Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Vascular endothelial growth factor (VEGF) receptor Flk-1/KDR in endothelial cells is activated during vasculogenesis and angiogenesis upon ligand-receptor interaction. Activated Flk-1/KDR has been shown to recruit Src homology 2 domain-containing signaling molecules that are known to serve as links to the activation of the mitogen-activated protein (MAP) kinase signaling pathway. To define the functional significance of phosphatidylinositol (PI) 3-kinase in VEGF signaling, we have examined its role in human umbilical vein endothelial cell (HUVEC) cycle progression. We show herein that p85, the regulatory subunit of PI 3-kinase, is constitutively associated with Flk-1/KDR. The treatment of HUVECs with VEGF promoted tyrosine autophosphorylation of Flk-1/KDR and also induced phosphorylation of p85. This was followed by an increase in the PI 3-kinase activity, which was sensitive to wortmannin, a potent PI 3-kinase inhibitor. VEGF also induced a striking activation of MAP kinase in a time-dependent manner. Inhibition studies with both a dominant-negative p85 mutant and the PI 3-kinase inhibitor, wortmannin, were employed to show for the first time that VEGF-stimulated PI 3-kinase modulates MAP kinase activation and nuclear events such as transcription from c-fos promoter and entry into the synthesis (S)-phase. Our data demonstrate the importance of PI 3-kinase as a necessary signaling component of VEGF-mediated cell cycle progression.
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PMID:The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. 1018 76

The signaling pathways activated by the macrophage colony-stimulating factor (M-CSF) to promote survival of monocyte and macrophage lineage cells are not well established. In an effort to elucidate these pathways, we have used two cell types responsive to M-CSF: NIH 3T3 fibroblasts genetically engineered to express human M-CSF receptors (3T3-FMS cells) and human monocytes. M-CSF treatment induced M-CSF receptor tyrosine phosphorylation and recruitment of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) to these receptors. These M-CSF receptor events correlated with activation of the serine/threonine kinase Akt. To clarify that PI3K products activate Akt in response to M-CSF, NIH 3T3 fibroblasts expressing mutant human M-CSF receptors (3T3-FMS(Y809F)) that fail to activate Ras in response to M-CSF also exhibit increased Akt kinase activity in response to M-CSF challenge. Furthermore, Akt appears to be the primary regulator of survival in 3T3-FMS cells, as transfection of genes encoding dominant-negative Akt isoforms into these fibroblasts blocked M-CSF-induced survival. In normal human monocytes, M-CSF increased the levels of tyrosine-phosphorylated proteins and induced Akt activation in a PI3K-dependent manner. The PI3K inhibitor LY294002 blocked M-CSF-mediated monocyte survival, an effect that was partially restored by caspase-9 inhibitors. These data suggest that M-CSF may induce cell survival through Akt-induced suppression of caspase-9 activation.
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PMID:Macrophage colony-stimulating factor promotes cell survival through Akt/protein kinase B. 1047 97

The involvement of tyrosine phosphorylation during macrophage infection with Leishmania amazonensis amastigotes was investigated. PTK antagonists such as genistein, herbimycin A, geldanamycin and tyrphostin 25 had no significant effect on adhesion to, or entry into, murine peritoneal macrophages, but increased parasite intracellular survival. LPS-induced tyrosine phosphorylation of target host proteins assessed by immunoprecipitation and Western blot was impaired or reversed by living amastigotes soon after 60 min-infection. Such reversion was not due to parasite-secreted molecules but was contact-dependent, as assessed by cytochalasin D treatment of macrophage monolayers prior to infection. Paraformaldehyde-fixed or sodium vanadate-treated amastigotes exerted no significant effect on overall macrophage tyrosine phosphorylation. Immunoprecipitation of proteins employing 4G10 anti-phosphotyrosine antibody followed by Western blotting revealed that tyrosine phosphorylation of 120, 85, 60, 44 and 35 kDa proteins was selectively reversed by amastigote infection. Inhibition, measured by densitometry was from about 66-100% of uninfected cells. None of these proteins was immunoprecipitated from amastigote-infected macrophage lysates but all of them except for p85 were recovered after treatment of parasites with 100 microM sodium orthovanadate prior to infection, a treatment that inhibits Leishmania amastigote protein ecto-phosphatase. The 44 kDa protein was identified as ERK1 MAP kinase (MAPK) by Western blot. Amastigote infection also decreased tyrosine phosphorylation induced by zymosan particles. Vanadate treatment of amastigotes prior to infection significantly decreased parasite intracellular survival. The action of a putative leishmanial ecto-protein phosphatase (PPase) is suggested.
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PMID:Altered tyrosine phosphorylation of ERK1 MAP kinase and other macrophage molecules caused by Leishmania amastigotes. 1047 71

Alternate splicing of mRNA encoding c-KIT results in isoforms which differ in the presence or absence of four amino acids (GNNK) in the juxtamembrane region of the extracellular domain of the receptor. In this study we show that these isoforms of human c-KIT, expressed at similar levels in NIH3T3 cells, display differential effects on various attributes of transformation. The GNNK- isoform strongly promoted anchorage independent growth (colony formation in semi-solid medium), loss of contact inhibition (focus formation), and led to tumorigenicity in nude mice. In contrast, the GNNK+ isoform elicited colony formation but relatively poor focus formation and no tumorigenicity. Saturation binding analysis indicated that the isoforms do not differ significantly in their affinity for the KIT ligand, Steel Factor (SLF). Negligible ligand-independent receptor phosphorylation was observed in either case but, after ligand stimulation, the GNNK- isoform displayed more rapid and extensive tyrosine autophosphorylation and faster internalization. Both isoforms recruited the p85 subunit of phosphatidylinositol 3-kinase and led to similar phosphorylation of its downstream effector c-Akt, but the GNNK- isoform gave rise to more MAP kinase phosphorylation. Thus the c-KIT isoforms display different signalling characteristics and have different transforming activity in NIH3T3 cells.
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PMID:Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts. 1052 34

Protein tyrosine phosphorylation is an integral part of cytokine-induced proliferation and differentiation of hematopoietic cells. The authors previously reported cloning and characterization of the receptor tyrosine kinase Tif, also termed Tyro3. Using the yeast 2-hybrid technology, they recently identified that the p85 subunit of phosphatidylinositol 3-kinase (PI3 kinase) interacted with the cytoplasmic domain of Tyro3. On treatment with epidermal growth factor (EGF), NIH3T3 cells expressed EGFR/Tyro3 (a fusion receptor with the extracellular domain from epidermal growth factor receptor and the transmembrane and cytoplasmic domains from Tyro3), and EGFR/Tyro3 was rapidly phosphorylated on tyrosine residues. The interaction between Tyro3 and p85 was also confirmed by glutathione S-transferase (GST) pull-down experiments. Co-immunoprecipitation followed by Western blot analysis revealed that PI3 kinase was associated with and phosphorylated by the activated Tyro3. Tyro3-associated PI3 kinase exhibited an enhanced kinase activity. In addition, EGF treatment of EGFR/Tyro3-expressing cells led to enhanced phosphorylation of Akt, a downstream component of PI3 kinase. Treatment of NIH3T3 cells expressing a full length of rat Tyro-3, but not NIH3T3 cells, with protein S also resulted in phosphorylation of Akt. Soft agar colony assays showed that the addition of EGF to EGFR/Tyro3-transfected cells, but not to the parental NIH3T3 cells, resulted in a concentration-dependent increase in the formation of anchorage-independent colonies. Tyro3-mediated transformation of NIH3T3 cells was significantly blocked by wortmannin, a PI3 kinase-specific inhibitor. Results of these combined studies strongly suggested that the oncogenic transforming ability of Tyro3 was mediated at least in part by the PI3 kinase pathway. (Blood. 2000;95:633-638)
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PMID:Transforming activity of receptor tyrosine kinase tyro3 is mediated, at least in part, by the PI3 kinase-signaling pathway. 1062 73

RON is a receptor tyrosine kinase that mediates cell scattering, migration, and tubular formation. This study focused on the function of two tyrosines, Y1330 and Y1337, in the C-terminus of RON in regulating epithelial cell scattering and migration. Substitution of both tyrosine residues with phenylalanine causes complete loss of cell scattering and migration in kidney 293 cells. In contrast, single mutation of either tyrosine residue has no effect. We found that mutation at Y1330 or Y1337 alone does not significantly affect the association of RON with PI-3 kinase, whereas a double mutation abolishes the recruitment of substrates. RON-mediated cell migration was inhibited by PI-3 kinase inhibitor wortmannin. This effect was also achieved by a dominant inhibitory p85 of PI-3 kinase. We conclude that Y1330 and Y1337 are required for RON-mediated cell motility. By associating with PI-3 kinase, the Y1330-Y1337 docking site plays a critical role in transducing motile signals of RON.
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PMID:Requirement of both tyrosine residues 1330 and 1337 in the C-terminal tail of the RON receptor tyrosine kinase for epithelial cell scattering and migration. 1063 Nov 20

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4, 5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.
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PMID:A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling. 1064 29

The RET gene codes for a receptor tyrosine kinase that plays a crucial role during the development of both the enteric nervous system and the kidney. Germ line missense mutations at one of six codons specifying extracytoplasmic cysteines are responsible for two related cancer disorders as follows: multiple endocrine neoplasia type2A (MEN2A) and familial medullary thyroid carcinoma (FMTC). MEN2A and FMTC mutations result in a constitutive catalytic activity and as a consequence convert RET into a dominantly acting transforming gene. Although it has been shown that RET-MEN2 mutants activate several transduction pathways, their respective contribution to the neoplastic phenotype remains poorly understood. Over the past few years, it has become increasingly clear that the transforming ability of several viral and cellular oncoproteins depends on their capacity to activate phosphatidylinositol 3-kinase (PI3K). We now report that RET carrying a representative MEN2A mutation at Cys-634 (termed RET-MEN2A) activates PI3K and its downstream effector, the serine/threonine kinase AKT/protein kinase B. Previous studies have demonstrated that mutation of Tyr-1062, which is the intracellular docking site for Shc and Enigma on RET, abolishes the RET-MEN2A transforming activity. We provide evidence that mutation of Tyr-1062 abrogates the binding of the p85 regulatory subunit of PI3K to RET-MEN2A and the subsequent stimulation of the PI3K/AKT pathway. Furthermore, infection of rat fibroblasts with a retrovirus expressing a dominant-interfering form of PI3K suppresses RET-MEN2A-dependent transformation, whereas overexpression of AKT enhances the RET-MEN2A oncogenic potential. In summary, these data are consistent with the notion that RET-mediated cell-transforming effect is critically dependent on the activation of the PI3K/AKT pathway.
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PMID:Transforming ability of MEN2A-RET requires activation of the phosphatidylinositol 3-kinase/AKT signaling pathway. 1065 52

Binding of platelet-derived growth factor receptor (PDGF) to its receptor (PDGFR) activates its receptor tyrosine kinase which autophosphorylates tyrosine residues. The p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) binds to specific phosphotyrosines on PDGFR-beta and through the associated p110 catalytic subunit of PI 3-kinase catalyzes the formation of lipids that are involved in intracellular signaling. We examined if GM1 affects interactions between PDGFR-beta and specific proteins involved in PDGFR-mediated signaling. U-1242 MG cells were studied under different growth conditions using immunoprecipitation and Western Blot analysis. PDGF-stimulated the association of PDGFR-beta with p85, ras GTPase-activating protein and PLC gamma. GM1 decreased these associations in parallel with decreased tyrosine phosphorylation of PDGFR. PDGF augmented the activity of PI 3-kinase associated with PDGFR-beta, and this was attenuated by GM1. However, GM1 did not alter SH2 domains of p85. GM1 probably inhibits PDGF-induced signaling proteins with PDGFR-beta by inhibiting phosphorylation of specific tyrosines on the receptor which bind to SH2-domains on signaling proteins.
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PMID:GM1 inhibits early signaling events mediated by PDGF receptor in cultured human glioma cells. 1069 3

The neu differentiation factors/heregulins (HRGs) comprise a family of polypeptide growth factors that activate p185(erbB-2) through direct binding to either erbB-3 or erbB-4 receptor tyrosine kinases. We have previously shown that HRG-beta is mitogenic for various human mammary epithelial cell lines that coexpress c-erbB-2 and c-erbB-3. Phosphatidylinositol 3-kinase (PI3K) is activated by p185(erbB-2) /erbB-3 heterodimers in cells stimulated by HRG, and PI3K is constitutively activated by p185(erbB-2) /erbB-3 in breast carcinoma cells that overexpress c-erbB-2. To better understand the relative abilities of HRGs, epidermal growth factor (EGF), or insulin to activate PI3K under normal physiological conditions, we compared the levels of recruitment of the 85-kDa regulatory subunit of PI3K when activated by the type I (erbB) or type II [insulin-like growth factor (IGF)] receptor tyrosine kinases in two different nontransformed human mammary epithelial cell lines. The nontransformed H16N-2 cells isolated from normal tissue express EGFR, p185(erbB-2), and erbB-3, and are highly responsive to the mitogenic effects of HRG-beta as well as to the combination of EGF and insulin in serum-free culture. We measured the stoichiometry of p85 recruited by tyrosine-phosphorylated proteins induced in H16N-2 cells by either the alpha or the beta isoform of HRG. HRG-beta was greater than 10-fold more potent in inducing p85 recruitment than was the less biologically active HRG-alpha isoform. HRG-beta was also a more potent inducer of p85 recruited by tyrosine-phosphorylated proteins than was either EGF, insulin, or EGF and insulin combined. Furthermore, erbB-3 principally mediated the direct recruitment of p85 in cells stimulated by HRG or EGF, indicating that, in addition to the high-level activation of PI3K by p185(erbB-2) / erbB-3, EGFR/erbB-3 heterodimer interaction is essential for the weak but significant level of PI3K activated by EGF in cells that express normal EGFR levels. Studies using the PI3K inhibitor wortmannin also indicated that PI3K activation was required for the proliferation of H16N-2 cells induced by either HRG-beta or EGF and insulin in serum-free culture. Finally, HRG-beta was also an especially potent inducer of PI3K in the nontransformed MCF-10A cells, which were derived spontaneously from normal reduction mammoplasty tissue. These data show, for the first time, a side-by-side quantitative comparison of the relative degree of PI3K activated by different growth factors in nontransformed growth factor-dependent cells under precisely defined conditions in culture.
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PMID:Heregulin-beta is especially potent in activating phosphatidylinositol 3-kinase in nontransformed human mammary epithelial cells. 1079 4


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