Protein tyrosine phosphorylation of the human sperm head during capacitation： immunolocalization and relationship with acquisition of sperm-fertilizing ability

The occurrence of tyrosine phosphorylation （TP） in the sperm head during capacitation has been poorly investigated, and no data exist on the relationship of its dynamics with the acquisition of sperm fertilizing ability. This study localized TP of head proteins in human spermatozoa during capacitation and explored its relationship with acquisition of the ability to display progesterone （P）-stimulated acrosome reactions （ARs） and to penetrate zona-free hamster oocytes. By immunofluorescence, TP immunoreactivity was revealed in the acrosomal region of formaldehyde-fixed/unpermeabilized samples, whereas it was abolished in fixed/permeabilized samples, in which TP immunoreactivity was high in the principal piece. No TP immunoreaetivity was detectable in unfixed spermatozoa. Head TP immunoreactivity was localized externally to the acrosome, close to the cytoplasmic membrane, as assessed by transmission electron microscopy. The increase in head TP was an early event during capacitation, occurring within 1 h in capacitating conditions. At this time, the P-stimulated ARs were also increased, whereas egg penetration was as poor as in uncapacitated spermatozoa. At 5 h of capacitation, the extent of neither head TP nor the P-induced ARs were greater than that at 1 h, whereas egg penetration had significantly increased. Seminal plasma inhibited head TP, P-induced ARs and egg penetration. None of these inhibitory effects, unlike those on tail TP, were prevented by the cAMP analogue dbcAMP （N,2-O-dibutyryladenosine 3＇,5＇-cyclic monophosphate）. In conclusion, head TP is a subsurface event occurring early during capacitation and is closely related to acquisition of the ability to display P-stimulated ARs, whereas the ability to fuse with oolemma and to decondense is a later capacitation-related event.

Tyrosine phosphorylation and bacterial virulence

Protein phosphorylation on tyrosine has emerged as a key device in the control of numerous cellular functions in bacteria.In this article,we review the structure and function of bacterial tyrosine kinases and phosphatases.Phosphorylation is catalyzed by autophosphorylating adenosine triphosphate-dependent enzymes（bacterial tyrosine（BY） kinases） that are characterized by the presence of Walker motifs.The reverse reaction is catalyzed by three classes of enzymes：the eukaryotic-like phosphatases（PTPs） and dual-specific phosphatases;the low molecular weight protein-tyrosine phosphatases（LMW-PTPs）;and the polymerase-histidinol phosphatases（PHP）.Many BY kinases and tyrosine phosphatases can utilize host cell proteins as substrates,thereby contributing to bacterial pathogenicity.Bacterial tyrosine phosphorylation/dephosphorylation is also involved in biofilm formation and community development.The Porphyromonas gingivalis tyrosine phosphatase Ltp1 is involved in a restraint pathway that regulates heterotypic community development with Streptococcus gordonii.Ltp1 is upregulated by contact with S.gordonii and Ltp1 activity controls adhesin expression and levels of the interspecies signal AI-2.

THE IMMUNOREGULATORY EFFECT OF TLSF_(JM) ON THE EXPRESSION OF T CELL IL-2R AND PROTEIN TYROSINE PHOSPHORYLATION

The immunoregulatory effect of TLSFJM on the expression of T cell IL- 2R and protein tyrosine phosphorylation ( PTP ) was investigated by immunohistochemistry technique. The results showed that TLSFJMcan markedly suppress the expression of IL-2R and PTP on PHA or TPA-stimulated human PBMC and murine IL-2 dependent cell line CTLL-2. However, there was no effect of TLSFJMon the production of IL-1, IL-2 and IL-6 that play an important role in the course of T lymphocyte proliferation and differentiation.

Lithium decreased NR2B tyrosine phosphorylation and interactions of NR2B and PSD-95 with Src in rat hippocampus following cerebral ischemia

Objective： To study the effects of chronic lithium on N-methyl-d-aspartate receptor subunit 2B （NR2B） tyrosine phosphorylation and the interactions of NR2B and PSD-95 with Src induced by cerebral ischemia/reperfusion （I/R）. Methods： Transient （15min） cerebral ischemia was induced by four-vessel occlusion procedure in SD rats. Immunoprecipitation （IP） and immunoblotting （IB） were performed to investigate the phosphorylation and interactions of proteins. The effects of lithium on tyrosine phosphorylation of NR2B and its interactions with PSD-95 and Src were examined. Results： Transient cerebral ischemia 15 rain followed by reperfusion 6h （I/R 6h） caused a significant increase in tyrosine phosphorylation of NR2B. Administration of LiCI for 7days before ischemia caused a profound decrease in tyrosine phosphorylation of NR2B. Similiarly. the interactions of NR2B and PSD-95 with Src were also enhanced by I/R 6h. moreover, these interactions were also inhibited by chronic lithium. Conclusion： Pretreatment with lithium decrease tyrosine phosphorylation of NR2B and interactions of NR2B and PSD-95 with Src during cerebral I/R.

Effect of Metabolites of Fusarium Solani on Tyrosine Phosphorylation in Cultivated Cells of Solarium Tuberosum

Elicitor-induced phosphorylation of tyrosine residues in proteins of potato was studied. Proteins of crude extract of suspension culture of potato were analyzed by one- and two-dimensional electrophoresis followed by Western blotting with monoclonal antibodies PY20 to phosphotyrosine proteins. One- and two-dimensional electrophoresis revealed l l and 25 tyrosine-phosphorylated proteins, respectively. Glycoprotein increased the phosphorylation level of most of these proteins.

Src mediatesβ-adrenergic receptor induced YAP tyrosine phosphorylation

The Hippo pathway is a newly identified pathway and evolutionarily conserved from flies to humans mainly regulating cell proliferation.Transcriptional co-activator Yes-associated protein(YAP)functions as a major downstream effector and key node of the Hippo pathway.Phosphorylation of YAP is critical to regulate YAP activity and its corresponding functions.β-adrenergic receptor(β-AR),a typical G protein coupled receptor(GPCR),mediates proliferation in various cell types and regulates multiple physical and pathological processes.However,the role ofβ-AR in regulating YAP remains elusive.Here,we report thatβ-AR can obviously stimulate YAP tyrosine phosphorylation.The mechanism is thatβ-AR stimulation results in tyrosine kinase Src activation and Src phosphorylates YAP tyrosine at Y357.Further studies demonstrate that inhibition of Src kinase activity can obviously alleviateβ-AR induced YAP tyrosine phosphorylation and cell proliferation.We conclude thatβ-AR can induce YAP tyrosine phosphorylation and also establish the Src/YAP pathway as a critical signaling branch downstream of GPCR.

Synaptic non-GluN2B-containing NMDA receptors regulate tyrosine phosphorylation of GluN2B 1472 tyrosine site in rat brain slices

Activation of N-methyl-D-aspartate receptors（NMDARs）mediates changes in the phosphorylation status of the glutamate receptors themselves.Previous studies have indicated that during synaptic activity,tyrosine kinases（Src and Fyn）or phosphatases（PTPαand STEP）are involved in regulating the phosphorylation of NMDARs.In this study,we used immunoblotting to investigate the role of an NMDAR subpopulation on the phosphorylation level of the GluN2B subunit at the Y1336 and Y1472sites in rat brain slices after NMDA treatment.We found that NMDA stimulation dramatically decreased the phosphorylation level of GluN2B at Y1472 in a dose-and time-dependent manner,but not at Y1336.Extrasynaptic NMDAR activation did not reduce the phosphorylation of GluN2B at Y1472.In addition,ifenprodil,a selective antagonist of GluN2Bcontaining NMDARs,did not abolish the decreased phosphorylation of GluN2B at Y1472 triggered by NMDA.These results suggest that the activation of synaptic GluN2A-containing NMDARs is required for the decreased phosphorylation of GluN2B at Y1472that is induced by NMDA treatment in rat brain slices.

Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gabl without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3（N） or the regulatory Y221 residue of CrkII, is critical for the induction of Gabl- Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gabl. In GST pull-down assay, Crk-SH2 bound to wild-type Gahl, whereas Crk-SH3（N） interacted with the Gabl mutant, which lacks the clus- tered tyrosine region （residues 242-410）. Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gabl-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory- lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

Effect of genistein on acrosome reaction and zona pellucida binding independent of protein tyrosine kinase inhibition in bull

Aim： To investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa. Methods： The effect of genistein upon motility was assessed by computer-assisted motion analysis. Hemizona assay was performed to detect the ability of spermatozoa binding to the zona pellucida. The inducibility of the acrosome reaction using progesterone and ZP3-6 peptide was analysed by fluorescein-conjugated Pisum sativum agglutinin （FITC-PSA）/Hoechst 33258 double staining. Capacitation after incubation with genistein was assessed by the chlortetracycline （CTC） assay. Immunoblots showed the pattern of protein tyrosine phosphorylation of cryopreserved bovine spermatozoa. Results： Immunodetection of tyrosine-phosphorylated proteins showed that genistein did not affect tyrosine phosphorylation in cryopreserved bovine spermatozoa. However, genistein significantly reduced the progesterone- and ZP3-6 peptide-mediated induction of the acrosome reaction and led to a dose-dependent inhibition of sperm-zona pellucida binding; while sperm motility and capacitation were not affected by this phytoestrogen, as indicated by computer-assisted sperm motion analysis and the CTC assay, respectively. Conclusion： Our results suggest that in cryopreserved bovine spermatozoa, genistein affects a protein tyrosine phosphorylation-independent signal transduction pathway that is involved in sperm capacitation, the acrosome reaction and sperm-zona pellucida binding.

ADHESION-INDUCE PROTEIN TYROSINE PHOSPHORY-LATION IS ASSOCIATED WITH INVASIVE AND METASTATIC POTENTIALS IN B16-BL6 MELANOMA CELLS

Objective: The interaction of cancer cell with extracellular matrix (ECM) happens as an earlier and specific event in the invasive and metastatic cascade. To explore the key element(s) in cancer metastasis and observe the cellECM interaction and its role. Methods: To interrupt the cellECM interaction by suppression of adhesioninduced protein tyrosine phosphorylation with protein tyrosine kinase inhibitor genistein in B16B16 mouse melanoma cells. Results: When B16BL6 cells attached to Matrigel, a solubilized basement membrane preparation from EHS sarcoma, a 125 kDa protein increased its phosphotyrosine content dramatically. In contrast, when the cells were pretreated with 20μM or 30 μM genistein for 3 days, it was revealed a less increase in the phosphotyrosine content of this 125 kDa protein in response to cell attachment to ECM was revealed with immunoblot analysis. Accompanied by the lower level of adhesioninduced protein tyrosine phosphorylation the genisteintreated cells exhibited a decrease in their capabilities of adhesion to Matrigel and invasion through reconstituted basement membrane. The potentials of and forming lung metastatic nodules were also shown to be decreased dramatically in these genisteintreated cells. Conclusion: It was suggested that protein tyrosine phosphorylation in cellECM interaction might be associated with invasive and metastatic potentials in cancer cells.

EphA3 functions are regulated by collaborating phosphotyrosine residues

Ephrin ligands interact with Eph receptors to regulate a wide variety of biological and pathological processes. Recent studies have identified several downstream pathways that mediate the functions of these receptors. Activation of the receptors by ephrin binding results in the phosphorylation of the receptor tyrosine residues. These phospho- rylated residues serve as docking sites for many of the downstream signaling pathways. However, the relative contributions of different phosphotyrosine residues remain undefined. In the present study, we mutated each individual tyrosine residues in the cytoplasmic domain of EphA3 receptor and studied the effects using cell migration, process retraction, and growth cone collapse assays. Stimulation of the EphA3 receptor with ephrin-A5 inhibits 293A cell mi- gration, reduces NG108-15 cell neurite outgrowth, and induces growth cone collapse in hippocampal neurons. Muta- tion of either Y602 or Y779 alone partially decreases EphA3-induced responses. Full abrogation can only be achieved with mutations of both Y602 and Y779. These observations suggest a collaborative model of different downstream pathways.

Overexpression of tyrosine phosphorylated proteins in reproductive tissues of polycystic ovary syndrome rats induced by letrozole

Objective:To identify the alteration of tyrosine phosphorylated protein expression in rats with polycystic ovary syndrome(PCOS).Methods:Sixteen female Sprague-Dawley rats were divided into the control and letrozole-induced PCOS groups.The oestrus cycle of rats was performed by vaginal smear.Sex hormones and morphology of the ovary,oviduct,and uterus were observed.Expressions and intensity of androgen receptor and tyrosine phosphorylated proteins of reproductive organs were investigated by Western blot.Results:Various polycysts and increased androgen receptor expression were present in the ovary of the PCOS group.The levels of follicle-stimulating hormone and testosteone were significantly higher in the PCOS group while progesterone and estradiol levels were significantly decreased as compared with the control group(P<0.05).Only the size of uterus in the PCOS group was significantly smaller than the control group.However,the density of collagen fibers observed in PCOS uterus was greater than the control group.Moreover,tyrosine phosphorylated proteins were significantly overexpressed in ovary(52,42,and 28 kDa),oviduct(72,56,42,and 28 kDa),and uterus(53 and 42 kDa)of the PCOS group compared to the control group.Conclusions:Presence of tyrosine phosphorylated proteins in the ovary,oviduct and uterus suggests that overexpression of tyrosine phosphorylated proteins may be involved in potential mechanism of female infertility especially in PCOS.

Therapeutic potential of targeting protein tyrosine phosphatases in liver diseases

Protein tyrosine phosphorylation is a post-translational modification that regulates protein structure to modulate demic organisms’homeostasis and function.This physiological process is regulated by two enzyme families,protein tyrosine kinases(PTKs)and protein tyrosine phosphatases(PTPs).As an important regulator of protein function,PTPs are indispensable for maintaining cell intrinsic physiology in different systems,as well as liver physiological and pathological processes.Dysregulation of PTPs has been implicated in multiple liver-related diseases,including chronic liver diseases(CLDs),hepatocellular carcinoma(HCC),and liver injury,and several PTPs are being studied as drug therapeutic targets.Therefore,given the regulatory role of PTPs in diverse liver diseases,a collated review of their function and mechanism is necessary.Moreover,based on the current research status of targeted therapy,we emphasize the inclusion of several PTP members that are clinically significant in the development and progression of liver diseases.As an emerging breakthrough direction in the treatment of liver diseases,this review summarizes the research status of PTP-targeting compounds in liver diseases to illustrate their potential in clinical treatment.Overall,this review aims to support the development of novel PTP-based treatment pathways for liver diseases.

Proteomic changes in mammalian spermatozoa during epididymal maturation

Epididymal maturation is associated with the activation of a cAMP-induced tyrosine phosphorylation cascade, which is ultimately associated with the expression of capacitation-dependent sperm functions, such as hyperactivated movement and acrosomal exocytosis. As spermatozoa progress through the epididymis they first acquire the capacity to phosphorylate tyrosine on targets on the principal piece, followed by the midpiece. By the time these cells have reached the cauda epididymidis they can phosphorylate the entire tail from neck to endpiece. This particular pattern of phosphorylation is associated with the ontogeny of fully functional spermatozoa that are capable of fertilizing the oocyte. Proteomic analyses indicate that this change is associated with the phosphorylation of several mitochondrial proteins, creation of a mitochondrial membrane potential and activation of mitochondrial free radical generation. At least in rodent species, activation of sperm mitochondria appears to be a particularly important part of epididymal maturation. （Asian J Androl 2007 July; 9： 554-564）

Characterization of CagA variable region of Helicobacter pylori isolates from Chinese patients

AIM: To characterize the CagA variable region of Helicobacter pylori isolates from Chinese patients.METHODS: DNA fragments in CagA variable region were amplified and sequenced respectively from genomic DNA of 19 isolates from patients with gastric cancer and 20isolates from patients with chronic gastritis. The tendency of phosphorylation in tyrosine(s) of CagA proteins was evaluated subsequently by phosphorylation assay in vivo and in vitro respectively.RESULTS: About 97.44% (38/39) H pylori isolates possessed CagA gene. CagA+ strains contained 2-4tandem five-amino-acid motifs EPIYA but only one EPIYA had repeated sequence in CagA variable region in different isolates. There was no significant difference between the number of EPIYA motifs in H pylori from patients with different diseases. However, only tyrosine site in EPIYA within repeated sequence could be phosphorylated by AGS cells in vivo although all tyrosine sites in EPIYA could be phosphorylated in vitro.CONCLUSION: CagA in Chinese has no functional difference in perturbing cellular signal pathway among different H pylori isolates.

Trichosanthin inhibits T cell activation by interfering with the recruitment of ZAP-70 to CD3 ζchain

Plant protein Trichosanthin (Tk) has been shown in our previous experiments to suppress antigenic response of T cells. Here we explored its inhibitory mechanisms on the proliferation of human Jurkat leukemia T cell triggered by anti-CD3 McAb. By examination of tyrosine phosphorylation of cell lysate, we were able to show that Tk could interfere with the PTK-related activity in the TCR/CD3initiated signal transduction in addition to blocking the phosphorylation of PKC. As shown in our experiment,the expression intensity of ZAP-70, a kind of protein tyrosine kinase, was not changed but its phosphorylation could be inhibited. When physical link between CD3(chain and ZAP-70 was further examined by using coimmunoprecipitation after pluse-treatment of the cell line with Tk, the anti-CD3 McAb-induced recruitment of ZAP70 to CD3 ζ chain was observed to be blocked in some extent. This may account for, at least in part, how Trichosanthin was able to inhibit the TCR-triggered T cell proliferation.

Regulation of gene expression, growth, and cell survival by IL-4: Contribution of multiple signaling pathways

Interleukin-4 is a cytokine produced by activated T cells, mast cells, and basophils that elicits many important biological responses[1] (see Tab 1). These responses range from the regulation of helper T cell differentiation[2] and the production of IgE[3] to the regulation of the adhesive properties of endothelial cells via VCAM-1[4]. In keeping with these diverse biological effects, high-affinity binding sites for IL-4 (Kd 20 to 300 pM) have been detected on many hematopoietic and non-hematopoietic cell types at levels ranging from 50 to 5000 sites per cell[5],This review will focus on the discrete signal transduction pathways activated by the IL-4 receptor and the coordination of these individual pathways in the regulation of a final biological outcome.

p120 Catenin Translocation is Involved in Enhancement of Hepatoma Cellular Malignant Features

OBJECTIVE To investigate the relationship between p 120^ctn translocation and hepatocellular carcinoma cell malignant features and the relationship between p 120^ctn and β-catenin translocation in cell signaling. METHODS Human hepatocellular carcinoma cells were over expressed with p120^ctn isoform 3A using a DNA transfection method. The effects of transfection and expression of p120^ctn and its binding capacity to E-cadherin were examined using immunoprecipitation and immunoblotting methods. p120^ctn subcellular localization and its relation with β-catenin were detected using immunofluorescent microscopy, p120^ctn phosphorylation was produced by EGF treatment. Cell adhesion, cell migration and cell proliferation were also examined in this study. RESULTS We found that p 120^ctn expression was increased after transfection and the binding capacity of p120^ctn to E-cadherin was enhanced. Tyrosine phosphorylation of p120^ctn increased after transfection and EGF treatment. p120^ctn and β-catenin cellular localization displayd a membrane and cytoplasmic expression pattern, but they translocated into the nucleus for relocalization after p120^ctn overexpression plus EGF stimulation. Cell adhesion ability was increased and migration ability reduced after transfection without EGF. Following transfection without EGF cellular proliferation was reduced, but increased after EGF treatment. CONCLUSION Our results suggest that p120^ctn plays an important role in hepatocellular carcinoma cell adhesion, migration and proliferation. In addition there is a relationship between p120^ctn and β-catenin subcellular localization and signaling.

Neuroprotective effects of tetrandrine against vascular dementia

Tetrandrine is one of the major active ingredients in Menispermaceae Stephania tetrandra S.Moore,and has specific therapeutic effects in ischemic cerebrovascular disease.Its use in vascular dementia has not been studied fully.Here,we investigated whether tetrandrine would improve behavioral and cellular impairments in a two-vessel occlusion rat model of chronic vascular dementia.Eight weeks after model establishment,rats were injected intraperitoneally with 10 or 30 mg/kg tetrandrine every other day for 4 weeks.Behavioral assessment in the Morris water maze showed that model rats had longer escape latencies in training trials,and spent less time swimming in the target quadrant in probe trials,than sham-operated rats.However,rats that had received tetrandrine showed shorter escape latencies and longer target quadrant swimming time than untreated model rats.Hematoxylin-eosin and Nissl staining revealed less neuronal necrosis and pathological damage,and more living cells,in the hippocampus of rats treated with tetrandrine than in untreated model rats.Western blot assay showed that interleukin-1β expression,and phosphorylation of the N-methyl-D-aspartate 2B receptor at tyrosine 1472,were lower in model rats that received tetrandrine than in those that did not.The present findings suggest that tetrandrine may be neuroprotective in chronic vascular dementia by reducing interleukin-1β expression,N-methyl-D-aspartate receptor 2B phosphorylation at tyrosine 1472,and neuronal necrosis.

Elucidating the various multi-phosphorylation statuses of protein functional regions by 193-nm ultraviolet photodissociation

Ultraviolet photodissociation is a high-energy fast excitation method in mass spectrometry and has beensuccessfully applied for the elucidation of sequences and structures of biomolecules. However, its abilityto distinguish the phosphorylation sites isomers of multi-phosphopeptides has been not systematicallyinvestigated until now. A 193-nm ultraviolet laser dissociation mass spectrometry system wasestablished in this study and applied to elucidate the complex multi-phosphorylation statuses mimickingthe functional regions of Sicl, Gli3 and Tau. The numbers of matched fragment ions and phosphorylationsite-determining ions were improved on average 123% and 104%, respectively, by utilizing the ultravioletphotodissociation strategy, comparing to the typically utilized collision induced dissociation strategy.Finally. 94% phosphorylation sites within various statuses were unambiguously elucidated.