Correlation between the activation of Neu tyrosine kinase and promotion of foci formation induced by selected organochlorine compounds in the MCF-7 model system.

Several studies have shown that some organochlorine compounds act like estrogen in certain animals and in vitro cell culture systems, and therefore, there is a possibility that they could promote the process of tumorigenesis in breast cancer cells. In our previous study, two representative organochlorines, 1,1,1-trichloro 2-o-chlorophenyl-2'-p-chlorophenyl ethane (o,p'-DDT) and beta-1,2,3,4,5,6-hexachlorocyclohexane (beta HCH), were found to directly activate the protein tyrosine kinase of Neu (c-erbB-2 proto-oncogene product) immunoprecipitates isolated from MCF-7 breast cancer cells. In the current study, we also found that 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 1 nM and alpha-HCH isomers at 100 nM could also significantly activate protein tyrosine kinase of Neu immunoprecipitates in a cell-free system. We also found that organochlorines result in an increase of Neu protein tyrosine kinase after intact cell treatment in estrogen-depleted medium. This Neu kinase activation by beta-HCH (100 nM) was blocked when the cells were pretreated with Neu mRNA antisense oligonucleotide (p < 0.07, Student's t-test). Endogenously added alpha-, beta-, and gamma-HCH, o,p'-DDT, 2,2'-dichlorobiphenyl (2,2'-PCB), and 2,4,5-T at 100 nM were found to promote foci formation in postconfluent cultures of this cell line. This stimulatory effect caused by 17beta-estradiol, o,p'-DDT, and beta-HCH on foci formation was inhibited by coincubation with Neu monoclonal antibody (p < 0.05). Those two events induced by organochlorines (i.e., Neu kinase activation and foci formation) seemed causally correlated.     

Interaction of the CC-chemokine RANTES with glycosaminoglycans activates a p44/p42 mitogen-activated protein kinase-dependent signaling pathway and enhances human immunodeficiency virus type 1 infectivity

The interaction of the CC-chemokine RANTES with its cell surface receptors transduces multiple intracellular signals: low concentrations of RANTES (1 to 10 nM) stimulate G-protein-coupled receptor (GPCR) activity, and higher concentrations (1 microM) activate a phosphotyrosine kinase (PTK)-dependent pathway. Here, we show that the higher RANTES concentrations induce rapid tyrosine phosphorylation of multiple proteins. Several src-family kinases (Fyn, Hck, Src) are activated, as is the focal adhesion kinase p125 FAK and, eventually, members of the p44/p42 mitogen-activated protein kinase (MAPK) family. This PTK signaling pathway can be activated independently of known seven-transmembrane GPCRs for RANTES because it occurs in cells that lack any such RANTES receptors. Instead, activation of the PTK signaling pathway is dependent on the expression of glycosaminoglycans (GAGs) on the cell surface, in that it could not be activated by RANTES in GAG-deficient cells. We have previously demonstrated that RANTES can both enhance and inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). Here we show that activation of both PTK and MAPK is involved in the enhancement of HIV-1 infectivity caused by RANTES in cells that lack GPCRs for RANTES but which express GAGs.     

Epidermal growth factor-specific protein tyrosine phosphorylation in preimplantation embryo development.

We examined whether epidermal growth factor (EGF)-induced preimplantation mouse embryo development and function are mediated by EGF-specific protein tyrosine phosphorylation (PTP). In situ cross-linking and autophosphorylation studies showed that EGF receptor (EGF-R) in Day 4 mouse blastocysts is a protein of approximately 170 kDa that is phosphorylated when exposed to EGF and ATP. Furthermore, EGF induced about a twofold increase in protein tyrosine kinase (PTK) activity in Day 4 blastocysts when incubated in the presence of a peptide substrate with a tyrosine moiety and ATP. RG 50864, a specific inhibitor of EGF-dependent PTK, diminished autophosphorylation of the 170-kDa protein and completely blocked PTK activity in the blastocyst induced by EGF. However, this inhibitor did not affect EGF binding to the embryonic cell surface. In contrast, an inactive tyrphostin compound, RG 50862, did not alter EGF-induced PTK activity in the blastocyst. These findings led us to examine the effects of these tyrphostin compounds on preimplantation mouse embryo development and blastocyst hatching in vitro. RG 50864, in a dose-dependent manner, inhibited EGF-dependent development of 2-cell embryos to blastocysts and the number of cells per blastocyst. This inhibitor also antagonized EGF-induced zona-hatching of blastocysts formed from 8-cell embryos in culture. However, the inhibitor was not effective in deterring transforming growth factor-beta 1-induced blastocyst formation. The inactive compound, RG 50862, had no effects on EGF-dependent blastocyst formation or zona-hatching. The data show that the effects of RG 50864 are specific and mediated by inhibition of EGF-specific PTK activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombospondin-1 opens the paracellular pathway in pulmonary microvascular endothelia through EGFR/ErbB2 activation

Thrombospondin-1 (TSP1) is a multidomain protein that contains epidermal growth factor (EGF)-like repeats that indirectly activate the EGF receptor (EGFR) and selected downstream signaling pathways. In these studies, we show that TSP1 opens the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls) in a dose-, time-, and protein tyrosine kinase (PTK)-dependent manner. TSP1 increased tyrosine phosphorylation of proteins enriched to intercellular boundaries including the zonula adherens (ZA) proteins, vascular endothelial-cadherin, γ-catenin, and p120 catenin. In HMVEC-Ls, EGFR and ErbB2 are expressed at low levels, and both heterodimerize and tyrosine autophosphorylate in response to TSP1. Prior EGFR-selective PTK inhibition with AG1478 or ErbB2-selective PTK inhibition with AG825 protected against TSP1-induced tyrosine phosphorylation of ZA proteins and barrier disruption. Preincubation of HMVEC-Ls with an EGFR ectodomain-blocking antibody also prevented TSP1-induced opening of the paracellular pathway. Therefore, in HMVEC-Ls, TSP1 increases tyrosine phosphorylation of ZA proteins and opens the paracellular pathway, in part, through EGFR/ErbB2 activation. Surprisingly, recombinant TSP1 EGF-like repeats 1-3 and the high-affinity EGFR ligands, EGF, TGF-α, and amphiregulin, each failed to increase paracellular permeability. However, HMVEC-Ls in which EGFR was overexpressed became responsive to the EGF-like repeats of TSP1 as well as to EGF. These studies indicate that TSP1 disrupts the endothelial barrier through EGFR/ErbB2 activation although additional signals are necessary in cells with low receptor expression.     

Inhibition of stimulus-dependent epidermal growth factor receptor and transforming growth factor-alpha mRNA accumulation by the protein kinase C inhibitor staurosporine

The ability of staurosporine, a potent inhibitor of protein kinase C, to block certain cellular events initiated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) was examined. Treatment of MDA468 breast cancer cells with TPA decreases EGF binding to the cell surface and this effect is blocked by pretreatment with staurosporine with an IC50 of 30 nM. Either 10(-9) M EGF or 100 ng/ml TPA stimulated the accumulation of both EGF receptor and TGF-alpha mRNA and staurosporine (50 nM) completely abolished these mRNA accumulations. Staurosporine did not block EGF-stimulated tyrosine phosphorylation of its receptor as measured by immunoblotting with anti-phosphotyrosine antibodies. The ability of staurosporine to block the mRNA responses of either EGF or TPA suggests that these two agents have common signaling pathways and it implies a role for protein kinase C in the control of EGF receptor and TGF-alpha expression.     

Peroxynitrite modulates the activation of p38 and extracellular regulated kinases in PC12 cells

Although peroxynitrite appears to contribute to neuronal dysfunction in several neurodegenerative disorders, little is known about how peroxynitrite affects cellular signaling processes. This study investigated if peroxynitrite affects the mitogen-activated protein kinases, extracellular-regulated kinases 1 and 2 (ERK1/2) and p38. Exposure of PC12 cells to 500 microM peroxynitrite activated ERK1/2 and p38 within 5 min and this was followed by gradual decreases in activation over the next 25 min. Activation of ERK1/2 by peroxynitrite was mediated by activation of the epidermal growth factor (EGF) receptor in a calcium/calmodulin-dependent kinase II- and src family tyrosine kinase-dependent manner, as it was blocked by the selective EGF receptor inhibitor AG1478, by KN62, an inhibitor of calcium/calmodulin-dependent kinase II, and by PP1, a src family tyrosine kinase inhibitor. Activation of p38 by peroxynitrite was independent of the EGF receptor, required activation of calcium/calmodulin-dependent kinase II and src family tyrosine kinases, and was modulated by nerve growth factor (NGF) in a time-dependent manner. Pretreatment with NGF (2 h) attenuated, whereas cotreatment with NGF potentiated, peroxynitrite-induced activation of p38. Thus, peroxynitrite activates ERK1/2 and p38, activation of EGF receptors, calcium/calmodulin-dependent kinase II, and src family tyrosine kinases participate in these signaling responses to peroxynitrite, and peroxynitrite- and NGF-induced signaling activities converge on p38.     

Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.     

Cell surface receptors activate p21-activated kinase 1 via multiple Ras and PI3-kinase-dependent pathways

p21-activated kinases (PAKs) were the first identified mammalian members of a growing family of Ste20-like serine–threonine protein kinases. In this study, we show that PAK1 can be stimulated by carbachol, lysophosphatidic acid (LPA), epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) by multiple independent and overlapping pathways. Dominant-negative Ras, Rac, and Cdc42 inhibited PAK1 activation by all of these agonists, while active Rac1 and Cdc42 were sufficient to maximally activate PAK1 in the absence of any treatment. Active Ras induced only a weak activation of PAK1 that could be potentiated by muscarinic receptor stimulation. Studies using inhibitors of the EGF receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI3-kinase) and protein kinase C (PKC) revealed that all of the cell surface agonists could activate PAK1 through pathways independent of PKC, that EGF stimulated a PI3-kinase dependent pathway to stimulate PAK1, and that muscarinic receptor stimulation of PAK1 was predominantly mediated through this EGF-R-dependent mechanism. Activation of PAK1 by LPA was independent of PI3-kinase and the EGF receptor, but was inhibited by dominant-negative RhoA. These results identify multiple Ras-dependent pathways to activation of PAK1.     

Tyrphostin AG556 increases the activity of large conductance Ca2+‐activated K+ channels by inhibiting epidermal growth factor receptor tyrosine kinase

The present study was designed to investigate whether large conductance Ca²⁺‐activated K⁺ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK‐HEK 293 cells expressing both functional α‐subunits and the auxiliary β1‐subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK‐HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α‐ and β1‐subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre‐contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.     

Identification and characterization of the tyrosine protein kinases of rat spleen

High levels of tyrosine protein kinase have been recently detected in the membranes of rat spleen. In the present report the tyrosine protein kinase activity of the 30,000 x g pellet of rat spleen has been solubilized and partially purified by ion exchange and gel permeation chromatography. Two peaks of tyrosine protein kinase of Mr 35,000 (TPK-I) and Mr 40,000 (TPK-II) have been resolved. These kinases were free of the EGF receptor and insulin receptor tyrosine protein kinases. Although TPK-I and TPK-II phosphorylated angiotensin II, casein, histone, tubulin, phosphorylase b, and p36 they differed from each other in preference for the substrates. Both tyrosine protein kinases did not phosphorylate anti-pp60v-src IgG.     

Discovery of novel imidazo[1,2-a]pyrazin-8-amines as Brk/PTK6 inhibitors

A series of substituted imidazo[1,2-a]pyrazin-8-amines were discovered as novel breast tumor kinase (Brk)/protein tyrosine kinase 6 (PTK6) inhibitors. Tool compounds with low-nanomolar Brk inhibition activity, high selectivity towards other kinases and desirable DMPK properties were achieved to enable the exploration of Brk as an oncology target.     

Angiotensin II stimulates rat astrocyte mitogen-activated protein kinase activity and growth through EGF and PDGF receptor transactivation

We showed that the intracellular tyrosine kinases src and pyk2 mediate angiotensin II (Ang II) stimulation of growth and ERK1/2 mitogen-activated protein (MAP) kinase phosphorylation in astrocytes. In this study, we investigated whether the membrane-bound receptor tyrosine kinases platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors mediate Ang II stimulation of ERK1/2 and astrocyte growth. Ang II significantly stimulated PDGF and EGF receptors in a dose- and time-dependent manner. The PDGF receptor and the EGF receptor were maximally stimulated with 100 nM Ang II (0.98+/-0.18- and 4.4+/-1.4-fold above basal, respectively). This stimulation occurred as early as 5 min, and was sustained for at least 15 min for both receptor tyrosine kinases. Moreover, 1 microM AG1478 and 0.25 microM PDGFRInhib attenuated Ang II stimulation of the EGF and PDGF receptors, respectively. Ang II-induced phosphorylation of ERK1/2 and astrocyte growth was mediated by both PDGF and EGF receptors. This report also provides novel findings that co-inhibiting EGF and PDGF receptors had a greater effect to decrease Ang II-induced ERK1/2 (90% versus 49% and 71% with PDGF receptor and EGF receptor inhibition, respectively), and astrocyte growth (60% versus 10% and 32% with PDGF receptor and EGF receptor inhibition, respectively). In conclusion we showed in astrocytes that the PDGF and the EGF receptors mediate Ang II-induced ERK1/2 phosphorylation and astrocyte growth and that these two receptors may exhibit synergism to regulate effects of the peptide in these cells.     

Vanadium salts stimulate mitogen-activated protein (MAP) kinases and ribosomal S6 kinases

Effect of several vanadium salts, sodium orthovanadate, vanadyl sulfate and sodium metavanadate on protein tyrosine phosphorylation and serine/threonine kinases in chinese hamster ovary (CHO) cells overexpressing a normal human insulin receptor was examined. All the compounds stimulated protein tyrosine phosphorylation of two major proteins with molecular masses of 42 kDa (p42) and 44 kDa (p44). The phosphorylation of p42 and p44 was associated with an activation of mitogen activated protein (MAP) kinase as well as increased protein tyrosine phosphorylation of p42^mapk and p44^mapk. Vanadinm salts also activated the 90 kDa ribosomal s6 kinase (p90^rsk) and 70 kDa ribosomal s6 kinase (p70^s6k). Among the three vanadium salts tested, vanadyl sulfate appeared to be slightly more potent than others in stimulating MAP kinases and p70^s6k activity. It is suggested that vanadium-induced activation of MAP kinases and ribosomal s6 kinases may be one of the mechanisms by which insulin like effects of this trace element are mediated.Abbreviations eIF-4 eukaryotic protein synthesis initiation factor-4 - GRB-2 growth factor receptor bound protein-2 - GSK-3 Glycogen Synthase Kinase-3 - IRS-1 insulin receptor substrate-1 - ISPK insulin stimulated protein kinase - MAPK mitogen activated protein kinase, also known as - ERK extracellular signal regulated kinase - MAPKK mitogen activated protein kinase kinase, also known as-MEK, MAPK or ERK kinase - PHAS-1 phosphorylated heat and acid stable protein regulated by insulin - PI3K phosphatidyl inositol 3-kinase - PP1-G protein phosphatase-glycogen bound form - PTK protein tyrosine kinase - PTPase protein tyrosine phosphatase - rsk ribosomal s6 kinases - shc src homology domain containing protein - SOS son of sevenless     

Expression of the human epidermal growth factor receptor in a murine T-cell hybridoma. A transmembrane protein tyrosine kinase can synergize with the T-cell antigen receptor.

Although the T-cell receptor for antigen (TCR) lacks intrinsic kinase activity, stimulation of this receptor induces tyrosine phosphorylation of multiple substrates. In contrast, the epidermal growth factor receptor (EGFR) has intrinsic cytoplasmic tyrosine kinase catalytic activity that is activated upon EGF binding. To compare the functional effects of the TCR and a transmembrane protein tyrosine kinase (PTK), we used retrovirus-mediated gene transduction to express the human c-erbB proto-oncogene, encoding the EGFR, in a murine T-cell hybridoma. Tyrosine phosphorylation induced by the TCR and the EGFR occurred on substrates unique to each receptor as well as on several shared substrates, including the zeta chain of the TCR. Stimulation of the EGFR induced calcium ion flux in these cells, suggesting that the heterologous tyrosine kinase can couple to the T-cell phospholipase signal transduction pathway, but this stimulus did not lead to interleukin 2 production. However, EGF stimulation of transduced cells significantly enhanced TCR signaling, as assessed by interleukin 2 production, indicating that cross talk can occur between the TCR and a transmembrane PTK.     

HER2/ErbB2-induced Breast Cancer Cell Migration and Invasion Require p120 Catenin Activation of Rac1 and Cdc42

Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.     

Tyrosine phosphorylation of a 120-kilodalton pp60src substrate upon epidermal growth factor and platelet-derived growth factor receptor stimulation and in polyomavirus middle-T-antigen-transformed cells.

The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.     

Interaction between the epidermal growth factor receptor and phosphoinositide kinases.

Ligand-activated epidermal growth factor (EGF) receptors are coupled to the phosphatidylinositol (PtdIns) pathway to stimulate formation of two second messengers, inositol trisphosphate and diacylglycerol. Investigation of the interaction between EGF receptors and phosphoinositide kinases identified PtdIns and PtdIns(4)P kinase activities in extensively washed EGF receptor immunoprecipitates. Studies using COOH-terminal truncation mutant EGF receptors and immunoisolation by an EGF receptor peptide anti-serum in the presence of peptide (residues 644-666) indicated that the phosphoinositide kinases were associated with the region located between the inner membrane boundary and the kinase domain of the EGF receptor. In vivo cross-linking identified four tyrosine phosphorylated proteins of approximately 135, 62, 55, and 47 kDa associated with the EGF receptor. After EGF stimulation, PtdIns and PtdIns(4)P kinase activities were markedly increased among proteins isolated by monoclonal antiphosphotyrosine antibodies. The activities associated with the EGF receptor and with tyrosine-phosphorylated proteins were identified as PtdIns4-and PtdIns(4)P 5-kinase. Tyrosine dephosphorylation did not alter the activity of the prominent PtdIns(4)P 5-kinase activity. These results indicate that the phosphoinositide kinases are associated with and tyrosine phosphorylated by the EGF receptor as part of the mechanism coordinating responses between signal transduction pathways but do not demonstrate that tyrosine phosphorylation of PtdIns(4)P 5-kinase is sufficient to activate the enzyme.