Marked Increase In Tyrosine Phosphorylation Research Articles

Fibrin activates GPVI in human and mouse platelets

AbstractThe glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet signaling receptor for collagen. Paradoxically, in a FeCl3 injury model, occlusion, but not initiation of thrombus formation, is delayed in GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that GPVI is a receptor for fibrin and speculate that this contributes to development of an occlusive thrombus. We observed a marked increase in tyrosine phosphorylation, including the FcRγ chain and Syk, in human and mouse platelets induced by thrombin in the presence of fibrinogen and the αIIbβ3 blocker eptifibatide. This was not seen in platelets stimulated by a protease activated receptor (PAR)-4 peptide, which is unable to generate fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar to that induced by activation of GPVI. Consistent with this, thrombin did not induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient mouse platelets. Mouse platelets underwent full spreading on fibrin but not fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in the absence of GPVI. Spreading on fibrin was associated with phosphatidylserine exposure (procoagulant activity), and this too was blocked in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to immobilized monomeric and polymerized fibrin. A marked increase in embolization was seen following FeCl3 injury in GPVI-deficient mice, likely contributing to the delay in occlusion in this model. These results demonstrate that GPVI is a receptor for fibrin and provide evidence that this interaction contributes to thrombus growth and stability.

Requirement for Daxx in mature T-cell proliferation and activation

The protein Daxx promotes Fas-mediated cell death through activation of apoptosis signal-regulating kinase 1, leading to the activation of the MAPKs JNK and p38. Owing to the in utero lethality of daxx-deficient mice, the in vivo role of Daxx has been so far difficult to analyze. We have generated transgenic mice expressing a dominant-negative form of Daxx (Daxx-DN) in the T-cell lineage. We show that Daxx is recruited to the Fas receptor upon FasL engagement and that Daxx-DN expression protects activated T cells from Fas-induced cell death, by preventing the death-inducing signal complex to be properly formed. Normal lymphocyte development and homeostasis are nevertheless observed. Interestingly, we report that both in vitro and in vivo stimulation of Daxx-DN T-lymphocytes leads to increased proliferative T-cell responses. This increased proliferation is associated with a marked increase in tyrosine phosphorylation of LAT and ZAP70 as Daxx-DN favor their recruitment to the T-cell receptor (TCR) complex. These findings identify Daxx as a critical regulator of T-lymphocyte homeostasis by decreasing TCR-induced cell proliferation and by promoting Fas-mediated cell death.

Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa

Fertilization of the mammalian oocyte depends on the ability of spermatozoa to undergo a process known as capacitation as they ascend the female reproductive tract. A fundamental feature of this process is a marked increase in tyrosine phosphorylation by an unusual protein kinase A (PKA)-mediated pathway. To date, the identity of the intermediate PKA-activated tyrosine kinase driving capacitation is still unresolved. In this study, we have identified SRC as a candidate intermediate kinase centrally involved in the control of sperm capacitation. Consistent with this conclusion, the SRC kinase inhibitor SU6656 was shown to suppress both tyrosine phosphorylation and hyperactivation in murine spermatozoa. Moreover, SRC co-immunoprecipitated with PKA and this interaction was found to lead to an activating phosphorylation of SRC at position Y416. We have also used difference-in-2D-gel-electrophoresis (DIGE) in combination with mass spectrometry to identify a number of SRC substrates that become phosphorylated during capacitation including enolase, HSP90 and tubulin. Our data further suggest that the activation of SRC during capacitation is negatively controlled by C-terminal SRC kinase. The latter was localized to the acrosome and flagellum of murine spermatozoa by immunocytochemistry, whereas capacitation was associated with an inactivating serine phosphosphorylation of this inhibitory kinase.

Cerebral ischemia enhances tyrosine phosphorylation of occludin in brain capillaries

Cerebral ischemia induces disruption of the blood–brain barrier (BBB), and this disruption can initiate the development of brain injuries. Although the molecular structure of tight junctional complexes in the BBB has been identified, little is known about alterations of tight junctional proteins after cerebral ischemia. Therefore, we investigated alterations of tight junctional proteins, i.e., occludin and zonula occludens (ZO)-1, in isolated rat brain capillaries after microsphere-induced cerebral embolism. We demonstrated that the levels of occludin and ZO-1 had decreased after the embolism. The embolism also resulted in a marked increase in tyrosine phosphorylation of occludin, which was coincident with an increase in the activity of c-Src. These results suggest that a decrease in the levels of occludin and ZO-1, and an increase in tyrosine phosphorylation of occludin may play an important role in the disruption of tight junctions, which may lead to dysfunction of the BBB after cerebral ischemia.

Interaction between leptin and insulin signaling pathways differentially affects JAK-STAT and PI 3-kinase-mediated signaling in rat liver.

Chronic leptin treatment markedly enhances the effect of insulin on hepatic glucose production unproportionally with respect to body weight loss and increased insulin sensitivity. In the present study the cross-talk between insulin and leptin was evaluated in rat liver. Upon stimulation of JAK2 tyrosine phosphorylation, leptin induced JAK2 co-immunoprecipitation with STAT3, STAT5b, IRS-1 and IRS-2. This phenomenon parallels the leptin-induced tyrosine phosphorylation of STAT3, STAT5b, IRS-1 and IRS-2. Acutely injected insulin stimulated a mild increase in tyrosine phosphorylation of JAK2, STAT3 and STAT5b. Leptin was less effective than insulin in stimulating IRS phosphorylation and their association with PI 3-kinase. Simultaneous treatment with both hormones yielded no change in maximal phosphorylation of STAT3, IRS-1, IRS-2 and Akt, but led to a marked increase in tyrosine phosphorylation of JAK2 and STAT5b when compared with isolated administration of insulin or leptin. This indicates that there is a positive cross-talk between insulin and leptin signaling pathways at the level of JAK2 and STAT5b in rat liver.

Direct Visualization of Internalization and Intracellular Trafficking of Dopamine-Releasing Protein-36aa

Dopamine-releasing protein (DARP) is a potent regulator of dopamine (DA) release known to be involved in the development of rat catecholaminergic systems. In the present study, we examined the internalization and transport of DARP-36aa, a synthetic peptide derived from the N-terminal sequence of DARP, in rat C6 glioma cells. A colloidal gold DARP-36aa conjugate (DARP-36aa: AU) and biotinylated DARP-36aa were employed to visualize internalization and intracellular transport of DARP-36aa. Electron microscopy demonstrated that DARP-36aa: AU was rapidly incorporated into C6 glioma cells. Internalization via clathrin-coated pits and vesicles was clearly observed followed by transport and sorting of DARP-36aa: AU into multivesicular bodies, tubulo-vesicular endosomes, and lysosomes. Internalization of DARP-36aa: AU was also examined in primary mesencephalic cell cultures where a similar pattern of internalization and transport via clathrin-coated pits and vesicles was observed. Fluorescence microscopy using a biotinylated DARP-36aa/avidin-rhodamine conjugate revealed that DARP-36aa is diffusely distributed on the plasmalemma prior to internalization at 4°C. Following a 30-min incubation at 37°C DARP-36aa was concentrated in the cytosol, particularly in areas surrounding cellular projections and the perikaryon. Immunocytochemical studies employing biotinylated DARP-36aa and an anti-clathrin heavy chain antibody demonstrated that DARP-36aa and clathrin colocalize during DARP-36aa internalization. We also observed a marked increase in tyrosine phosphorylation of a 45-kD protein in response to DARP-33a stimulation in C6 glioma cells. Genistein, a specific tyrosine kinase inhibitor, significantly inhibited DARP-36aa: AU internalization and transport in C6 glioma cells. These findings suggest that tyrosine kinase activity may result in DARP-36aa receptor-mediated endocytosis.

Coincubation of human spermatozoa with Chlamydia trachomatis in vitro causes increased tyrosine phosphorylation of sperm proteins.

Elementary bodies (EBs) of the obligate intracellular bacterium Chlamydia trachomatis are responsible for the first step of attachment to host cells. We have studied the effects of EBs on human sperm protein tyrosine phosphorylation, which is important to sperm function. Indirect immunofluorescence using antiphosphotyrosine antibodies showed that serovar E, but not LGV, caused increased tyrosine phosphorylation which was localized to the sperm tail region. Immunoblotting revealed that serovar E caused a marked increase in tyrosine phosphorylation of 80- and 95-kDa sperm proteins, whereas serovar LGV caused increased phosphorylation of only the 80-kDa moiety. Considering the importance of tyrosine phosphorylation for sperm capacitation and other aspects of sperm function, we conclude that EBs may affect these events.

ATP depletion increases tyrosine phosphorylation of beta-catenin and plakoglobin in renal tubular cells.

This study examines the hypothesis that the loss of integrity of the junctional complex induced by ATP depletion is related to alterations in tyrosine phosphorylation of the adherens junction proteins beta-catenin and plakoglobin. ATP depletion of cultured mouse proximal tubular (MPT) cells induces a marked increase in tyrosine phosphorylation of both beta-catenin and plakoglobin. The tyrosine phosphatase inhibitor vanadate has the same effect in ATP-replete (control) monolayers, whereas genistein, a tyrosine kinase inhibitor, reduces phosphorylation of both proteins in ATP-replete monolayers and prevents the hyperphosphorylation of these proteins with ATP depletion. This study also demonstrates that the fall in the transepithelial resistance of MPT monolayers induced by ATP depletion can be reproduced by treatment of ATP-replete monolayers with vanadate, whereas genistein substantially ameliorates the fall in transepithelial resistance induced by ATP depletion. Also, using immunofluorescence microscopy it was demonstrated that ATP depletion results in a marked diminution of E-cadherin staining in the basolateral membrane of MPT cells. Vanadate mimics this effect of ATP depletion, whereas genistein ameliorates the reduction in the intensity of E-cadherin staining induced by ATP depletion. Because it is has been well established that hyperphosphorylation of the catenins leads to dissociation of the adherens junction and to dysfunction of the junctional complex, it is proposed that the increase in tyrosine phosphorylation of catenins observed in MPT cells during ATP depletion contributes to the loss of function of the junctional complex associated with sublethal injury.

LYMPHOCYTE-MEDIATED DENDRITIC CELL TYROSINE PHOSPHORYLATION AND MAP KINASE ACTIVATION CONTRIBUTE TO ALLOGENEIC RESPONSES IN VITRO

819 The cytokine release which follows the interaction of recipient dendritic cells (DC) and donor lymphocytes (TL) initiates the immune processes which culminate in graft rejection. Cell-cell contact between DC and TL results from a number of surface proteins, including CD86 and CD80 on DC and the receptor CD28 on TL. While CD28 co-stimulatory signals can be mediated via tyrosine phosphorylation signaling, the nature of the DC response following TL contact remains to be defined. We postulated that such contact leads to DC tyrosine phosphorylation and therefore to activation of key signaling cascades, such as the Mitogen Activated Protein (MAP) kinases. To test this hypothesis splenic TL harvested from C3H mice were layered over confluent DC isolated from C57BL/6 mice and co-cultured. TL and DC were separated at various times, and the two cell populations studied individually for signaling events. As determined by Western blot analysis, the TL stimulated a marked increase in tyrosine phosphorylation within DC that peaked by 90 minutes and then faded. By contrast, tyrosine phosphorylation in the TL population over the same time span was weak. Phosphorylated, active ERK MAP kinase isoforms were consistently upregulated in DC, with less phosphorylation of the related p38 MAP kinase. The physiologic importance of DC tyrosine phosphorylation was suggested by a 67±11% (mean±SEM, n=4) inhibition of 24-hour TNFα and 18±9% inhibition of IL-2 production when the TL/DC co-culture was exposed to the broad-spectrum tyrosine kinase inhibitor Tyrphostin AG126 (50 μM). Intriguingly, selective ERK MAP kinase inhibition with PD98059 (50 μM) inhibited production of both TNFα (45±10%) and IL-2 (39±6%), while selective p38 inhibition with SB203580 (30 μM) inhibited only TNFα (52±8%), with no effect on IL-2 release (<1%). We have previously noted that blockade of the CD86/CD28 and CD80/CD28 interaction with specific monoclonal antibodies against CD86 and CD80 abrogates cytokine generation in this model. Pretreatment of DC with the same antibodies markedly attenuated TL-induced DC tyrosine phosphorylation. These data argue that contact with allogeneic TL stimulates a time-dependent increase in DC tyrosine phosphorylation that is mediated at least in part via CD86 and CD80 and is crucial for the subsequent induction of immune responses. The ERK MAP Kinase in particular plays a key role in the allogeneic immune response, suggesting that future immunomodulatory therapies could be targeted to this pathway.

Involvement of reactive oxygen species in human sperm arcosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates.

Although recent evidence indicated that the production of reactive oxygen species (ROS) by human spermatozoa may be involved in the regulation of capacitation, very little is known about the role of ROS in the acrosome reaction. To address this issue, Percoll-washed spermatozoa were incubated in Ham's F-10 medium in the absence (no capacitation) or presence (capacitation) of fetal cord serum ultrafiltrate (FCSu) or progesterone. The effects of the ROS scavengers, superoxide dismutase (SOD), and catalase were then tested on the acrosome reaction induced by lysophosphatidylcholine (LPC), A23187, and ultrafiltrates from follicular fluid (FFu) and FCSu, as well as on the protein tyrosine phosphorylation associated with this process. 2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA)-amplified chemiluminescence was used to determine the extracellular superoxide (O2.-) production from spermatozoa. The observations that both SOD and catalase reduced (in the case of LPC) or totally prevented (in the other cases) the acrosome reaction of capacitated spermatozoa and that hydrogen peroxide (H2O2) or ROS generated by the combination of xanthine and xanthine oxidase (O2.-, which dismutates to H2O2) triggered the acrosome reaction indicated the involvement of ROS in this process. In fact, capacitated spermatozoa in which the acrosome reaction was induced by LPC, A23187, and FFu produced more O2.- than noncapacitated spermatozoa treated with the same agents. A23187 and LPC had minor effects on protein tyrosine phosphorylation of noncapacitated spermatozoa. However, these inducers caused a decrease in tyrosine phosphorylation of Triton-soluble proteins (mainly those of 37, 42, and 47 kDa) from capacitated spermatozoa, a decrease more pronounced in the presence of SOD. On the other hand, there was a marked increase in tyrosine phosphorylation of few proteins (70 to 105 kDa) from the Triton-insoluble fraction, which was partly reversed by SOD (in the case of LPC and A23187) or catalase (in the case of A23187), or abolished in the presence of the two antioxidants (in the case of A23187). These data indicate that the acrosome reaction is associated with an extracellular O2.- generation by spermatozoa and that both O2.- and H2O2 may be involved in the regulation of this process. The mechanism by which these ROS act is unknown but may involve tyrosine phosphorylation of sperm proteins.

Free Radical Stimulation of Tyrosine Kinase and Phosphatase Activity in Human Peripheral Blood Mononuclear Cells

Human lymphocytes were challenged with reactive oxygen species (ROS) generated by xanthine/xanthine oxidase leading to an increase in tyrosine phosphorylation, together with an increase in tyrosine phosphatase activity. In the presence of 50 μM vanadate and xanthine/xanthine oxidase, tyrosine phosphatase activity was inhibited and a marked increase in tyrosine phosphorylation was observed. The addition of catalase abolished the increase in tyrosine phosphorylation while the addition of superoxide dismutase had no effect. This suggests that vanadate together with hydrogen peroxide derived from xanthine/xanthine oxidase activity, interact to produce an agent that is an effective inhibitor of tyrosine phosphatase activity. When human lymphocytes were challenged with xanthine/xanthine oxidase in the presence of 50 μM CuCl2, an increase in both tyrosine phosphatase and kinase activity was observed. Cupric ions inhibited xanthine oxidase activity by 84%; neither superoxide or hydroxyl radicals could be detected, but traces of hydrogen peroxide were detected in the medium. We conclude that unbound metals can interact with ROS and readily influence signalling mechanisms in human lymphocytes.

Induction of tyrosine phosphorylation and Na+/H+ exchanger activation during shrinkage of human neutrophils.

The ubiquitous isoform of the Na+/H+ exchanger (NHE1) is essential for the regulation of cellular volume. The underlying molecular mechanism, which is poorly understood, was studied in human polymorphonuclear leukocytes (PMN). Suspension of PMN in hypertonic media induced rapid cellular shrinkage and activation of NHE1, which is measurable as a cytosolic alkalinization. Concomitantly, hypertonic stress also induced extensive tyrosine phosphorylation of several proteins. Pretreatment of PMN with genistein, a tyrosine kinase inhibitor, prevented not only the tyrosine phosphorylation in response to a hypertonic shock but also the activation of NHE1. The signal elicited by hyperosmolarity that induces activation of tyrosine kinases and NHE1 was investigated. Methods were devised to change medium osmolarity without altering cell volume and vice versa. Increasing medium and intracellular osmolarity in normovolemic cells failed to activate tyrosine kinases or NHE1. However, shrinkage of cells under iso-osmotic conditions stimulated both tyrosine phosphorylation and NHE1 activity. These findings imply that cells detect alterations in cell size but not changes in osmolarity or ionic strength. The identity of the proteins that were tyrosine-phosphorylated in response to cell shrinkage was also investigated. Unexpectedly, the mitogen-activated protein kinases SAPK, p38, erk1, and erk2 were not detectably phosphorylated or activated. In contrast, the tyrosine kinases p59(fgr) and p56/59(hck) were phosphorylated and activated upon hypertonic challenge. We propose that cells respond to alterations in cell size, but not to changes in osmolarity, with increased tyrosine phosphorylation, which in turn leads to the activation of NHE1. The resulting changes in ion content and cytosolic pH contribute to the restoration of cell volume in shrunken cells.

Vascular Endothelial Growth Factor Stimulates Tyrosine Phosphorylation and Recruitment to New Focal Adhesions of Focal Adhesion Kinase and Paxillin in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulated the tyrosine phosphorylation of multiple components in confluent human umbilical vein endothelial cells (HUVECs) including bands of Mr 205,000, corresponding to the VEGF receptors Flt-1 and KDR, and Mr 145,000, 120,000, 97,000, and 65,000-70,000. VEGF caused a striking and transient increase in mitogen-activated protein (MAP) kinase activity and stimulated phospholipase C-gamma tyrosine phosphorylation, but it had no effect on phosphatidylinositol 3'-kinase activity. VEGF caused a marked increase in tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)), which was both rapid and concentration-dependent. VEGF produced similar effects on p125(FAK) in the endothelial cell line ECV.304. VEGF stimulated tyrosine phosphorylation of the 68-kDa focal adhesion-associated component, paxillin, with similar kinetics and concentration dependence to that for p125(FAK). Thrombin and the phorbol ester, phorbol 12-myristate 13-acetate, also increased p125(FAK) tyrosine phosphorylation in HUVECs. The effect of VEGF on p125(FAK) tyrosine phosphorylation was completely inhibited by the actin filament-disrupting agent cytochalasin D and was partially inhibited by the protein kinase C inhibitor GF109203X. Inhibition of the MAP kinase pathway using a specific inhibitor of MAP kinase kinase had no effect on p125(FAK) tyrosine phosphorylation. VEGF stimulated migration and actin stress fiber formation in confluent HUVEC, and VEGF-induced p125(FAK)/paxillin tyrosine phosphorylation was accompanied by increased immunofluorescent staining of p125(FAK), paxillin, and phosphotyrosine in focal adhesions in confluent cultures of HUVECs. These findings identify p125(FAK) and paxillin as components in a VEGF-stimulated signaling pathway and suggest a novel mechanism for VEGF regulation of endothelial cell functions.

Insulin stimulates the phosphorylation of the 66- and 52-kilodalton Shc isoforms by distinct pathways.

In contrast to the 52-kDa Shc isoform, insulin stimulation caused a quantitative, time-dependent decrease in the SDS-PAGE mobility of 66-kDa Shc in both Chinese hamster ovary/IR cells and 3T3L1 adipocytes. Alkaline phosphatase treatment and direct phosphoamino acid analysis demonstrated that insulin stimulated an increase in serine phosphorylation of the 66-kDa isoform but not 52-kDa Shc, although the latter displayed a marked increase in tyrosine phosphorylation. To identify the responsible kinase pathway, we compared the effects on 66-kDa Shc serine phosphorylation by insulin, anisomycin, and osmotic shock, agents that specifically activate the ERK, JNK, or both pathways, respectively. Insulin and osmotic shock both stimulated a decrease in 66-kDa Shc mobility, whereas anisomycin had no effect. Furthermore, expression of a dominant-interfering Ras mutant (N17Ras) prevented the insulin-stimulated, but not the osmotic shock-induced serine phosphorylation of 66-kDa Shc. Consistent with a MEK-dependent pathway mediating 66-kDa Shc serine phosphorylation, the specific MEK inhibitor (PD98059) and expression of a dominant-interfering MEK mutant partially inhibited both the insulin and osmotic shock-induced reduction in 66-kDa Shc mobility. In contrast, expression of the MAP kinase phosphatase (MKP-1) completely prevented ERK activation but did not inhibit the serine phosphorylation of 66-kDa Shc. These data demonstrate that insulin stimulates the serine phosphorylation of the 66-kDa Shc isoform through a MEK-dependent mechanism.

Starvation-increased insulin-dependent tyrosine phosphorylation of the 195-kDa protein in intact rat liver.

Insulin stimulates tyrosine phosphorylation of 175-195 kDa proteins including insulin receptor substrate-1 (IRS-1) in various tissues and cell types. In intact rat livers, starvation increased the insulin-dependent tyrosine phosphorylation of the insulin receptor and IRS-1 as has been described by others. Surprisingly, starvation greatly increased the tyrosine phosphorylation of the 195-kDa protein induced by insulin, indicating that this protein may be a new substrate of the insulin receptor kinase. The marked increase in tyrosine phosphorylation of the 195-kDa protein may have a physiological role in signal transmission in response to insulin under starvation conditions.

Stimulation of Fc(alpha) receptors induces tyrosine phosphorylation of phospholipase C-gamma(1), phosphatidylinositol phosphate hydrolysis, and Ca2+ mobilization in rat and human mesangial cells.

Although knowledge of IgA Fc receptor (Fc(alpha)R) structure and gene organization has progressed in the past few years, signal transduction pathways elicited by its activation have hardly been studied. Previously, we have demonstrated that mesangial cells (MC) possess Fc(alpha)R stimulation triggers several biologic responses. In this work, we studied the early biochemical signals triggered by Fc(alpha)R stimulation in MC. MC incubation with aggregated IgA (AIgA) elicited a dose-dependent increase in cytosolic Ca2+ ([Ca2+]i). The response was rapid and transient, and slowly fell to the original baseline. Ca2+ mobilization was dependent on the Fc region of the IgA, because Fc, but neither Fab fragment nor carbohydrates, inhibited the [Ca2+] rise. The initial induction of [Ca2+]i rise was due to Ca2+ mobilization from inositol trisphosphate (IP3)-sensitive intracellular stores, while sustained levels were maintained through extracellular Ca2+ influx. Stimulation of Fc(alpha)R also resulted in production of IP3, temporally correlated with Ca2+ mobilization. Protein tyrosine kinase inhibitors abolished [Ca2+]i rise, indicating that tyrosine phosphorylation of some substrates is required for Ca2+ mobilization. Stimulation through Fc(alpha)R gave rise to a marked increase in tyrosine phosphorylation of several proteins, including the 147-kDa band, similar in size to phospholipase C-gamma(1) (PLC-gamma(1)). Tyrosine phosphorylation of PLC-gamma(1) reached a maximum 30 s after stimulation, as determined by immunoprecipitation and Western blot. Collectively, these results indicate that the Fc(alpha)R signaling pathway in MC involves PLC-(gamma(1) activation, IP3 formation, and Ca2+ mobilization, and is linked to activation of tyrosine kinases.

Extracellular Zinc Ions Induces Mitogen-Activated Protein Kinase Activity and Protein Tyrosine Phosphorylation in Bombesin-Sensitive Swiss 3T3 Fibroblasts

The growth factor-like effect of zincin vitroandin vivo,which has long been recognized was investigated with respect to its mechanisms of action. Addition of zinc chloride to bombesin-sensitive Swiss 3T3 mouse fibroblasts induced a fourfold stimulation in the cytosolic myelin basic protein kinase activity. The response was dose- and time-dependent, with an ED50of around 100 μMand a peak at 5 min. The kinase activity coeluted with p42 MAP kinase using chromatography on Mono-Q ion exchanger. Intracellular loading of cells with the heavy metal chelator BTC-5N did not attenuate the response to zinc. The action of zinc was not suppressed by long-term pretreatment with 4β-phorbol dibutyrate (48 h). Addition of 0.3 mMvanadate alone did not increase the kinase activity, but prolonged the action of zinc when added simultaneously. Addition of zinc (0.3 mM) or epidermal growth factor for 1 min resulted in a marked increase in tyrosine phosphorylation of proteins with apparent molecular weights of ≈100, 105–120, 215, and 240 kDa in whole cell extracts. Immunoprecipitation against the p85 subunit of phosphatidylinositol 3-kinase resulted in the appearance of two phosphotyrosine-containing proteins, 100 and 115 kDa, in extracts from cells treated with zinc or epidermal growth factor, indicating that the tyrosine phosphorylation was recognized by the corresponding SH2-domains. The present study demonstrates that extracellular zinc has the potential to partially mimic the action of growth factors on intracellular MAP kinase activation and protein tyrosine phosphorylation.

The Yeast Immunophilin Fpr3 Is a Physiological Substrate of the Tyrosine-specific Phosphoprotein Phosphatase Ptp1

The tyrosine-specific phosphoprotein phosphatase encoded by the Saccharomyces cerevisiae PTP1 gene dephosphorylates artificial substrates in vitro, but little is known about its functions and substrates in vivo. The presence of Ptp1 resulted in dephosphorylation of multiple tyrosine-phosphorylated proteins in yeast expressing a heterologous tyrosine-specific protein kinase, indicating that Ptp1 can dephosphorylate a broad range of substrates in vivo. Correspondingly, several proteins phosphorylated at tyrosine by endogenous protein kinases exhibited a marked increase in tyrosine phosphorylation in ptp1 mutant cells. One of these phosphotyrosyl proteins (p70) was also dephosphorylated in vitro when incubated with recombinant Ptp1. p70 was purified to homogeneity; analysis of four tryptic peptides revealed that p70 is identical to the recently described FPR3 gene product, a nucleolarly localized proline rotamase of the FK506- and rapamycin-binding family. The identity of p70 with Fpr3 was confirmed in the demonstration that the abundance of tyrosine-phosphorylated p70 in ptp1 mutants was strictly correlated with the level of FPR3 expression; immobilized phosphotyrosyl Fpr3 was directly dephosphorylated by recombinant Ptp1. Site-directed mutagenesis demonstrated that the site of tyrosine phosphorylation is Tyr-184, which resides within the nucleolin-like amino-terminal domain of Fpr3. Protein kinase activities from yeast cell extracts can bind to and phosphorylate the immobilized amino-terminal domain of Fpr3 on serine, threonine, and tyrosine. Fpr3 represents the first phosphotyrosyl protein identified in S. cerevisiae that is not itself a protein kinase and is as yet the only known physiological substrate of Ptp1.

Modulation of protein tyrosine phosphorylation in rat insular cortex after conditioned taste aversion training.

Protein tyrosine phosphorylation is a major signal transduction pathway involved in cellular metabolism, growth, and differentiation. Recent data indicate that tyrosine phosphorylation also plays a role in neuronal plasticity. We are using conditioned taste aversion, a fast and robust associative learning paradigm subserved among other brain areas by the insular cortex, to investigate molecular correlates of learning and memory in the rat cortex. In conditioned taste aversion, rats learn to associate a novel taste (e.g., saccharin) with delayed poisoning (e.g., by LiCl injection). Here we report that after conditioned taste aversion training, there is a rapid and marked increase in tyrosine phosphorylation of a set of proteins in the insular cortex but not in other brain areas. A major protein so modulated, of 180 kDa, is abundant in a membrane fraction and remains modulated for more than an hour after training. Exposure of the rats to the novel taste alone results in only a small modulation of the aforementioned proteins whereas administration of the malaise-inducing agent per se has no effect. To the best of our knowledge, this is the first demonstration of modulation of protein tyrosine phosphorylation in the brain after a behavioral experience.

Unique and selective mitogenic effects of vanadate on SV40-transformed cells.

Vanadate and insulin both function as unique complete mitogens for SV40-transformed 3T3T cells, designated CSV3-1, but not for nontransformed 3T3T cells. The mitogenic effects induced by vanadate and insulin in CSV3-1 cells are mediated by different signaling mechanisms. For example, vanadate does not stimulate the tyrosine phosphorylation of the insulin receptor beta-subunit nor the 170 kDa insulin receptor substrate-1. Instead, vanadate induces a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins that is not observed in insulin-stimulated CSV3-1 cells. Perhaps most interestingly, vandate-induced mitogenesis is associated with the selective induction of c-jun and junB expression without significantly inducing c-fos or c-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation and c-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation and c-jun and junB expression may serve the critical roles in mediating vandate-induced mitogenesis in SV40-transformed cells.