in human B cells.

Mechanical Strain Increases Protein Tyrosine Phosphorylation in Airway Smooth Muscle Cells

Mechanical stress contributes to normal structure and function of the lung as well as pathology in such diseases as bronchopulmonary dysplasia and adult respiratory distress syndrome. Stress-related increases in airway smooth muscle (ASM) quantity are reflectedin vitrowhere cultured ASM cells respond to cyclic deformational strain with increased proliferation, cell reorientation, protein production, stress fibers, and focal adhesions. To understand the mechanisms of mechanical signaling in ASM cells, we investigated whether strain increased tyrosine phosphorylation of focal adhesion-related proteins. ASM cells were grown to confluence on collagen type I and subjected to 30 min of cyclic deformation strain (2 s of 25% deformation of the substratum, 2 s relaxation) and compared at various time points with identical cells not subjected to strain for phosphotyrosine content of three focal adhesion-concentrated proteins (pp125FAK, paxillin, and talin) by Western blotting. Strain caused a rapid increase in tyrosine phosphorylation of pp125FAKand paxillin. Tyrosine phosphorylation decreased by 4 h in pp125FAKafter discontinuing strain but remained elevated in paxillin at 24 h. Increases in tyrosine phosphorylation of talin were not found. In separate studies, when cells were strained in the presence of tyrosine kinase inhibitors (genistein and herbimycin A), strain-induced reorientation and elongation were inhibited. Mechanochemical signal transduction appears to mediate cell morphologic changes through quantitative and possibly qualitative changes in tyrosine phosphorylation of adhesion-related proteins.

Treatment of human spermatozoa with seminal plasma inhibits protein tyrosine phosphorylation.

It has long been known that seminal plasma contains factors that influence the fertilizing capacity of spermatozoa in many different ways. However, little is understood of the biochemical cascades triggered when spermatozoa and seminal plasma interact. In this study, we examined how incubation with seminal plasma affected protein tyrosine phosphorylation in human spermatozoa. Increased protein tyrosine phosphorylation is a hallmark of sperm capacitation in several mammalian species, including human. Seminal plasma blocks protein tyrosine phosphorylation when added to washed, non-capacitated spermatozoa. Removal of seminal plasma and incubation in capacitating medium led to partial recovery of the tyrosine phosphorylation cascade. Addition of seminal plasma to a suspension of spermatozoa previously incubated for 5 h under capacitating conditions decreased the level of tyrosine phosphorylation on all proteins in a dose-dependent manner. In this case, the phosphotyrosine signal did not increase upon removal of seminal plasma followed by overnight incubation in fresh capacitating media, indicating that removal of seminal plasma was necessary but not sufficient for protein tyrosine phosphorylation to occur. These results indicate that human seminal plasma contains factors that influence the tyrosine phosphorylation status of human spermatozoa.

Pervanadate-induced adhesion of CD4+ T cell to fibronectin is associated with tyrosine phosphorylation of paxillin.

The initial stages of T cell activation involve tyrosine protein kinase-mediated intracellular signaling events. Integrin-mediated adhesion of CD4+ T lymphocytes to extracellular matrix glycoproteins, such as fibronectin, is an activation-dependent process. The involvement of tyrosine protein kinases in the adhesion of CD4+ T cells to fibronectin was examined using pervanadate, a protein-tyrosine phosphatase inhibitor. Pervanadate induced the adhesion of human CD4+ T cells to immobilized fibronectin in a beta1 integrin-mediated fashion, and adhesion was associated with an increase of protein tyrosine phosphorylation. Tyrosine protein kinase inhibitors abrogated both T cell adhesion and intracellular protein tyrosine phosphorylation. Participation of cytoskeletal proteins in the pervanadate-induced T cell adhesion was indicated because cytoskeleton disruption by cytochalasin B inhibited cell adhesion to fibronectin. We demonstrate that the cytoskeletal protein paxillin underwent time-dependent tyrosine phosphorylation simultaneously with pervanadate-induced T cell adhesion to fibronectin. Tyrosine phosphorylation of paxillin was related to cell adhesion, since pretreatment of T cells with cytochalasin B abrogated both adhesion and phosphorylation. This study demonstrates a correlation between activation of protein tyrosine kinases, tyrosine phosphorylation of paxillin, and integrin-mediated T cell adhesion to extracellular matrix glycoproteins.

Phosphatase inhibitors potentiate 4-hydroxynonenal-induced phospholipase D activation in vascular endothelial cells.

We have previously reported that endothelial cell phospholipase D (PLD), activated by 4-hydroxynonenal (4-HNE), was independent of protein kinase C activation. To determine whether PLD stimulation by 4-HNE is related to protein tyrosine phosphorylation, the effects of tyrosine kinase (Tyrk) and protein tyrosine phosphatase (PTPase) inhibitors on PLD activation were investigated. Pretreatment of bovine pulmonary artery endothelial cells (BPAEC) with Tyrk inhibitors, such as genistein, erbstatin, and herbimycin attenuated 4-HNE-induced PLD activation. Furthermore, vanadate, phenylarsine oxide, and diamide, inhibitors of PTPases, markedly increased the 4-HNE-induced PLD activation. The effects of Tyrk and PTPase inhibitors were specific towards the 4-HNE, as these agents had no effect on the agonist- or TPA-induced PLD activation. In addition to PLD activation, treatment of BPAEC with 4-HNE increased tyrosine phosphorylation of proteins including bands of molecular weights 40,000-60,000, 70,000-90,000, and 110,000-130,000. The 4-HNE-mediated increase in protein tyrosine phosphorylation was partly inhibited by genistein (100 microM). Vanadate (10 microM) pretreatment also potentiated 4-HNE-induced protein tyrosine phosphorylation. These data suggest that 4-HNE-mediated stimulation of PLD may occur as a result of activation of tyrosine kinases.

Rapid tyrosine phosphorylation of HS1 in the response of mouse lymphoma L5178Y-R cells to photodynamic treatment sensitized by the phthalocyanine Pc 4.

The ability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to activate cellular signal transduction pathways in murine lymphoma L5178Y-R cells has been assessed by observing increases in protein tyrosine phosphorylation at early times post-PDT. Western blot analysis with an anti-phosphotyrosine antibody revealed a dramatic increase in phosphorylation of two major protein bands of Mr approximately 80,000 and approximately 55,000 in response to PDT. The increase was PDT dose-dependent, occurred as early as 20 s after initiation of light exposure of Pc 4-preloaded cells and was amplified by the presence of the protein tyrosine phosphatase inhibitor, sodium orthovanadate (NaVO4). By immunoprecipitation, one of the Mr approximately 80,000 phosphorylated proteins has been identified as HS1, a substrate of nonreceptor-type protein tyrosine kinases. Although vanadate greatly enhanced the level and extent of PDT-induced phosphorylation, it had no influence on overall photocytotoxicity or on the rate of apoptotic DNA fragmentation. Genistein, an inhibitor of protein tyrosine kinases, diminished tyrosine phosphorylation of the Mr approximately 80,000 and other proteins and dramatically potentiated cell killing induced by PDT but did not significantly affect PDT-induced apoptosis. The results suggest that PDT rapidly activates a membrane-associated src family kinase(s) in L5178Y-R cells, one substrate of which is HS1, and that protein tyrosine phosphorylation is part of a stress response, protecting a portion of the cells from the lethal effects of PDT but not altering the mechanism by which they die.

Amyloid Fibrils Activate Tyrosine Kinase-Dependent Signaling and Superoxide Production in Microglia

Alzheimer's disease (AD) is a devastating neurological disorder characterized by loss of cognitive skills and progressive dementia. The pathological hallmark of AD is the presence of numerous senile plaques throughout the hippocampus and cerebral cortex associated with degenerating axons, neurofibrillary tangles, and gliosis. The core of the senile plaque primarily is composed of the 39-43 amino acid beta-amyloid peptide (Abeta), which forms fibrils of beta-pleated sheets. Although considerable genetic evidence implicates Abeta in the pathogenesis of AD, a direct causal link remains to be established. Senile plaques are foci of local inflammatory processes, as evidenced by the presence of numerous activated microglia and acute phase proteins. Abeta has been shown to elicit inflammatory responses in microglia; however, the intracellular events mediating these effects are largely unknown. We report that exposure of microglia and THP1 monocytes to fibrillar Abeta led to time- and dose-dependent increases in protein tyrosine phosphorylation of a population of proteins similar to that elicited by classical immune stimuli such as immune complexes. The tyrosine kinases Lyn, Syk, and FAK were activated on exposure of microglia and THP1 monocytes to Abeta, resulting in the tyrosine kinase-dependent generation of superoxide radicals. The present data support a role for oxidative damage in the pathogenesis of AD, provide an important mechanistic link between Abeta and the generation of reactive oxygen intermediates, and identify molecular targets for therapeutic intervention in AD.

Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3'5'-monophosphate-dependent pathway.

Mammalian sperm capacitation, defined as an obligatory maturational process leading to the development of the fertilization-competent state, results from a poorly understood series of morphological and molecular events. We report here that ejaculated bovine sperm, incubated under conditions that support capacitation in vitro, display a reproducible pattern of protein tyrosine phosphorylations that are regulated by a cAMP-dependent pathway. The appearance of these tyrosine phosphorylated proteins correlated temporally with the time course of capacitation induced by heparin, and these phosphorylations displayed a similar heparin concentration dependence. Glucose, which inhibits capacitation, inhibited these protein tyrosine phosphorylations in media containing heparin. The biologically active cAMP analogues (dibutyryl cAMP [db-cAMP], 8-bromo cAMP, sp-cAMPS) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced the same protein tyrosine phosphorylation patterns as seen with heparin. Moreover, these cAMP agonists could overcome the inhibition of the heparin-induced tyrosine phosphorylations by glucose. In contrast, Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS), a protein kinase A (PK-A) antagonist, blocked the capacitation-associated increases in protein tyrosine phosphorylation. This cAMP regulation of the protein tyrosine phosphorylation pattern is mediated by PK-A since N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide-dihydrochloride (H89), another inhibitor of PK-A, inhibited the heparin-induced protein tyrosine phosphorylation pattern in a concentration-dependent manner in either the absence or presence of db-cAMP, IBMX, and glucose. These data support a model for sperm capacitation that includes protein tyrosine phosphorylation as an important regulatory pathway, and a role for cAMP/PK-A in the regulation of this pathway leading to capacitation. These studies are the first to report a unique interrelationship between tyrosine kinase/phosphatase and cAMP signaling pathways at the level of PK-A in bovine sperm capacitation.

Bovine oocyte activation after intracytoplasmic sperm injection

Spermatozoa must undergo capacitation prior to fertilization. In humans, this process appears regulated by oxidoreduction reactions. We investigated the possibility that these reactions involved the sulfhydryl-disulfide pair, which offers a reversible regulation of cellular processes. The effects of reagents targeted for sulfhydryl and disulfide groups on human sperm capacitation, superoxide (O2•−) generation and protein tyrosine phosphorylation were evaluated. The sulfhydryl targeted agents, phenylarsine oxide (PAO), diamide, dithiopyridine (DTP), N-ethylmaleimide (NEM), maleimidylpropionyl biocytin (MPB), p-chloromercuribenzoic acid (PCMB), and bromobimane analogs (mBBr and qBBr) triggered sperm capacitation to levels comparable to those observed with a biological inducer, fetal cord serum ultrafiltrate (FCSu). Capacitation induced by NEM, MPB, PCMB, and PAO was prevented by superoxide dismutase (SOD) and associated with an increased sperm production of O2•−. However, SOD did not affect the increase in protein tyrosine phosphorylation of spermatozoa treated with NEM, PAO, or MPB. Disulfide reductants, dithiothreitol (DTT), thioredoxin (TRX), glutathione (GSH), tris-(2-carboxyethyl) phosphine (TCEP), and tris-(2-cyanoethyl) phosphine (TCP) partially to totally inhibited FCSu-induced sperm capacitation and O2•− production. TCEP, DTT, and TRX decreased the capacitation-associated tyrosine phosphorylation of sperm proteins. The strong time-dependent increase of sperm membrane sulfhydryl groups exposed to the extracellular space occurring during the first hour of capacitation could indicate an important rearrangement of sulfhydryl carrying proteins during the initiation of capacitation. Therefore, protein sulfhydryl-disulfide status may be important for the regulation of human sperm capacitation and the mechanisms involved may be complex and multifactorial.

Reactive oxygen species and sperm physiology.

Although high concentrations of reactive oxygen species (ROS) cause sperm pathology (ATP depletion leading to insufficient axonemal phosphorylation, lipid peroxidation and loss of motility and viability), recent evidence demonstrates that low and controlled concentrations of these ROS play an important role in sperm physiology. Reactive oxygen species, such as the superoxide anion, hydrogen peroxide and nitric oxide, induce sperm hyperactivation, capacitation or the acrosome reaction in vitro. The ROS involved in these processes may vary depending on experimental conditions, but all the evidence converges to describe these events as 'oxidative' or 'redox regulated'. Human sperm capacitation and acrosome reaction are associated with extracellular production of a superoxide anion that is thought to originate from a membrane 'oxidase'. The enzymes responsible for tyrosine phosphorylation-dephosphorylation of sperm proteins are possible targets for ROS since mild oxidative conditions cause increases in protein tyrosine phosphorylation and acrosome reaction. The lipid peroxidation resulting from low concentrations of ROS promotes binding to the zona pellucida and may trigger the release of unesterified fatty acids from the sperm plasma membrane. The fine balance between ROS production and scavenging, as well as the right timing and site for ROS production are of paramount importance for acquisition of fertilizing ability.

Differential Regulation of Proline-rich Tyrosine Kinase 2/Cell Adhesion Kinase β (PYK2/CAKβ) and pp125FAK by Glutamate and Depolarization in Rat Hippocampus

The mechanisms by which stimuli that raise cytosolic free Ca2+ concentrations in neurons can increase protein tyrosine phosphorylation are not known. Using rat hippocampal slices and cortical synaptosomes, we have examined the regulation of two highly related cytoplasmic tyrosine kinases, pp125 focal adhesion kinase (pp125(FAK)) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta). Membrane depolarization increased tyrosine phosphorylation of PYK2/CAKbeta and pp125(FAK). These effects were blocked by EGTA or by protein kinase C inhibitors (RO31-8220; GF109203X) and mimicked by ionomycin or phorbol 12-myristate 13-acetate, in the case of pp125(FAK), or their combination in the case of PYK2/CAKbeta. Glutamate and specific agonists of ionotropic (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and N-methyl-D-aspartate) or metabotropic (trans-1-aminocyclopentane-1,3, -dicarboxylate) glutamate receptors stimulated the phosphorylation of pp125(FAK), but not of PYK2/CAKbeta. Glutamate effects were prevented by GF109203X. Thus, in hippocampal slices, tyrosine phosphorylation of pp125(FAK) and PYK2/CAKbeta are regulated differentially by pathways involving Ca2+ and protein kinase C. pp125(FAK) and PYK2/CAKbeta may provide specific links between neuronal activity, increases in cytosolic Ca2+ and protein tyrosine phosphorylation, which may be important for neuronal survival, and synaptic plasticity.

Tyrosine kinase inhibitors enhance a Ca(2+)‐activated K+ current (IAHP) and reduce IAHP suppression by a metabotropic glutamate receptor agonist in rat dentate granule neurones.

1. Activation of metabotropic glutamate receptors (mGluRs) inhibits a transient Ca(2+)-activated K+ current (IAHP) responsible for the slow after-hyperpolarization that follows depolarizations of dentate granule neurones in rat hippocampal brain slices. Here we show for the first time that this physiological consequence of mGluR stimulation is selectively attenuated by blockers of protein tyrosine kinases (PTKs). 2. Several distinct types of PTK blockers, including genistein, tyrphostin-B42 and lavendustin-A, reduced the inhibition of IAHP by the selective mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). Inhibition of IAHP by 5-HT was unaffected. The PTK blockers by themselves doubled the duration of IAHP suggesting that there exists a tonic inhibitory influence on IAHP that is reduced by PTK antagonists. 3. Inclusion of EGTA (1 mM) in the patch pipette also potentiated the IAHP and reduced the inhibitory action of ACPD on IAHP, consistent with the observation of others that chelation of intracellular Ca2+ prevents protein tyrosine phosphorylation induced by ACPD. 4. we propose that mGluR-initiated inositol 1,4,5-trisphosphate (InsP3) production mobilizes intracellular Ca2+ and leads to increased protein tyrosine phosphorylation which in turn leads to inhibition of IAHP.

Regulation of a neuronal form of focal adhesion kinase by anandamide.

Anandamide is an endogenous ligand for central cannabinoid receptors and is released after neuronal depolarization. Anandamide increased protein tyrosine phosphorylation in rat hippocampal slices and neurons in culture. The action of anandamide resulted from the inhibition of adenylyl cyclase and cyclic adenosine 3', 5'-monophosphate-dependent protein kinase. One of the proteins phosphorylated in response to anandamide was an isoform of pp125-focal adhesion kinase (FAK+) expressed preferentially in neurons. Focal adhesion kinase is a tyrosine kinase involved in the interactions between the integrins and actin-based cytoskeleton. Thus, anandamide may exert neurotrophic effects and play a role in synaptic plasticity.