Regulation Of Protein Tyrosine Phosphorylation Research Articles

Linoleic acid exerts antidiabetic effects by inhibiting protein tyrosine phosphatases associated with insulin resistance

The regulation of protein tyrosine phosphorylation by protein tyrosine kinases and protein tyrosine phosphatases (PTPs) is involved in intracellular signaling pathways, such as cell proliferation, differentiation, migration, and metabolism. As inhibitors of certain PTPs associated with insulin resistance have been shown to improve insulin sensitivity, the use of inhibitors against PTPN1, PTPN9 or PTPN11 is considered an effective strategy for treating type 2 diabetes. Herein, for the first time, we revealed that linoleic acid (LA) inhibited the catalytic activity of PTPN1, PTPN9 and PTPN11 in vitro, indicating that LA targeted PTPN1, PTPN9, and PTPN11. Additionally, 40 μM LA treatment enhanced glucose uptake through activation of the AMPK and Akt signaling pathways. Taken together, these findings indicate that LA, a multi-targeting inhibitor of PTPN1, PTPN9, and PTPN11, may exert antidiabetic effects to prevent type 2 diabetes.

A Functional Interplay Between Pyk2 and Src Family Kinases Links Ca2+ Signaling To Protein Tyrosine Phosphorylation In Thrombin-Stimulated Platelets

IntroductionThrombin and other soluble agonists promote platelet activation by binding to G-protein-coupled receptors, which typically recruit phospholipase C and trigger cytosolic Ca2+ increase and protein kinase C stimulation. Platelets also express several tyrosine kinases, including different members of the Src family (mainly Src and Lyn) and of the focal adhesion family (such as FAK and Pyk2), which are known to be activated upon stimulation with thrombin. Both Src family kinases (SFKs) and Pyk2 are required for thrombin-induced aggregation and thromboxane A2 production, and both of them have been shown to be regulated by intracellular Ca2+ (Xiang et al, J Biol Chem 2012; Canobbio et al, Blood 2013). However, the hierarchy of protein tyrosine kinases activation, as well as the molecular mechanism linking agonist-induced increase of cytosolic Ca2+ to protein tyrosine phosphorylation is poorly understood. In this study, we investigated the interplay between intracellular Ca2+, Pyk2 and SFKs in the regulation of protein tyrosine phosphorylation in thrombin-stimulated platelets. ResultsIn the presence of the ADP scavenger apyrase, thrombin caused the time-dependent tyrosine phosphorylation of several proteins in aspirin-treated human, as well as mouse platelets. This effect was severely reduced by the intracellular Ca2+ chelator BAPTA-AM and by the SFKs inhibitor PP2, and was totally suppressed only when BAPTA-AM and PP2 were used in combination, indicating that cytosolic Ca2+ and SFKs activity cooperate in the regulation of the whole process of protein tyrosine phosphorylation elicited by thrombin. Analysis of SFKs activity in human and mouse platelets with an anti-pY416Src antibody, that detects the autophosphorylation site in the catalytic domain, revealed that cytosolic Ca2+ is upstream SFKs activation upon thrombin stimulation. Accordingly, SFKs phosphorylation in the catalytic domain was also directly promoted by the Ca2+ ionophore A23187. Immunoblotting analysis on high resolution gels allowed to distinguish between Src and Lyn, and revealed that PP2 prevented phosphorylation of both kinases, but BAPTA-AM only inhibited Src , but not Lyn phosphorylation caused by thrombin. The Ca2+ ionophore A23187 also triggered the phosphorylation and activation of the Ca2+-dependent focal adhesion kinase Pyk2, and stimulation with thrombin promoted Pyk2 activation by a Ca2+-dependent mechanism. However, thrombin- or A23187-induced activation of Pyk2 was also reduced by the SFKs inhibitor PP2, and both PP2 and BAPTA-AM were required to completely suppress Pyk2 activation. In platelets from Pyk2 knockout mice, stimulation of SFKs by thrombin was strongly reduced, as it was the overall pattern of protein tyrosine phosphorylation. Importantly, also SFKs activation caused by Ca2+ionophore was significantly impaired in Pyk2-deficient platelets. Finally, in the absence of Pyk2, thrombin-induced aggregation was reduced to a level comparable to that of wild type platelets treated with PP2, although inhibition of SFKs by PP2 further reduced aggregation of Pyk2-deficient platelets. ConclusionsThese results indicate that in thrombin-stimulated platelets intracellular Ca2+ increase promotes Pyk2 stimulation, which initiates SFKs activation and establishes a positive loop which reinforces the Pyk2/SFKs axis to allow the subsequent massive tyrosine phosphorylation of multiple platelet substrates. Disclosures:No relevant conflicts of interest to declare.

The calcium-sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa.

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium-sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3-phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H-89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract.

Co-Expression of Protein Tyrosine Kinases EGFR-2 and PDGFRβ with Protein Tyrosine Phosphatase 1B in Pichia pastoris

The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Coexpression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and PDGFRβ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and PDGFRβ fusion proteins were purified by Ni(2+) affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and PDGFRβ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics.

Calcium–calmodulin and pH regulate protein tyrosine phosphorylation in stallion sperm

The mechanisms leading to capacitation in stallion sperm are poorly understood. The objective of our study was to define factors associated with regulation of protein tyrosine phosphorylation in stallion sperm. Stallion sperm were incubated for 4 h in modified Whitten's media with or without bicarbonate, calcium, or BSA. When sperm were incubated in air at 30×10⁶/ml at initial pH 7.25, protein tyrosine phosphorylation was detected only in medium containing 25 mM bicarbonate alone; calcium and BSA inhibited phosphorylation. Surprisingly, this inhibition did not occur when sperm were incubated at 10×10⁶/ml. The final pH values after incubation at 30×10⁶ and 10×10⁶ sperm/ml were 7.43 ± 0.04 and 7.83 ± 0.07 (mean ± s.e.m.) respectively. Sperm were then incubated at initial pH values of 7.25, 7.90, or 8.50 in either air or 5% CO₂. Protein tyrosine phosphorylation increased with increasing final medium pH, regardless of the addition of bicarbonate or BSA. An increase in environmental pH was observed when raw semen was instilled into the uteri of estrous mares and retrieved after 30 min (from 7.47 ± 0.10 to 7.85 ± 0.08), demonstrating a potential physiological role for pH regulation of capacitation. Sperm incubated in the presence of the calmodulin (CaM) inhibitor W-7 exhibited a dose-dependent increase in protein tyrosine phosphorylation, suggesting that the inhibitory effect of calcium was CaM mediated. These results show for the first time a major regulatory role of external pH, calcium, and CaM in stallion sperm protein tyrosine phosphorylation.

Global Proteomic Assessment of the Classical Protein-Tyrosine Phosphatome and “Redoxome”

Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H(2)O(2). Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.

Sildenafil Inhibits Superoxide Formation and Prevents Endothelial Dysfunction in a Mouse Model of Secondhand Smoke Induced Erectile Dysfunction

We determined the effect of passive secondhand cigarette smoke on 1) erectile function in vivo, 2) molecular mechanisms involved in penile vascular function, and 3) erectile function and penile molecular signaling in the presence of phosphodiesterase type 5 inhibitor therapy. Four groups of mice were used, including group 1--controls, group 2--mice exposed to 3 weeks of secondhand smoke (5 hours per day for 5 days per week), group 3--control plus sildenafil (100 mg/kg per day) and group 4--smoke exposed plus sildenafil (100 mg/kg per day). Cavernous nerve electrical stimulation and intracavernous injection of acetylcholine were done to assess erectile function. Constitutive and inducible nitric oxide synthase activity, reactive oxygen species generation, nitrotyrosine formation and superoxide anion levels were assessed. Decreased erectile responses to cavernous nerve electrical stimulation and impaired endothelium dependent erectile responses to ACh in mice exposed to secondhand smoke were observed. Superoxide anion was increased in endothelial and corporeal smooth muscle cells of smoking mouse penises. In mice exposed to secondhand smoke constitutive nitric oxide synthase activity was decreased, and inducible nitric oxide synthase activity, reactive oxygen species generation and nitrotyrosine formation increased. Sildenafil therapy restored constitutive nitric oxide synthase activity in the penis of smoking mice, decreased inducible nitric oxide synthase activity, reactive oxygen species generation and nitrotyrosine formation, and improved erectile responses to cavernous nerve electrical stimulation and acetylcholine. Short-term exposure to secondhand smoke impairs erectile function through excessive penile reactive oxygen species signaling and inducible nitric oxide synthase activity. Decreased penile constitutive nitric oxide synthase activity may be attributable to the decreased endothelial nitric oxide synthase activity resulting from increased oxidative stress. Sildenafil therapy restored nitric oxide synthase activity and decreased reactive oxygen species signaling, resulting in improved erectile function.

Centrosome separation driven by actin-microfilaments during mitosis is mediated by centrosome-associated tyrosine-phosphorylated cortactin

The regulation of protein tyrosine phosphorylation is an important aspect during the cell cycle. From G2-M transition to mitotic anaphase, phosphorylation of Tyr421, Tyr466 and Tyr482 of cortactin, an actin-filament associated protein, is dramatically induced. The phosphorylated cortactin is almost exclusively associated with centrosomes or spindle poles during mitosis. At G2-M transition prior to the breakdown of the nuclear envelope, two duplicated centrosomes migrate towards opposite ends of the nucleus to form the spindle poles. This centrosome-separation process and also the start of mitosis are inhibited or delayed by the depolymerization of actin filaments. Also inhibited is the separation of centrosomes when a truncated form of cortactin is expressed, whose C-terminus contains the tyrosine phosphorylation region but lacks the actin-binding domains. We introduced mutations at the tyrosine phosphorylation sites in the truncated C-terminus of cortactin and found that the C-terminus could no longer interfere with centrosome separation process. Our study shows that, cortactin phosphorylated at Tyr421, Tyr466 and Tyr482 mediates the actin-filament-driven centrosome separation at G2-M transition by providing a bridge between the centrosome and actin-filaments.

The Phenotypic Consequences of an Activating Mutation in the Juxtamembrane Wedge of CD45 Is Sensitive to Genetic Modifiers.

The dynamic regulation of protein tyrosine phosphorylation represents a powerful control point for integration of environmental signals into cellular responses. By modulating the activity of Src family protein tyrosine kinases, the receptor-like protein tyrosine phosphatase CD45 plays a critical role in regulating phosphotyrosine levels in hematopoietic cells. We previously generated mice containing a single point mutation (denoted CD45E613R) in the juxtamembrane wedge of CD45. Demonstrating the critical negative regulatory function of the wedge, in a mixed 129/Sv-C57Bl/6 (B6) genetic background, the CD45E613R mutation led to T and B cell activation, a lymphoproliferative disorder (LPD) characterized by splenomegaly and lymphadenopathy, and a lupus-like syndrome with autoantibody production and glomerulonephritis (GN). To determine which components of this phenotype were due to the direct effects of the wedge mutation and which were the consequence of genetic modifiers known to segregate between different murine strains, we backcrossed the CD45E613R mice at least 9 generations onto B6, 129/Sv, and BALB/c genetic backgrounds. Here, we use cellular and biochemical approaches to demonstrate that the CD45E613R mutation leads to cell autonomous hyperresponsiveness in B, T, and myeloid cells in each genetic background. However, the phenotypic consequence of this hyperresponsiveness is sensitive to strain-specific genetic modifiers. Despite similar signaling defects at a biochemical level, evaluation of cohorts of F9 CD45E613R and control mice (n=20 mice/genotype/strain) indicate that the LPD and autoimmune phenotypes can be genetically separated in homogenous inbred strains. For example, F9 B6 CD45E613R mice develop a very mild LPD and no evidence of autoimmunity (autoantibodies or GN) when examined serially to 18 months of age. In contrast, 100% of F9 BALB/c CD45E613R mice develop autoantibodies by 12 weeks of age. Despite autoantibody production, these animals fail to develop end-organ damage with age. The phenotype of F9 129/Sv CD45E613R mice is also modest with all animals developing a mild LPD with age and only 15% of the animals developing autoantibodies and GN. Confirming the influence of genetic modifiers in the CD45E613R phenotype, generation of F1 B6-129 mice recapitulated the original phenotype by developing a serve LPD, elevated white blood cell counts, production of autoantibodies, and GN. Disease was also more pronounced in B6-BALB/c F1 mice. To begin to identify modifying loci, 262 F2 CD45E613R B6-BALB mice were generated and characterized at 4 months of age. Isolated autoantibody production was observed in 15.6%, isolated LPD in 7.7%, and both LPD and autoantibodies in 6.5% of F2 mice. Analysis of 94 CD45E613R N2 backcross (B6-BALB/c F1 backcrossed to BALB/c) progeny revealed autoantibody in 64.9% of mice. Quantitative trait loci (QTL) mapping using 358 SNP markers to differentiate B6, BALB/c, and 129/Sv strains is being utilized to identify loci that either enhance or prevent lymphoproliferation and autoimmunity in the context of cell-intrinsic lymphocyte hyperactivity conferred by the CD45E613R mutation.

Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z

Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro. The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We report here a genetic method to screen for PTP substrates which we have named the “yeast substrate-trapping system.” This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This method is probably applicable to all the PTPs, because it is based on PTP–substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.

Involvements of mitochondrial thioredoxin reductase (TrxR2) in cell proliferation

Mammalian cells contain two forms of thioredoxin reductase (TrxR), cytosolic TrxR1 and mitochondrial TrxR2. To investigate the biological roles of TrxR2, we generated stable HeLa cell lines expressing a dominant negative form of TrxR2 (TrxR2DN) under the control of the tetracycline-off system. We observed that TrxR2DN-induced cells, following stimulation with EGF, produced more hydrogen peroxide than uninduced cells. The extent of protein tyrosine phosphorylation of many proteins including ERK was higher in TrxR2DN-induced cells than in uninduced cells when stimulated with fetal bovine serum or EGF. Induction of TrxR2DN also resulted in the increased rate of progression of G 1 to S phase in cell cycle and cell proliferation and affected the expression of many proteins involved in cell cycle. These results suggest that TrxR2 participates in the regulation of protein tyrosine phosphorylation and cell growth as a component of the mitochondria specific H 2O 2-eliminating system that includes peroxiredoxin III and thioredoxin 2.

VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts.

VE-cadherin is the essential adhesion molecule in endothelial adherens junctions, and the regulation of protein tyrosine phosphorylation is thought to be important for the control of adherens junction integrity. We show here that VE-PTP (vascular endothelial protein tyrosine phosphatase), an endothelial receptor-type phosphatase, co-precipitates with VE-cadherin, but not with beta-catenin, from cell lysates of transfected COS-7 cells and of endothelial cells. Co-precipitation of VE-cadherin and VE-PTP required the most membrane-proximal extracellular domains of each protein. Expression of VE-PTP in triple-transfected COS-7 cells and in CHO cells reversed the tyrosine phosphorylation of VE-cadherin elicited by vascular endothelial growth factor receptor 2 (VEGFR-2). Expression of VE-PTP under an inducible promotor in CHO cells transfected with VE-cadherin and VEGFR-2 increased the VE-cadherin-mediated barrier integrity of a cellular monolayer. Surprisingly, a catalytically inactive mutant form of VE-PTP had the same effect on VE-cadherin phosphorylation and cell layer permeability. Thus, VE-PTP is a transmembrane binding partner of VE-cadherin that associates through an extracellular domain and reduces the tyrosine phosphorylation of VE-cadherin and cell layer permeability independently of its enzymatic activity.

Reciprocal regulation of lymphocyte activation by tyrosine kinases and phosphatases.

The dynamic regulation of protein tyrosine phosphorylation is crucial for a number of cellular processes including cell growth, differentiation, migration, and death. It thus represents a powerful control point for integration of environmental signals into cellular responses. Regulation of tyrosine phosphorylation is determined by the balance between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). In the immune system, precise and coordinated regulation of this equilibrium allows for rapid responses to foreign antigens, whereas an imbalance between PTKs and PTPs can have pathologic consequences, including autoimmunity, immunodeficiency, and malignancy (1). In this review, we highlight recent advances in our understanding of key PTKs and PTPs that play critical roles in modulating cellular levels of phosphotyrosine in resting T cells, during T cell activation, and during downregulation of an immune response.

Protein tyrosine phosphorylation in sperm during gamete interaction in the mouse: the influence of glucose.

A key intracellular event during capacitation is protein tyrosine phosphorylation, but its involvement during sperm interaction with the oocyte has not been investigated. Glucose is necessary to achieve fertilization and thus may have an influence on sperm protein tyrosine phosphorylation. The objectives of this study were to 1) visualize protein tyrosine phosphorylation patterns in sperm during capacitation and interaction with the oocyte and 2) determine the influence of glucose. Protein tyrosine phosphorylation was investigated by Western analysis and immunofluorescence. Protein tyrosine phosphorylation was increased during capacitation, and immunofluorescence revealed that zona binding and gamete fusion were correlated with an increase in tyrosine phosphorylation of proteins in the midpiece. During capacitation, the absence of glucose led to a delay in the appearance of protein tyrosine phosphorylation. Following binding to the zona pellucida and the oolemma, tyrosine phosphorylation in the flagellum was also delayed in the absence of glucose and resulted in a significant inhibition of the midpiece phosphorylation. The correlation between successful gamete fusion and the tyrosine phosphorylation of midpiece proteins suggests that the effect of glucose on sperm-oocyte interaction is mediated through regulation of protein tyrosine phosphorylation in a specific area of the fertilizing sperm.

Increased expression of iNOS and c-fos via regulation of protein tyrosine phosphorylation and MEK1/ERK2 proteins in terminal bronchiole lesions in the lungs of rats exposed to cigarette smoke.

Epidemiological evidence suggests that smoking is a major cause of human lung cancer. However, the mechanism by which cigarette smoke induces the cancer remains unestablished. To evaluate the effects of cigarette smoke on the expression of inducible nitric oxide synthase (iNOS), nuclear protooncogenes and related mitogen-activated protein kinases (MAPKs) in rat lung tissue, a histopathological study of the effects of gas-phase cigarette smoke on rat lung tissue were carried out. The terminal bronchioles were found to be infiltrated predominantly by lymphocytes in the peribronchiolar region and a mild to moderate degree of emphysema was noted in the alveolar spaces. The terminal bronchioles also showed marked lipid peroxidation, dilatation, and peribronchiolar fibrosis. Immunohistochemical evaluation showed that the expression of iNOS, NF-kappa B, MAPKs (MEK1, ERK2), phosphotyrosine protein and c-fos was increased in the terminal bronchioles but protein kinase C (PKC), MEKK-1, c-jun, p38 and c-myc showed no change. These results provide evidence to suggest that exposure to cigarette smoke results in oxidant stress which leads to the stimulation of iNOS and c-fos together with the induction of protein tyrosine phosphorylation and MEK1/ERK2 which in turn may promote lung pathogenesis.

Regulation of sperm function by protein tyrosine phosphorylation in diverse wild felid species.

Protein tyrosine phosphorylation is associated with sperm capacitation and the acrosome reaction in several mammalian species. Changes in phosphorylation of a 95-kDa protein in human, mouse, and domestic cat spermatozoa are known to be influenced by capacitation and exposure to zona pellucida (ZP) proteins. We previously reported diminished phosphorylation of 95- and 160-kDa proteins in spermatozoa from teratospermic cats, compared with normospermic domestic cats. To determine if these proteins and mechanisms are present in other species in the phenotypically diverse Felidae family, we examined the relationship between tyrosine-phosphorylated sperm proteins and sperm morphology in the leopard cat (approximately 65% normal sperm/ejaculate), tiger (approximately 65%), clouded leopard (approximately 15%), and cheetah (approximately 30%). Furthermore, we investigated the involvement of cyclic adenosine monophosphate (cAMP) in the regulation of sperm protein tyrosine phosphorylation. Specifically, we assessed the following: 1) presence of tyrosine-phosphorylated proteins in sperm extracts; 2) changes in protein tyrosine phosphorylation after sperm capacitation and swim-up separation; 3) impact of tyrosine kinase inhibition on leopard cat sperm protein phosphorylation and ZP penetration; and 4) involvement of a cAMP-dependent pathway in the regulation of protein tyrosine phosphorylation. Immunoblotting analysis with anti-phosphotyrosine antibody (PY20) indicated that a 95-kDa protein was present in all four species. Additional phosphorylated proteins were detected in the leopard cat (145- and 175-kDa proteins), tiger (185-kDa protein), clouded leopard (160- and 190-kDa proteins), and cheetah (115- and 155-kDa proteins). Sperm capacitation in vitro increased phosphorylation of one or more proteins in the leopard cat, tiger and clouded leopard, but not in the cheetah. Although swim-up separation increased the proportion of morphologically normal spermatozoa in the clouded leopard and cheetah, no changes were observed in phosphorylation of the 95-kDa sperm protein. Thus, phosphorylation of the 95-kDa protein appeared to be related to the condition of teratospermia. Exposing leopard cat spermatozoa to the tyrosine kinase inhibitor, tyrphostin, reduced (P < 0.05) phosphorylation of the 95- and 145-kDa proteins, as well as ZP penetration, without affecting sperm motility. Similarly, when spermatozoa were incubated in the presence of cAMP analogs or active and inactive stereoisomers of cAMP, phosphorylation of sperm proteins was either stimulated or inhibited. Together, these data suggest that protein tyrosine kinase mechanisms appear conserved within the family Felidae and are regulated by a cAMP/protein kinase A pathway.

Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP‐dependent pathway

Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP‐dependent pathway

Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP‐dependent pathway

Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP‐dependent pathway

Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP-dependent pathway.

Changes of protein tyrosine phosphorylation in ejaculated boar sperm incubated in vitro were examined with the use of antiphosphotyrosine antibodies and immunoblotting. The intracellular levels of cAMP were modulated by treatment with various combinations of caffeine, 3-isobutyl-1-methylxanthine (IBMX), and dibutyryl cyclic AMP (dbcAMP), and acrosome reactions (ARs) were induced via treatment with divalent cation ionophore A23187. Proteins of Mr 34, 38, 40, and 44 (p34 ... p44) were strongly phosphorylated on tyrosine residues in freshly prepared sperm samples and at the same level during all subsequent treatments. Incubation of sperm in vitro for various periods of time induced an increase of tyrosine phosphorylation of p20, p93, and p175. The tyrosine phosphorylation of p93, p175, and several other sperm proteins was up-regulated in a concentration-dependent manner following treatment of the sperm with dbcAMP, caffeine, or IBMX alone, or with combinations of caffeine and IBMX, respectively, with dbcAMP; the tyrosine phosphorylation of p20 was not correlated with treatment of sperm with cAMP-elevating reagents. The percentage of sperm cells undergoing spontaneous ARs was not affected by the manipulation of cAMP levels and was not correlated with protein tyrosine phosphorylation. In contrast, the addition of calcium to the incubation media decreased protein tyrosine phosphorylation and elevated percentage of spontaneous ARs. The induction of ARs with A23187 caused a significant decrease of tyrosine phosphorylation of p93, p175, and p220/230, indicating that dephosphorylation on protein tyrosine residues might be associated with calcium influx during physiological ARs as well. Proteins p93 and p175 were effectively solubilized in greater than 9M urea/1% triton and in SDS sample buffer, but to only a small extent in triton, while p20 was virtually completely extractable with triton. In conjunction with the previously reported isolation of active tyrosine kinase sp42 from triton extracts of noncapacitated boar sperm cells (Berruti and Porzio, 1992: Biochim Biophys Acta 1118: 149-154), our results suggest that a cAMP-dependent event is required for tyrosine phosphorylation of triton-insoluble proteins such as p93 and p175. On the other hand, the tyrosine phosphorylation of p20 (and potentially other triton-soluble substrates) might not strictly require such cAMP up-regulation. We discuss the differences in the regulation of cAMP-dependent tyrosine phosphorylation in mouse, human, and boar sperm, and suggest that sensitivity to calcium and distinct basal levels of cyclic nucleotide PDE might correspond to species-specific reproduction strategies in mammals.

Reactive Oxygen Species Signaling through Regulation of Protein Tyrosine Phosphorylation in Endothelial Cells

Reactive Oxygen Species Signaling through Regulation of Protein Tyrosine Phosphorylation in Endothelial Cells