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)

PD 089828, a novel protein tyrosine kinase inhibitor of a new structural class, the 6-aryl-pyrido-[2,3-d]pyrimidines, was identified by screening a compound library with assays that measured protein tyrosine kinase activity. PD 089828 was found to inhibit human full-length fibroblast growth factor (FGF) receptor-1 (FGFR-1), platelet-derived growth factor (PDGF) receptor beta subunit (PDGFR-beta), Src nonreceptor tyrosine kinase (c-Src) and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases with half-maximal inhibitory potencies (IC50 values) of 0.15 +/- 0.02 (n = 4), 0.18 +/- 0.04 (n = 3), 1.76 +/- 0.28 (n = 4) and 5.47 +/- 0.78 (n = 6) microM, respectively. PD 089828 was further characterized as an ATP competitive inhibitor of the growth factor receptor tyrosine kinases (FGFR-1, PDGFR-beta and EGFR) but a noncompetitive inhibitor of c-Src tyrosine kinase with respect to ATP. In addition, PD 089828 inhibited PDGF- and EGF-stimulated receptor autophosphorylation in vascular SMC (VSMC) and basic FGF-mediated tyrosine phosphorylation in A121 cells with IC50 values similar to the potencies observed for inhibition of receptor tyrosine kinase activity. The inhibition of PDGF receptor autophosphorylation in VSMC by PD 089828 occurred rapidly, with maximal effects reached within 5 min of drug exposure. Inhibition after single exposure was long lasting but also rapidly reversible, occurring within 5 min after drug removal. The PDGF-induced association of downstream signaling proteins, including phosphoinositide-3-kinase (PI-3K), growth factor receptor binding protein-2 (GRB2), SH-2 domain and collagen like (Shc) and phospholipase Cgamma (PLCgamma), with VSMC PDGF receptors was also blocked as a result of the inhibition of PDGF-stimulated receptor autophosphorylation by PD 089828. PD 089828 also inhibited the PDGF-induced tyrosine phosphorylation of the 44- and 42-kDa mitogen-activated protein kinase isoforms. Moreover, the effects of PD 089828 were demonstrated in functional assays in which PDGF-stimulated DNA synthesis, PDGF-directed migration and serum-stimulated growth of VSMC were all inhibited to the same extent as PDGF receptor autophosphorylation (IC50 = 0.8, 4.5 and 1.8 microM, respectively). These results highlight the biological characteristics of PD 089828 as a novel, broadly active protein tyrosine kinase inhibitor with long-lasting but reversible cellular effects. The potential therapeutic use of these broadly acting, nonselective inhibitors as antiproliferative and antimigratory agents could extend to such diseases as cancer, atherosclerosis and restenosis in which redundancies in growth-signaling pathways are known to exist.
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PMID:Inhibition of growth factor-mediated tyrosine phosphorylation in vascular smooth muscle by PD 089828, a new synthetic protein tyrosine kinase inhibitor. 919 Aug 82

Growth factor receptor-binding protein-2 (Grb2) plays a key role in signal transduction initiated by Bcr/Abl oncoproteins and growth factors, functioning as an adaptor protein through its Src homology 2 and 3 (SH2 and SH3) domains. We found that Grb2 was tyrosine-phosphorylated in cells expressing BCR/ABL and in A431 cells stimulated with epidermal growth factor (EGF). Phosphorylation of Grb2 by Bcr/Abl or EGF receptor reduced its SH3-dependent binding to Sos in vivo, but not its SH2-dependent binding to Bcr/Abl. Tyr209 within the C-terminal SH3 domain of Grb2 was identified as one of the tyrosine phosphorylation sites, and phosphorylation of Tyr209 abolished the binding of the SH3 domain to a proline-rich Sos peptide in vitro. In vivo expression of a Grb2 mutant where Tyr209 was changed to phenylalanine enhanced BCR/ABL-induced ERK activation and fibroblast transformation, and potentiated and prolonged Grb2-mediated activation of Ras, mitogen-activated protein kinase and c-Jun N-terminal kinase in response to EGF stimulation. These results suggest that tyrosine phosphorylation of Grb2 is a novel mechanism of down-regulation of tyrosine kinase signaling.
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PMID:Tyrosine phosphorylation of Grb2 by Bcr/Abl and epidermal growth factor receptor: a novel regulatory mechanism for tyrosine kinase signaling. 1172 15

E2 rapidly activates MAPK in breast cancer cells, and the mechanism for this effect has not been fully identified. Since growth factor-induced MAPK activation involves signaling via the adapter protein Shc (Src-homology and collagen homology) and its association with membrane receptors, we hypothesized that breast cancer cells utilize similar signaling mechanisms in response to E2. In the present study, we demonstrated that E2 rapidly induced Shc phosphorylation and Shc-Grb2 (growth factor receptor binding protein 2)-Sos (son of sevenless) complex formation in MCF-7 cells. Overexpression of dominant negative Shc blocked the effect of E2 on MAPK, indicating a critical role of Shc in E2 action. Using selective inhibitors, we also demonstrated that ERalpha and Src are upstream regulators of Shc. A rapid physical association between ERalpha and Shc upon E2 stimulation further evidenced the role of ERalpha on Shc activation. Mutagenesis studies showed that the phosphotyrosine binding and SH2 domains of Shc are required to interact with the activation function 1, but not activation function 2, domain of ERalpha. Using a glutathione-S-transferase-Shc pull-down assay, we demonstrated that this ERalpha-Shc association was direct. Biological consequences of this pathway were further investigated at the genomic and nongenomic levels. E2 stimulated MAPK-mediated Elk-1 transcriptional activity. Confocal microscopy studies showed that E2 rapidly induced formation of membrane ruffles, pseudopodia, and ERalpha membrane translocation. The E2-induced morphological changes were prevented by antiestrogen. Together our results demonstrate that ERalpha can mediate the rapid effects of E2 on Shc, MAPK, Elk-1, and morphological changes in breast cancer cells
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PMID:Linkage of rapid estrogen action to MAPK activation by ERalpha-Shc association and Shc pathway activation. 1177 43

Grb-2-related adaptor protein (Grap) is a Grb2-like SH3-SH2-SH3 adaptor protein with expression restricted to lymphoid tissues. Grap(-/-) lymphocytes isolated from targeted Grap-deficient mice exhibited enhanced proliferation, interleukin-2 production, and c-fos induction in response to mitogenic T-cell receptor (TCR) stimulation, compared to wild-type cells. Ectopic expression of Grap led to a suppression of Elk-1-directed transcription induced by the Ras/Erk pathway, without having effects on gene expression mediated by Jnk and p38 mitogen-activated protein kinases. Together, these data suggest that Grap, unlike Grb2, acts as a negative regulator of TCR-stimulated intracellular signaling by downregulating signal relay through the Ras/Erk pathway.
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PMID:Grap negatively regulates T-cell receptor-elicited lymphocyte proliferation and interleukin-2 induction. 1197 56

OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia.
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PMID:Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930. 1609 34

RET/papillary thyroid carcinoma (PTC) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase with protein dimerization motifs encoded by heterologous genes. Here, we show that RET/PTC1 activates the Rap1 small GTPase. The activation of Rap1 was dependent on the phosphorylation of RET Tyr(1062). RET/PTC1 recruited a complex containing growth factor receptor binding protein 2-associated binding protein 1 (Gab1), CrkII (v-crk sarcoma virus CT10 oncogene homologue II), and C3G (Rap guanine nucleotide exchange factor 1). By using dominant-negative and small interfering duplex (small interfering RNA) oligonucleotides, we show that RET/PTC1-mediated Rap1 activation was dependent on CrkII, C3G, and Gab1. Activation of Rap1 was involved in the RET/PTC1-mediated stimulation of the BRAF kinase and the p42/p44 mitogen-activated protein kinases. Proliferation and stress fiber formation of RET/PTC1-expressing PC Cl 3 thyroid follicular cells were inhibited by the dominant-negative Rap1(N17) and by Rap1-specific GTPase-activating protein. Thus, Rap1 is a downstream effector of RET/PTC and may contribute to the transformed phenotype of RET/PTC-expressing thyrocytes.
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PMID:RET/papillary thyroid carcinoma oncogenic signaling through the Rap1 small GTPase. 1721 Jul 21

Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration.
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PMID:The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration. 2697 94

Glial cell derived neurotrophic factor (GDNF)-dependent receptor tyrosine kinase RET activity is required for proper development of the nervous system and genitourinary tract. Loss-of-function mutations in RET are associated with enteric nervous system abnormalities (Hirschsprung disease) and renal deficits (Potter's syndrome), whereas activating mutations lead to hereditary cancer syndromes (multiple endocrine neoplasia type 2A and type 2B). RET activation is crucial for the proper regulation of a variety of cellular processes including cell migration, proliferation and neurite outgrowth. By analyzing a series of RET mutants we found that Y1062 was critical for stimulating GDNF-mediated proliferation as well as proliferation stimulated by GDNF-independent oncogenic forms of RET. Studies using small interfering RNA driven by lentivirus to knock-down expression of particular adaptor proteins that interact with RET phospho-Y1062, demonstrated that only Src-homology 2 and growth factor receptor binding protein 2 were necessary for RET-mediated proliferation by wild type and oncogenic forms of RET. Interestingly, we discovered that Y1062 was also required for GDNF-stimulated neurite outgrowth. However, small interfering RNAs to either Src-homology 2 or growth factor receptor binding protein 2 or a panel of other adaptor proteins known to interact with RET Y1062 were incapable of blocking GDNF-stimulated neurite formation, indicating that differential use of intracellular adaptors is responsible for regulating alternative RET-stimulated cellular events such as proliferation versus a differentiation response like neurite outgrowth.
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PMID:Deciphering adaptor specificity in GFL-dependent RET-mediated proliferation and neurite outgrowth. 1766 53

ErbB-2 overexpression and amplification occurs in 15% to 30% of human invasive breast carcinomas associated with poor clinical prognosis. Previously, we have shown that four ErbB-2/Neu tyrosine-autophosphorylation sites within the cytoplasmic tail of the receptor recruit distinct adaptor proteins and are sufficient to mediate transforming signals in vitro. Two of these sites, representing the growth factor receptor binding protein 2 (Grb2; Neu-YB) and the Src homology and collagen (Shc; Neu-YD) binding sites, can induce mammary tumorigenesis and metastasis. Here, we show that transgenic mice bearing the two other ErbB-2 autophosphorylation sites (Neu-YC and Neu-YE) develop metastatic mammary tumors. A detailed comparison of biological profiles among all Neu mutant mouse models revealed that Neu-YC, Neu-YD, and Neu-YE mammary tumors shared similar pathologic and transcriptional features. By contrast, the Neu-YB mouse model displayed a unique pathology with a high metastatic potential that correlates with a distinct transcriptional profile, including genes that promote malignant tumor progression such as metalloproteinases and chemokines. Furthermore, Neu-YB tumor epithelial cells showed abundant intracellular protein level of the chemokine CXCL12/SDF-1alpha, which may reflect the aggressive nature of this Neu mutant mouse model. Taken together, these findings indicate that activation of distinct Neu-coupled signaling pathways has an important impact on the biological behavior of Neu-induced tumors.
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PMID:Distinct ErbB-2 coupled signaling pathways promote mammary tumors with unique pathologic and transcriptional profiles. 1769 61

Targeting disease-causing proteins for ubiquitination and degradation by chimeric molecules represents a promising alternative therapeutic strategy in cancer. Here, several Cbl-based chimeric ubiquitin ligases were recombined to achieve effective down-regulation of HER2. These chimeric molecules consisted of the Cbl NH(2)-terminal tyrosine kinase binding domain, linker, and RING domain, with the Src homology 2 domain replaced with that from growth factor receptor binding protein 2 (Grb2), Grb7, p85, or Src. The chimeric proteins not only interacted with HER2 but also enhanced the down-regulation of endogenous overexpressed HER2. After the chimeric proteins were introduced into HER2-overexpressing breast cancer SK-BR-3 cells or ovarian cancer SK-OV-3 cells, they effectively promoted HER2 ubiquitination and degradation in a RING finger domain-dependent manner. Consequently, expression of these chimeric molecules led to an inhibition of colony formation, increased the proportion of cells in the G(1) cycle, and suppressed tumorigenicity. Collectively, our findings suggest that the Cbl-based chimeric ubiquitin ligases designed in the present study may represent a novel approach for the targeted therapy of HER2-overexpressing cancers.
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PMID:Degradation of HER2 by Cbl-based chimeric ubiquitin ligases. 1787 12


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