Enhanced Protein Tyrosine Phosphorylation Research Articles

Redox Pioneer: Professor Sue Goo Rhee.

Dr. Sue Goo Rhee is recognized as a Redox Pioneer because he has published five articles in the field of antioxidants and redox signaling that have been cited >1000 times and 69 of his articles in this field have been cited between 100 and 1000 times. Dr. Rhee is known for his discovery of the first three prototypical members of the phospholipase C family, and for the discovery of the ubiquitously expressed peroxiredoxins. Peroxiredoxin catalyzes the thiol-mediated reduction of H2O2. These enzymes protect cellular molecules from oxidative damage. Importantly, they also regulate cell signaling by modulating the intracellular levels of H2O2 that are induced by signaling agonists. He elucidated the mechanism by which the peroxiredoxins participate in signaling by H2O2: Dr. Rhee demonstrated that growth agonists such as epidermal growth factor induce a transient elevation of intracellular H2O2 that oxidize the catalytically essential cysteine residue of protein tyrosine phosphatases. The oxidation inactivates the phosphatases, allowing enhanced protein tyrosine phosphorylation to mediate cell signaling. In addition, he established that peroxiredoxins are exquisitely regulated through phosphorylation, glutathionylation, and hyperoxidation of their active site cysteine to cysteine sulfinic acid. Dr. Rhee showed that cysteine oxidation to its sulfinic acid derivative is not irreversible as previously thought. The reduction of hyperoxidized peroxiredoxin is catalyzed by sulfiredoxin. His further investigations implicated cyclic hyperoxidation and reduction of peroxiredoxin in the regulation of certain circadian rhythms.

The Interaction of Pneumocystis with the C-Type Lectin Receptor Mincle Exerts a Significant Role in Host Defense against Infection.

Pneumocystis pneumonia (PCP) remains a major cause of morbidity and mortality within immunocompromised patients. In this study, we examined the potential role of macrophage-inducible C-type lectin (Mincle) for host defense against Pneumocystis Binding assays implementing soluble Mincle carbohydrate recognition domain fusion proteins demonstrated binding to intact Pneumocystis carinii as well as to organism homogenates, and they purified major surface glycoprotein/glycoprotein A derived from the organism. Additional experiments showed that rats with PCP expressed increased Mincle mRNA levels. Mouse macrophages overexpressing Mincle displayed increased binding to P. carinii life forms and enhanced protein tyrosine phosphorylation. The binding of P. carinii to Mincle resulted in activation of FcRγ-mediated cell signaling. RNA silencing of Mincle in mouse macrophages resulted in decreased activation of Syk kinase after P. carinii challenge, critical in downstream inflammatory signaling. Mincle-deficient CD4-depleted (Mincle-/-) mice showed a significant defect in organism clearance from the lungs with higher organism burdens and altered lung cytokine responses during Pneumocystis murina pneumonia. Interestingly, Mincle-/- mice did not demonstrate worsened survival during PCP compared with wild-type mice, despite the markedly increased organism burdens. This may be related to increased expression of anti-inflammatory factors such as IL-1Ra during infection in the Mincle-/- mice. Of note, the P. murina-infected Mincle-/- mice demonstrated increased expression of known C-type lectin receptors Dectin-1, Dectin-2, and MCL compared with infected wild-type mice. Taken together, these data support a significant role for Mincle in Pneumocystis modulating host defense during infection.

SH2 Domain-Containing Protein Tyrosine Phosphatase 2 and Focal Adhesion Kinase Protein Interactions Regulate Pulmonary Endothelium Barrier Function

Enhanced protein tyrosine phosphorylation is associated with changes in vascular permeability through formation and dissolution of adherens junctions and regulation of stress fiber formation. Inhibition of the protein tyrosine phosphorylase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) increases tyrosine phosphorylation of vascular endothelial cadherin and β-catenin, resulting in disruption of the endothelial monolayer and edema formation in the pulmonary endothelium. Vascular permeability is a hallmark of acute lung injury (ALI); thus, enhanced SHP2 activity offers potential therapeutic value for the pulmonary vasculature in diseases such as ALI, but this has not been characterized. To assess whether SHP2 activity mediates protection against edema in the endothelium, we assessed the effect of molecular activation of SHP2 on lung endothelial barrier function in response to the edemagenic agents LPS and thrombin. Both LPS and thrombin reduced SHP2 activity, correlated with decreased focal adhesion kinase (FAK) phosphorylation (Y(397) and Y(925)) and diminished SHP2 protein-protein associations with FAK. Overexpression of constitutively active SHP2 (SHP2(D61A)) enhanced baseline endothelial monolayer resistance and completely blocked LPS- and thrombin-induced permeability in vitro and significantly blunted pulmonary edema formation induced by either endotoxin (LPS) or Pseudomonas aeruginosa exposure in vivo. Chemical inhibition of FAK decreased SHP2 protein-protein interactions with FAK concomitant with increased permeability; however, overexpression of SHP2(D61A) rescued the endothelium and maintained FAK activity and FAK-SHP2 protein interactions. Our data suggest that SHP2 activation offers the pulmonary endothelium protection against barrier permeability mediators downstream of the FAK signaling pathway. We postulate that further studies into the promotion of SHP2 activation in the pulmonary endothelium may offer a therapeutic approach for patients suffering from ALI.

Active SHP2 protects the pulmonary endothelium through a FAK‐mediated mechanism

Enhanced protein tyrosine phosphorylation is associated with increased vascular permeability. We have previously shown that chemical and molecular inhibition of the protein tyrosine phosphatase, SHP2, promotes edema formation in the pulmonary endothelium (EC) in vitro and in vivo; thus implicating SHP2 in a protective role of pulmonary EC barrier function. To study whether this function of SHP2 translates to therapeutic value, we assessed the effect of molecular activation of SHP2 on EC barrier function in response to edemagenic agents, lipopolysaccharide (LPS) and thrombin. Both LPS and thrombin reduced SHP2 activity, which correlated with an elevation in focal adhesion kinase (FAK) Y397 phosphorylation and decrease in SHP2 protein‐protein associations with FAK, p120RasGAP, and p190RhoGAP. In contrast, the barrier protective agent S1P elevated SHP2 activity, enhanced FAK Y576 phosphorylation and increased FAK‐SHP2 association. In vivo, overexpression of constitutively active SHP2 (caSHP2) enhanced EC monolayer resistance at basal conditions and attenuated LPS and thrombin‐induced permeability, however had no effect on s1P‐induced barrier protection. In vivo, overexpression of caSHP2 completely attenuated LPS‐induced pulmonary edema formation. Our data demonstrated that the protective role for SHP2 in maintaining pulmonary EC barrier function may be mediated via FAK.

Suicide Plasmid-Dependent IS1-Element Untargeted Integration into Aeromonas veronii bv. sobria Generates Brown Pigment-Producing and Spontaneous Pelleting Mutant

Foodborne Gram-negative pathogens belonging to the genus Aeromonas are variable in harboring insertion sequence (IS) elements that play an important role in the generation of dysfunctional relatives of known genes. Using suicide plasmids carrying an IS1-element, untargeted integration is a common problem during experimental trials to generate specific mutations by homologous recombination. In this work, different strains of Aeromonas veronii bv. sobria (AeG1 and ATCC 9071T), A. hydrophila ATCC 19570, and A. sobria ATCC 43979T are examined for acquisition of IS1-element from pYAK1 suicide plasmid. It was found that untargeted integration of IS1-element is encountered only in ATCC 9071T strain. Such untargeted integration generates a novel brown pigment-producing and spontaneous pelleting (BP(+)SP(+)) mutant. Furthermore, BP(+)SP(+) mutant strain secretes significantly higher quantity of PilF homologous protein than the wild-type strain and displays an enhanced protein tyrosine phosphorylation activity. Thus, current work shows that Aeromonas spp. strains are variable in their susceptibility for suicide plasmid-dependent IS1-element untargeted integration as well as the susceptible strain is changed to mimic pigment-producing and spontaneous pelleting strains that are naturally occurring among heterogeneous group of foodborne aeromonads.

PCSK4-null sperm display enhanced protein tyrosine phosphorylation and ADAM2 proteolytic processing during in vitro capacitation

To study the molecular basis for the accelerated capacitation rate in PCSK4-null sperm. Comparative and controlled experimental research study. Academic medical institute. Male mice C57BL/6J wild-type or null congenics for the Pcsk4 allele. Cauda and epididymal sperm were capacitated for varying times. Differences in sperm protein tyrosine phosphorylation and proteolytic processing of sperm-egg ligands ADAM2 and ADAM3. The PCSK4-null sperm proteins are hyper-tyrosine phosphorylated during capacitation. This hyperphosphorylation is dependent on protein kinase A (PKA), albumin, and calcium. There is also more ADAM2 proteolytic processing from a 46-kDa form of ADAM2 to a 27-kDa form in PCSK4-null sperm than in wild-type sperm. This processing is dependent on cholesterol efflux. Lack of PCSK4 is associated with quantitative changes in the phosphorylation and proteolysis of sperm proteins during capacitation; therefore, alterations in signal transduction and proteolytic processing during capacitation may underlie the fertilization incompetence of PCSK4-null sperm. More investigation is needed to determine how and to what extent these changes might contribute to the loss of fertilizing ability of PCSK4-null sperm.

Extracellular Signal-Regulated Kinase Activation during Renal Ischemia/Reperfusion Mediates Focal Adhesion Dissolution and Renal Injury

Acute renal failure due to ischemia/reperfusion involves disruption of integrin-mediated cellular adhesion and activation of the extracellular signal-regulated kinase (ERK) pathway. The dynamics of focal adhesion organization and phosphorylation during ischemia/reperfusion in relation to ERK activation are unknown. In control kidneys, protein tyrosine-rich focal adhesions, containing focal adhesion kinase, paxillin, and talin, were present at the basolateral membrane of tubular cells and colocalized with short F-actin stress fibers. Unilateral renal ischemia/reperfusion caused a reversible protein dephosphorylation and loss of focal adhesions. The focal adhesion protein phosphorylation rebounded in a biphasic manner, in association with increased focal adhesion kinase, Src, and paxillin tyrosine phosphorylation. Preceding phosphorylation of these focal adhesion proteins, reperfusion caused increased phosphorylation of ERK. The specific mitogen-activated protein kinase kinase 1/2 inhibitor U0126 prevented ERK activation and attenuated focal adhesion kinase, paxillin, and Src phosphorylation, focal adhesion restructuring, and ischemia/reperfusion-induced renal injury. We propose a model whereby ERK activation enhanced protein tyrosine phosphorylation during ischemia/reperfusion, thereby driving the dynamic dissolution and restructuring of focal adhesions and F-actin cytoskeleton during reperfusion and renal injury.

Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca2+ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca2+ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyryl-cAMP and the Epac-selective 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca2+. This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca2+, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-alpha-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca2+ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca2+ in exocytosis.

Focal adhesion kinase (pp125 FAK) phosphorylation is linked to force generation in F9 cells

Acute renal failure due to ischemia/reperfusion involves disruption of integrin-mediated cellular adhesion and activation of the extracellular signal-regulated kinase (ERK) pathway. The dynamics of focal adhesion organization and phosphorylation during ischemia/reperfusion in relation to ERK activation are unknown. In control kidneys, protein tyrosine-rich focal adhesions, containing focal adhesion kinase, paxillin, and talin, were present at the basolateral membrane of tubular cells and colocalized with short F-actin stress fibers. Unilateral renal ischemia/reperfusion caused a reversible protein dephosphorylation and loss of focal adhesions. The focal adhesion protein phosphorylation rebounded in a biphasic manner, in association with increased focal adhesion kinase, Src, and paxillin tyrosine phosphorylation. Preceding phosphorylation of these focal adhesion proteins, reperfusion caused increased phosphorylation of ERK. The specific mitogen-activated protein kinase kinase 1/2 inhibitor U0126 prevented ERK activation and attenuated focal adhesion kinase, paxillin, and Src phosphorylation, focal adhesion restructuring, and ischemia/reperfusion-induced renal injury. We propose a model whereby ERK activation enhanced protein tyrosine phosphorylation during ischemia/reperfusion, thereby driving the dynamic dissolution and restructuring of focal adhesions and F-actin cytoskeleton during reperfusion and renal injury.

Hyper-osmotic condition enhances protein tyrosine phosphorylation and zona pellucida binding capacity of human sperm

The aim of this study was to determine the effect of culture medium osmolality, in the range known to occur in the male and female reproductive tracts, on human sperm tyrosine phosphorylation and sperm-zona pellucida (ZP) interaction in vitro. Motile sperm (2x10(6)), selected by swim-up from semen of normozoospermic men with normal sperm-ZP binding, were incubated with or without four oocytes in 1 ml human tubal fluid (HTF) medium with different osmolalities (150, 200, 280, 350, 400 mOsm/kg) adjusted by variation of the NaCl concentration. After 2 h incubation, the number of sperm bound to the four ZP was examined, sperm motility and velocities were assessed by Hamilton-Thorn Motility Analyzer (IVOS 10) and sperm tyrosine phosphorylation was assessed by both western immunoblotting and immunofluorescence with an anti-phosphotyrosine monoclonal antibody (PY20). The effect of hyper-osmolality (400 mOsm/kg) on the ZP-induced acrosome reaction (AR) was also determined. Incubation of human sperm in hyper-osmotic medium significantly increased tyrosine phosphorylation and the number of sperm bound to the ZP. In contrast, hypo-osmotic medium significantly decreased both tyrosine phosphorylation and sperm-ZP binding. Medium with high osmolality (400 mOsm/kg) significantly reduced the ZP-induced AR. Both hypo- and hyper-osmotic media significantly decreased average sperm percentage progressive motility and velocities. Incubation of human sperm in hyper-osmotic media was associated with significantly increased tyrosine phosphorylation and ZP-binding ability but severely reduced the ZP-induced AR.

Loss of c-Cbl RING finger function results in high-intensity TCR signaling and thymic deletion

Signaling from the T-cell receptor (TCR) in thymocytes is negatively regulated by the RING finger-type ubiquitin ligase c-Cbl. To further investigate this regulation, we generated mice with a loss-of-function mutation in the c-Cbl RING finger domain. These mice exhibit complete thymic deletion by young adulthood, which is not caused by a developmental block, lack of progenitors or peripheral T-cell activation. Rather, this phenotype correlates with greatly increased expression of the CD5 and CD69 activation markers and increased sensitivity to anti-CD3-induced cell death. Thymic loss contrasts the normal fate of the c-Cbl-/- thymus, even though thymocytes from both mutant mice show equivalent enhancement in proximal TCR signaling, Erk activation and calcium mobilization. Remarkably, only the RING finger mutant thymocytes show prominent TCR-directed activation of Akt. We show that the mutant c-Cbl protein itself is essential for activating this pathway by recruiting the p85 regulatory subunit of PI 3-kinase. This study provides a unique model for analyzing high-intensity TCR signals that cause thymocyte deletion and highlights multiple roles of c-Cbl in regulating this process.

Role of Hydrogen Peroxide in Sperm Capacitation and Acrosome Reaction1

The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H(2)O(2)) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H(2)O(2) in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 microM H(2)O(2). At much higher concentrations of H(2)O(2) (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H(2)O(2) after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H(2)O(2). These two agents, but not heparin, induced Ca(2+)-dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H(2)O(2) (50 microM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H(2)O(2) are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H(2)O(2) activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.

Towards a physiological role for cytochrome P450 aromatase in ejaculated human sperm.

Advances in the definition of the function and the mechanism of estrogen action in different tissues have come from human and animal models of estrogen insufficiency. Recently we have demonstrated that aromatase is present and biologically active in human ejaculated sperm, suggesting that autonomous estradiol sperm production may influence sperm functions. In the present study we investigate a possible physiological role for enzymatically active P450 aromatase in human ejaculated sperm. To confirm the presence of mRNA coding for P450 aromatase, total RNA isolated from human sperm underwent RT-PCR and then Southern blot analysis. In non-capacitating medium, we observed that only estradiol and aromatizable steroids were able to increase sperm motility/migration; concomitantly they enhanced protein tyrosine phosphorylation and increased p-44/42 extracellular signal-regulated kinase activity. When we tested acrosin activity, it emerged that estradiol and aromatizable androgens were also able to induce the acrosome reaction evaluated by two different cytological staining techniques (triple-stain and fluorescein isothiocyanate-Pisum sativum agglutinin). All these events were enhanced by the 2'-O-dibutyryladenosine-3',5'-cyclic monophosphate and inhibited in the presence of the specific aromatase inhibitor, letrozole. From this study, it appears that a link exists between the locally produced estradiol (from ejaculated sperm), sperm capacitation and the acrosome reaction. The induction of both events by aromatizable androgens in the absence of exogenous mediators suggests that estrogen biosynthesis in ejaculated sperm is a process that may influence the intrinsic sperm fertilizing capability.

Acidic fibroblast growth factor (FGF-1) signaling inhibits peroxynitrite-induced cell death during pancreatic tumorigenesis

Previous immunohistochemical studies have demonstrated enhanced appearance of FGF-1 and nitrotyrosine, a footprint of reactive nitrogen species peroxynitrite (ONOO −), in human pancreatic adenocarcinoma. We have examined the consequences of constitutive exposure to FGF-1 in nontumorigenic rat ductal epithelial cells (ARIP). ARIP cells were transduced with either a secreted chimera of FGF-1, ARIP(FGF-1), or a control plasmid, 65 RIP(βg). These cells were evaluated for alteration in growth and morphology, responses to ONOO − (protein tyrosine nitration/phosphorylation), and in vivo tumor formation. ARIP(FGF-1) cells, in contrast to 65 RIP(βg), demonstrated a transformed morphology, a 2-fold increased growth rate, and enhanced protein tyrosine phosphorylation. Treatment with 150 μM ONOO − resulted in 86 and 7% ( p < .01) death of ARIP(βg) and ARIP(FGF-1), respectively. Exposure of 65 RIP(βg) cells to ONOO − enhanced tyrosine phosphorylation and tyrosine nitration of several polypeptides. Cell signaling by FGF-1 enhanced both phosphorylation and nitration of tyrosine residues in target proteins modified by ONOO −. ARIP(βg) cells failed to exhibit tumor formation in nude mice, but at d 7 in vivo cells were TUNEL and nitrotyrosine positive and FGF-1 negative. ARIP(FGF-1) cells readily formed tumor nodules, exhibiting features of pancreatic adenocarcinoma and demonstrating FGF-1-positive, nitrotyrosine-positive, and TUNEL-negative epithelium. These results suggest an interdependent role between FGF-1 and ONOO − during the development and progression of pancreatic adenocarcinoma.

Regulation of intestinal tyrosine phosphorylation and programmed cell death by peroxovanadate.

Cell suspensions of ileal mucosa undergo a rapid and synchronized form of programmed cell death when cultured in a simple medium at 37 degrees C. Because tyrosine phosphorylation of proteins plays a crucial role in the signal transduction of many cellular processes, we examined its role in intestinal programmed cell death by use of immunoblot and immunohistochemical methods. We observed a 50-70% reduction in tyrosine phosphorylation during the initial 10 min of intestinal epithelial cell culture. We hypothesized that the inhibition of protein tyrosine phosphatases would increase protein tyrosine phosphorylation in these suspensions and decrease programmed cell death. A strong inhibitor of these phosphatases (peroxovanadate) but not a weaker one (sodium orthovanadate) abolished the DNA fragmentation/laddering normally seen in dying enterocytes. Peroxovanadate enhanced protein tyrosine phosphorylation of many intestinal proteins, dramatically increasing the dually phosphorylated and active form of mitogen-activated protein kinase. Immunohistochemistry revealed a particularly high level of increased tyrosine phosphorylation in the intestinal crypts in peroxovanadate-treated mucosa. Kinetic studies indicated that the pivotal time for protein tyrosine phosphatase inhibition occurred within 5 min of ex vivo culture, precisely when protein tyrosine phosphorylation declined. Our data suggest that tyrosine kinase inactivation or tyrosine phosphatase activation may initiate intestinal epithelial cell death.

Phospholipase D activation in endothelial cells is redox sensitive.

Reactive oxygen species (ROS) are implicated in the pathophysiology of a number of vascular disorders, including atherosclerosis. Recent studies indicate that ROS modulate signal transduction in mammalian cells. Previously, we have shown that ROS (hydrogen peroxide, fatty acid hydroperoxide, diperoxovanadate, and 4-hydroxynonenal) enhance protein tyrosine phosphorylation and activate phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). In the present study, our aim was to investigate the role of exogenous thiol agents on ROS-induced PLD activation in conjunction with the role of cellular thiols--glutathione (GSH) and protein thiols--on PLD activation and protein tyrosine phosphorylation. Pretreatment of BPAECs with N-acetyl-L-cysteine (NAC) or 2-mercaptopropionylglycine (MPG) blocked ROS-induced changes in intracellular GSH and PLD activation. Also, pretreatment with NAC attenuated diperoxovanadate-induced protein tyrosine phosphorylation. Pretreatment of BPAECs with diamide or L-buthionine-(S,R)-sulfoximine (BSO), agents that lower intracellular GSH and thiols, enhanced PLD activity. Furthermore, NAC blocked diamide- or BSO-mediated changes in GSH levels, PLD activity, and protein tyrosine phosphorylation. NAC also attenuated diamide-induced tyrosine phosphorylation of proteins between 69 and 118 KDa. These results support the hypothesis that modulation of thiol-redox status (cellular nonprotein and protein thiols) may contribute to the regulation of ROS-induced protein tyrosine phosphorylation and PLD activation in vascular endothelium.

Cisplatin-induced activation of murine bone marrow-derived macrophages require protein tyrosine phosphorylation

The aim of the present study is to evaluate the involvement of tyrosine phosphorylation in the signal transduction mechanism of cisplatin-induced macrophage activation in vitro. Stimulation of bone marrow-derived macrophages (BMDM) with cisplatin (CP) resulted in a time- and dose-dependent phosphorylation of several proteins having estimated molecular weights of approximately 18, 20, 21, 30, 33, 35, 39, 41, 44, 58 and 123 kD, detected by immunoblot using anti-phosphotyrosine antibody. CP-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor genistein. Using this inhibitor, we were able to correlate tyrosine phosphorylation with several functional effects of CP on murine bone marrow-derived macrophages (BMDM) . Treatment of macrophages with genistein before incubation with CP completely inhibited the CP-induced tumoricidal activation of macrophages as well as production of TNF and NO, whereas pre-treatment of macrophages with phosphatase inhibitor sodium vanadate upregulated macrophage activation in addition to enhanced protein tyrosine phosphorylation. Taken together, these data suggest that tyrosine phosphorylation play a critical regulatory role in the activation of macrophages with CP.

Genistein Sensitivity of Calcium Transport Pathways in Serotonin-Activated Vascular Smooth Muscle Cells

Recent studies showed that serotonin-activated increases in intracellular Ca2+in vascular smooth muscle cells are associated with enhanced protein tyrosine phosphorylation. These responses were blocked by inhibition of tyrosine kinase activity with genistein, suggesting that the increases in Ca2+and tyrosine phosphorylation are functionally coupled. Therefore, we sought to characterize genistein-sensitive Ca2+transport pathways in rat aortic A10 cells loaded with fura-2. In the presence of extracellular Ca2+, serotonin evoked a transient increase in [Ca2+]ithat was followed by a smaller sustained increase. The transient was inhibited 25–40% by L-type Ca2+channel antagonists and inhibited 90–95% by genistein. The sustained response was unaffected by L-channel antagonists and only slightly inhibited by genistein. In the absence of extracellular Ca2+, the transient was reduced by 50%, while the sustained component was virtually abolished. These results suggest that influx and release pathways are major contributors to the transient component, whereas the lower sustained component is largely limited to influx pathways. The influx pathway during the transient probably involves an L-type Ca2+channel that is regulated by tyrosine kinase activity. The pathways that participate in the sustained response are different because they are insensitive tol-channel antagonists and only slightly inhibited by genistein. The transient evoked in Ca2+-free media was blocked by genistein, inhibited by caffeine, and prevented by thapsigargin. Ionomycin-induced release of Ca2+was unaffected by genistein, reduced by caffeine, and essentially eliminated by thapsigargin. Therefore, thapsigargin-mediated suppression of serotonin-activated release probably reflects depletion of Ca2+from the sarcoplasmic reticulum, whereas genistein-mediated suppression probably reflects inhibition of tyrosine kinase linked release. Caffeine-mediated suppression appears to involve both partial depletion of Ca2+and interference with release. Each A10 cell expressed at least two different ryanodine receptors and two different receptors for inositol 1,4,5-trisphosphate.

Polycythemia Vera V. Enhanced Proliferation and Phosphorylation Due to Vanadate Are Diminished in Polycythemia Vera Erythroid Progenitor Cells: A Possible Defect of Phosphatase Activity in Polycythemia Vera

Erythropoietin (EP) and stem cell factor (SCF ) are essential growth factors for erythroid progenitor cell proliferation and differentiation in serum-free culture. It has been previously shown that burst-forming units-erythroid and colony-forming units-erythroid from patients with polycythemia vera (PV) have enhanced sensitivity to EP and SCF compared with normal erythroid progenitors, but little is known about the mechanism for this difference. In the present investigation, the effect of EP and SCF on protein tyrosine phosphorylation in day-8 normal and PV erythroid colony-forming cells, which give rise to colonies of 2-49 hemoglobinized cells, was studied. EP rapidly induced tyrosine phosphorylation of the EP receptor, whereas the most prominent phosphorylated protein induced by SCF was identified as the SCF receptor. No additional phosphorylated proteins were evident when PV cells were compared with normal cells. Culture of normal erythroid progenitors with orthovanadate, an inhibitor of protein tyrosine phosphatases, resulted in an increased number of erythroid colonies and enhanced protein tyrosine phosphorylation. However, in contrast, little enhancement was evident with PV cells. These results indicate that, although vanadate may be acting in normal erythroid progenitors as a phosphatase inhibitor that potentiates the kinase activity induced by SCF and EP, this function is diminished in PV cells. Because erythropoiesis is regulated by a balance between protein tyrosine kinase activity and protein tyrosine phosphatase activity, PV patients may have an abnormal phosphatase activity allowing increased cell proliferation.

Polycythemia Vera V. Enhanced Proliferation and Phosphorylation Due to Vanadate Are Diminished in Polycythemia Vera Erythroid Progenitor Cells: A Possible Defect of Phosphatase Activity in Polycythemia Vera

AbstractErythropoietin (EP) and stem cell factor (SCF ) are essential growth factors for erythroid progenitor cell proliferation and differentiation in serum-free culture. It has been previously shown that burst-forming units-erythroid and colony-forming units-erythroid from patients with polycythemia vera (PV) have enhanced sensitivity to EP and SCF compared with normal erythroid progenitors, but little is known about the mechanism for this difference. In the present investigation, the effect of EP and SCF on protein tyrosine phosphorylation in day-8 normal and PV erythroid colony-forming cells, which give rise to colonies of 2-49 hemoglobinized cells, was studied. EP rapidly induced tyrosine phosphorylation of the EP receptor, whereas the most prominent phosphorylated protein induced by SCF was identified as the SCF receptor. No additional phosphorylated proteins were evident when PV cells were compared with normal cells. Culture of normal erythroid progenitors with orthovanadate, an inhibitor of protein tyrosine phosphatases, resulted in an increased number of erythroid colonies and enhanced protein tyrosine phosphorylation. However, in contrast, little enhancement was evident with PV cells. These results indicate that, although vanadate may be acting in normal erythroid progenitors as a phosphatase inhibitor that potentiates the kinase activity induced by SCF and EP, this function is diminished in PV cells. Because erythropoiesis is regulated by a balance between protein tyrosine kinase activity and protein tyrosine phosphatase activity, PV patients may have an abnormal phosphatase activity allowing increased cell proliferation.