Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro

Green tea extract and its polyphenolic components have been found to possess anticarcinogenic, antimutagenic, antihypertensive and antihepatotoxic effects, and several mechanisms have been proposed for these effects. In this study, the effects of five tea polyphenols, (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−) epicatechin-3-gallate (ECG), (−) epicatechin (EC) and (+)-catechin (C), were examined on the viability of Ehrlich ascites tumor cells in vitro and a possible relationship with tyrosine phosphorylation was determined. Proteins extracted from the cells treated with the tea polyphenols were separated by SDS-PAGE, and tyrosine-phosphorylated proteins were detected by immunoblotting with anti-phosphotyrosine antibody and the extent of phosphorylation determined. EGC (100 μM) caused a significant decrease in cell viability to 4.1±0.2% of the control value, and this correlated with a stimulation of protein tyrosine kinase (PTK) activity. EGCG (100 μM) also caused a slight decrease in cell viability (70% of the control value) but this and the other polyphenols, which had no effect on cell viability likewise, had no effect on tyrosine phosphorylation. Tyrosine phosphorylations of 42 and 45 kDa proteins were also observed for EGC. Further evaluation of the effect of EGC showed that the activity of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis in cells, decreased significantly as well. A significant correlation has therefore been observed between a cellular event, namely, a reduction in the viability of Ehrlich ascites tumor cells and an association with a tyrosine phosphorylation of 42 and 45 kDa proteins by the polyphenol EGC.     

Peroxynitrite signaling in human erythrocytes: synergistic role of hemoglobin oxidation and band 3 tyrosine phosphorylation

Peroxynitrite crosses the red blood cell (RBC) membrane and reacts with hemoglobin (Hb) producing mainly metHb, which is reduced back to ferrousHb by NADH- and NADPH-dependent reductases. Peroxynitrite also induces band 3 (B3) tyrosine phosphorylation, a signaling pathway believed to activate glucose metabolism. This study was aimed to decipher the relationship between these two peroxynitrite-dependent processes. Peroxynitrite induced a burst of the hexose monophosphate shunt (HMS), revealed by NMR studies, and a burst of the glycolytic pathway, measured by lactate production. The HMS plays a prominent role in membrane signaling, as demonstrated by B3 phosphotyrosine inhibition by the glycolytic pathway inhibitor 2-deoxy-glucose (2DG) and activation by dehydroepiandrosterone (DHEA), an inhibitor of HMS. Peroxynitrite-induced B3 tyrosine phosphorylation was paralleled by the inhibition of membrane-associated phosphotyrosine phosphatase (PTP) activity, which was protected by 2DG but not DHEA. Interestingly, heme poisoning with CO inhibited peroxynitrite-dependent Hb oxidation and lactate production but did not affect PTP down regulation. These results suggest two distinct and concurrent effects of peroxynitrite: one mediated by Hb which, likely in its oxidized state, binds more strongly to B3, and another mediated by PTP-dependent B3 phosphorylation. Both effects are directed towards a surge in glucose utilization.     

Involvement of Src family tyrosine kinase in apoptosis of human neutrophils induced by protozoan parasite Entamoeba histolytica

Tyrosine kinases are one of the most important regulators for intracellular signal transduction related to inflammatory responses. However, there are no reports describing the effects of tyrosine kinases on neutrophil apoptosis induced by Entamoeba histolytica. In this study, isolated human neutrophils from peripheral blood were incubated with live trophozoites in the presence or absence of tyrosine kinase inhibitors. Entamoeba-induced receptor shedding of CD16 and PS externalization in neutrophils were inhibited by pre-incubation of neutrophils with the broad-spectrum tyrosine kinase inhibitor genistein or the Src family kinase inhibitor PP2. Entamoeba-induced ROS production was also inhibited by genistein or PP2. Moreover, genistein and PP2 blocked the phosphorylation of ERK and p38 MAPK in neutrophils induced by E. histolytica. These results suggest that Src tyrosine kinases may participate in the signaling event for ROS-dependent activation of MAPKs during neutrophil apoptosis induced by E. histolytica.     

Large‐scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome‐wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high‐accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high‐confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine‐specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho‐residues in Arabidopsis is similar to that in humans, where over 90 tyrosine‐specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants.     

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [³²P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca²⁺ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC and , the major isotypes of PKC in BPAECs, by TPA ( 100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [³²P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.     

Integrin-Mediated Signal Transduction in Human Endothelial Cells: Analysis of Tyrosine Phosphorylation Events

Adhesion of human umbilical endothelial cells to fibronectin resulted in increased tyrosine phosphorylation of a group of proteins with molecular mass ranging from 100 to 130 kDa and of a 70 kDa protein. This pattern of tyrosine phosphorylation was also observed when endothelial cells adhered to vitronectin, collagen IV, collagen I and laminin or to culture dishes coated with antibodies directed to either βl, α3, α5, α6 or β3 integrin subunits. Increased phosphorylation of the 100–130 kDa proteins was detectable as early as 30 sec after adhesion, reached maximal level after 15 min, and remained high as long as the cells adhere to culture dishes. The 70 kDa protein was phosphorylated with a slower kinetics and its phosphorylation increased over a period of 3 h. Using specific monoclonal antibodies, the major component of the 100–130 kDa complex was identified as the focal adhesion tyrosine kinase p125^FAK. The phosphorylation of the pl25^FAK was also observed by inducing βl integrin clustering in rum adherent HEC, indicating that this is a primary signalling event induced by integrins. Using tyrosine kinase inhibitors, we show a direct correlation between integrin-stimulated tyrosine kinases and assembly of focal adhesions and actin fibres.     

A critical step for JNK activation: isomerization by the prolyl isomerase Pin1

c-Jun N-terminal kinase (JNK) is activated by dual phosphorylation of both threonine and tyrosine residues in the phosphorylation loop of the protein in response to several stress factors. However, the precise molecular mechanisms for activation after phosphorylation remain elusive. Here we show that Pin1, a peptidyl-prolyl isomerase, has a key role in the JNK1 activation process by modulating a phospho-Thr-Pro motif in the phosphorylation loop. Pin1 overexpression in human breast cancer cell lines correlates with increased JNK activity. In addition, small interfering RNA (siRNA) analyses showed that knockdown of Pin1 in a human breast cancer cell line decreased JNK1 activity. Pin1 associates with JNK1, and then catalyzes prolyl isomerization of the phospho-Thr-Pro motif in JNK1 from trans- to cis-conformation. Furthermore, Pin1 enhances the association of JNK1 with its substrates. As a result, Pin1(-/-) cells are defective in JNK activation and resistant to oxidative stress. These results provide novel insights that, following stress-induced phosphorylation of Thr in the Thr-Pro motif of JNK1, JNK1 associates with Pin1 and undergoes conformational changes to promote the binding of JNK1 to its substrates, resulting in cellular responses from extracellular signals.     

Cloning of the doding cDNA sequence of the gene for Csk tyrosine kinase from human lymphocytes

Tyrosine kinases of the Csk family play an important role in cell growth regulation and normal cell differentiation. They are also involved in carcinogenesis as oncoproteins. The main function of these tyrosine kinases is phosphorylation of tyrosine kinases of the Src family at their C-terminal regions to negatively regulate their activity. Disturbance of csk expression increases the Src tyrosine kinase activity. The full-length coding sequence of the csk cDNA was cloned from human lymphocytes. The 1624-bp cDNA consists of 12 exons and encodes a protein that has conserved SH2 and SH3 domains and is similar to human Csk tyrosine kinase by 99%. The full-length cDNA can be used to analyze the csk structure in normal or illdefined human cells.     

Selenazoles (selenium compounds) facilitate survival of cultured rat pheochromocytoma PC12 cells after serum-deprivation and stimulate their neuronal differentiation via activation of Akt and mitogen-activated protein kinase, respectively

The activation of extracellular receptor kinase (ERK) is one of the checkpoints to assess the activation of the classical Ras/mitogen-activated protein kinase (MAPK) cascade. Therefore, we tested more than 100 selenium-containing compounds for their ability to activate the MAPK signal pathway. Among them, we found that three selenazoles, 5-chloroacetyl-2-piperidino-1,3-selenazole (CS1), 5-chloroacetyl-2-morpholino-1,3-selenazole (CS2), and 5-chloroacetyl-2-dimethylamino-1,3-selenazole (CS3), induced the phosphorylation of ERK. These compounds also enhanced the phosphorylation of Akt, a signal transducing protein kinase for cell survival; and this phosphorylation was followed by suppression of cell death, thus suggesting that they had anti-apoptotic effects. Moreover, CSs 1-3 induced neurite outgrowth and facilitated the expression of neurofilament-M of PC12 cells, demonstrating that they induced neuronal differentiation of these cells. On the other hand, the CS-induced phosphorylation of MAPK was enhanced by buthionine sulfoximine (BSO), an activator of protein tyrosine phosphatases (PTPs), but inhibited by N-acetyl-l-cysteine (NAC), an inhibitor of receptor tyrosine kinase. These results imply that activation of some receptor tyrosine kinase(s) is involved in the mechanism of action of CSs 1-3. The activation of MAPK by CSs 1-3 was suppressed by U0126, a MEK inhibitor, but not by K252a, an inhibitor of TrkA; AG1478, an antagonist of epidermal growth factor receptor (EGFR); or by pertussis toxin. These results demonstrate that the CS-induced phosphorylation of Akt and MAP kinase (receptor tyrosine kinase(s)-MEK1/2-ERK1/2) cascades was responsible for suppression of apoptosis and facilitation of neuronal differentiation of PC12 cells, respectively. Our results suggest that CSs 1-3 are promising candidates as neuroprotective and/or neurotrophic agents for the treatment of various neurodegenerative neurological disorders.     

Fyn tyrosine kinase reduces the ethanol inhibition of recombinant NR1/NR2A but not NR1/NR2B NMDA receptors expressed in HEK 293 cells

NMDA receptors are potentiated by phosphorylation in a subunit- and kinase-specific manner. Both native and recombinant NMDA receptors are inhibited by behaviorally relevant concentrations of ethanol. Whether the phosphorylation state of individual subunits modulates the ethanol sensitivity of these receptors is not known. In this study, the effects of Fyn tyrosine kinase on the ethanol sensitivity of specific recombinant NMDA receptors expressed in HEK 293 cells were investigated. Whole-cell mode patch clamp and ratiometric calcium imaging demonstrated that the degree of ethanol inhibition of NR1/NR2B receptors was unaffected by Fyn tyrosine kinase. In contrast, the inhibition of NR1/NR2A receptors by ethanol (100 mM) was significantly reduced under conditions of enhanced Fyn-mediated tyrosine phosphorylation of the NR2A subunit. This effect was not observed at lower concentrations of ethanol (< or = 50 mM). These results suggest that tyrosine phosphorylation of specific NMDA receptors by Fyn tyrosine kinase may regulate the sensitivity of these receptors to the sedative/hypnotic concentrations of ethanol.     

Src tyrosine kinase-dependent migratory effects of antithrombin in leukocytes

Tyrosine kinases are known to play a critical role in the regulation of leukocyte function. Antithrombin mediates its effects via syndecan-4 which is known to be linked to the Src tyrosine kinases. In this study, we investigated the role of Src tyrosine kinases in antithrombin-regulated leukocyte migration and Src tyrosine kinase phosphorylation in response to stimulation with antithrombin. Neutrophils and monocytes obtained from forearm venous blood were pre-treated by various Src-family selective tyrosine kinase inhibitors with or without antithrombin followed by washing and assessment of their migratory response toward antithrombin, interleukin-8, or RANTES using Boyden microchemotaxis chambers. Activation status of the two major Src tyrosine kinase phosphorylation sides Tyr416 and Tyr527 was tested using Western blot analysis. Dose-dependent reversal of the antithrombin-mediated effects on neutrophil and monocyte migration was induced by the selective Src kinase inhibitors PP1 and PP2. In Western blot analyses, antithrombin increased Tyr416 and decreased Tyr527 phosphorylation of Src tyrosine kinases in a time- and dose-dependent manner. Moreover, co-incubation with antithrombin lowered the level of RANTES-induced Tyr416 phosphorylation. Therefore, Src tyrosine kinases linked to signaling of antithrombin-binding sites on leukocytes may play an important role in modulating effects on cells function.     

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to approximately 200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation.     

蛋白酪氨酸磷酸酶-PEST在生理调节及相关疾病中的作用

蛋白质分子中酪氨酸残基可逆性的磷酸化是细胞内信号分子传导的基本方式。两类作用相反的酶参与磷酸化的调节:蛋白酪氨酸激酶(protein tyrosinekinase,PTK)和蛋白酪氨酸磷酸酶(protein tyrosine phosphatase,PTP)。含脯氨酸-谷氨酸-丝氨酸-苏氨酸(P-E-S-T)结构域的蛋白酪氨酸磷酸酶(PTP-PEST)属于非受体型酪氨酸磷酸酶类,其本身能与多种蛋白质相互作用,并在细胞迁移、免疫细胞活化和胚胎发育等生理过程中发挥重要作用。本文对PTP-PEST的结构特点、生理功效、介导的信号传导途径和近年来PTP-PEST在疾病中的作用作一综述。     

Glutamate-stimulated activation of DNA synthesis via mitogen-activated protein kinase in primary astrocytes: involvement of protein kinase C and related adhesion focal tyrosine kinase

Glutamate is the major excitatory neurotransmitter in the CNS. Although its role in neurons has been studied extensively, little is known about its function in astrocytes. We studied the effects of glutamate on signaling pathways in primary astrocytes. We found that the tyrosine kinase related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated in response to glutamate in a time- and dose-dependent manner. This phosphorylation was pertussis toxin (PTX) sensitive and could be attenuated by the depletion of Ca2+ from intracellular stores. RAFTK tyrosine phosphorylation was mediated primarily by class I/II metabotropic glutamate receptors and depends on protein kinase C (PKC) activation. Glutamate treatment of primary astrocytes also results in a significant increase in the activity of the mitogen-activated protein kinases [extracellular signal-related kinases 1/2 (ERK1/2)]. Like RAFTK phosphorylation, ERK1/2 activation is PTX sensitive and can be attenuated by the depletion of intracellular Ca2+ and by PKC inhibition, suggesting that RAFTK might mediate the glutamate-dependent activation of ERK1/2. Furthermore, we demonstrated that glutamate stimulation of primary astrocytes leads to a significant increase in DNA synthesis. Glutamate-stimulated DNA synthesis is PTX sensitive and can be inhibited by the MAP kinase kinase inhibitor PD98059, suggesting that in primary astrocytes, glutamate might signal via RAFTK and MAP kinase to promote DNA synthesis and cell proliferation.     

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 -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, vanadate-induced mitogenesis is associated with the selective induction ofc-jun andjunB expression without significantly inducingc-fos orc-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation andc-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation andc-jun andjunB expression may serve the critical roles in mediating vanadate-induced mitogenesis in SV40-transformed cells.     

Enhanced phosphorylation and enzymatic activity of phosphoglucomutase by the Btk29A tyrosine kinase in Drosophila

The Drosophila Btk29A tyrosine kinase is suggested to be involved in diverse processes, although its target proteins are unknown. In the present study, we investigated substrates of Btk29A tyrosine kinase by expressing a catalytically activated form of Btk29A-P1 (Btk-EG) in Drosophila compound eyes. Expression in eye disks led to the development of the rough-eye phenotype and increased tyrosine phosphorylation of a 65-kDa protein. Partial amino acid sequence analysis of this protein showed that it was phosphoglucomutase. Phosphoglucomutase activity in heads from Btk-EG-expressing flies was higher than that in controls, suggesting that the levels of tyrosine phosphorylation and activity of the enzyme are associated with Btk29A tyrosine kinase activity.     

PTP inhibitor IV protects JNK kinase activity by inhibiting dual-specificity phosphatase 14 (DUSP14)

Protein phosphorylation plays critical roles in the regulation of protein activity and cell signaling. The level of protein phosphorylation is controlled by protein kinases and protein tyrosine phosphatases (PTPs). Disturbance of the equilibrium between protein kinase and PTP activities results in abnormal protein phosphorylation, which has been linked to the etiology of several diseases, including cancer. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by bis (4-trifluoromethyl-sulfonamidophenyl, TFMS)-1,4-diisopropylbenzene (PTP inhibitor IV). PTP inhibitor IV inhibited DUSP14 phosphatase activity. Kinetic studies with PTP inhibitor IV and DUSP14 revealed a competitive inhibition, suggesting that PTP inhibitor IV binds to the catalytic site of DUSP14. PTP inhibitor IV effectively and specifically inhibited DUSP14-mediated dephosphorylation of JNK, a member of the mitogen-activated protein kinase (MAPK) family.     

酪氨酸磷酸化蛋白质组学技术研究进展及其在生物医学研究中的应用

在酪氨酸磷酸化蛋白质组学的研究过程中,酪氨酸磷酸化位点的富集是最重要的一步。目前常用的富集方法是抗体亲和富集或SH2 superbinder富集。此外,通过质谱与生物信息学等技术,可实现大规模酪氨酸磷酸化位点的鉴定。对酪氨酸磷酸化蛋白质组学进行深度覆盖研究,揭示癌症发生发展过程中失调的激酶,将有助于深入理解癌症的发生发展过程;且由于75%的致癌基因是酪氨酸激酶基因,酪氨酸激酶抑制剂作为抗癌药物受到了越来越多的关注。应用酪氨酸磷酸化蛋白质组学技术,可以鉴定与癌症等重大疾病相关的酪氨酸激酶,从而帮助找到酪氨酸激酶抑制剂。总之,酪氨酸磷酸化蛋白质组学技术可以在酪氨酸激酶鉴定、酪氨酸激酶抑制剂研究及酪氨酸磷酸化信号通路研究等生物医学领域中得到很好的应用。     

Quantification of the Dynamic Phosphorylation Process of ERK Using Stable Isotope Dilution Selective Reaction Monitoring Mass Spectrometry

Mitogen‐activated protein (MAP) kinase signaling is critical for various cellular responses, including cell proliferation, differentiation, and cell death. The MAP kinase cascade is conserved in the eukaryotic kingdom as a three‐tiered kinase module—MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase—that transduces signals via sequential phosphorylation upon stimulation. Dual phosphorylation of MAP kinase on the conserved threonine‐glutamic acid‐tyrosine (TEY) motif is essential for its catalytic activity and signal activation; however, the molecular mechanism by which the two residues are phosphorylated remains elusive. In the present study, the pattern of dual phosphorylation of extracellular signal‐regulated kinase (ERK) is profiled on the TEY motif using stable isotope dilution (SID)‐selective reaction monitoring (SRM) mass spectrometry (MS) to elucidate the order and magnitude of endogenous ERK phosphorylation in cellular model systems. The SID‐SRM‐MS analysis of phosphopeptides demonstrates that tyrosine phosphorylation in the TEY motif is dynamic, while threonine phosphorylation is static. Analyses of the mono‐phosphorylatable mutants ERK^T202A and ERK^Y204F indicate that phosphorylation of tyrosine is not affected by the phosphorylation state of threonine, while threonine phosphorylation depends on tyrosine phosphorylation. The data suggest that dual phosphorylation of ERK is a highly ordered and restricted mechanism determined by tyrosine phosphorylation.     

Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked IFN-γ. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, 5 µM) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine (5 µM) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, 20 µM) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, 10 µM) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.