90 resultados para tyrosine kinase receptor


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To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.

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Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK.

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Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein-coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function.

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Brain tumors comprise a wide variety of neoplasia classified according to their cellular origin and their morphological and histological characteristics. The transformed phenotype of brain tumor cells has been extensively studied in the past years, achieving a significant progress in our understanding of the molecular pathways leading to tumorigenesis. It has been reported that the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is frequently altered in grade IV brain tumors resulting in uncontrolled cell growth, survival, proliferation, angiogenesis, and migration. This aberrant activation can be explained by oncogenic mutations in key components of the pathway or through abnormalities in its regulation. These alterations include overexpression and mutations of receptor tyrosine kinases (RTKs), mutations and deletions of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene, encoding a lipid kinase that directly antagonized PI3K activity, and alterations in Ras signaling. Due to promising results of preclinical studies investigating the PI3K/AKT pathway in grade IV brain tumors like glioblastoma and medulloblastoma, the components of this pathway have emerged as promising therapeutic targets to treat these malignant brain tumors. Although an arsenal of small molecule inhibitors that target specific components of this signaling pathway is being developed, its successful application in the clinics remains a challenge. In this article we will review the molecular basis of the PI3K/AKT signaling pathway in malignant brain tumors, mainly focusing on glioblastoma and medulloblastoma, and we will further discuss the current status and potential of molecular targeted therapies.

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Lung cancer is the leading cause of cancer-related mortality worldwide and more than 1 million people annually die in consequence of lung cancer. Although an improvement in lung cancer treatment could be achieved, especially in the last decade, the development of additional therapeutic strategies is urgently required in order to provide improved survival benefit for patients. Lung cancer formation is caused by genetic modifications commonly caused by tobacco smoking. Numerous studies have demonstrated the role of extracellular growth factors in lung cancer cell proliferation, metastasis, and chemoresistance. Mutations and amplifications in molecules related to receptor tyrosine signalling, such as EGFR, ErbB2, c-Met, c-Kit, VEGFR, PI3K, and PTEN are only some of the alterations known to contribute to the development of lung cancer. The phosphoinositide 3-kinase (PI3K) pathway, fundamental for cell development, growth, and survival, is known to be frequently altered in neoplasia, including carcinomas of the lung. Based on the high frequency of alterations, which include mutations and amplifications, leading to over-activation of certain upstream/downstream mediators, targeting components of the PI3K signalling pathway is considered to be a promising therapeutic approach in cancer treatment. In this article we will summarize the current knowledge about the involvement of PI3K signalling in lung cancer and discuss the development of targeted therapies involving PI3K pathway inhibitors.

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Alterations of the epidermal growth factor receptor (EGFR) can be observed in a significant subset of esophageal adenocarcinomas (EACs), and targeted therapy against EGFR may become an interesting approach for the treatment of these tumors. Mutations of KRAS, NRAS, BRAF, and phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) and deregulation of PTEN expression influence the responsiveness against anti-EGFR therapy in colorectal carcinomas. We investigated the prevalence of these events in a collection of 117 primary resected EACs, correlated the findings with EGFR expression and amplification, and determined their clinicopathologic impact. KRAS mutations were detected in 4 (3%) of 117 tumors (3× G12D and 1 G12V mutation). One tumor had a PIK3CA E545K mutation. Neither NRAS nor BRAF mutations were detected. Sixteen (14%) of 117 cases were negative for PTEN expression, determined by immunohistochemistry. Loss of PTEN was observed predominantly in advanced tumor stages (P = .004). There was no association between PTEN and EGFR status. Loss of PTEN was associated with shorter overall and disease-free survival (P < .001 each) and also an independent prognostic factor in multivariate analysis (P = .015). EGFR status had no prognostic impact in this case collection. In summary, loss of PTEN can be detected in a significant subset of EAC and is associated with an aggressive phenotype. Therefore, PTEN may be useful as a prognostic biomarker. In contrast, mutations of RAS/RAF/PIK3CA appear only very rarely, if at all, in EAC. A possible predictive role of PTEN in anti-EGFR treatment warrants further investigations, whereas determination of RAS/RAF/PIK3CA mutations may only have a minor impact in this context.

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G-protein-coupled receptor kinase 2 (GRK2) is a primary regulator of β-adrenergic signaling in the heart. G-protein-coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study.

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Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

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Recent studies have implied that GPIb-IX-V as well as functioning as an adhesion receptor may also induce signaling to mediate binding of platelets to damaged vessel wall to prevent bleeding. Reorganization of the cytoskeleton and redistribution of platelet structural proteins and signaling molecules are thought to be important in this early activation process, though the molecular mechanisms remain to be fully defined. In this study, we have used mucetin, a snake venom lectin protein that activates platelets via GPIb, to study the redistribution of GPIb in platelets. In unstimulated platelets, a minor portion of GPIb localized to Triton-insoluble cytoskeleton fractions (TIC). This portion increased considerably after platelet activation by mucetin. We also find increased contents of the FcRgamma chain in TIC. Anti-GPIb antibodies, mocarhagin or cytochalasin D completely inhibited the cytoskeletal translocation. In addition, BAPTA-AM, a cytoplasmic calcium chelator, strongly inhibited this process. On the other hand, inhibitors of alphaIIbbeta3, PLCgamma, PKC, tyrosine kinases, ADP receptor, PI3-kinase or EDTA are effective in preventing GPIb relocation in convulxin- but not in mucetin-activated platelets. We propose that cytoskeletal translocation of GPIb is upstream of alphaIIbbeta3 activation and cross-linking of GPIb is sufficient to induce this event in mucetin-activated platelets.

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To investigate mechanisms by which angiotensin converting enzyme (ACE)-inhibition increases insulin sensitivity, spontaneously hypertensive (SH) rats were treated with or without ramipril (1 mg/kg per day) for 12 weeks. Insulin binding and protein levels of insulin receptor substrate-1 (IRS-1), p85-subunit of phosphatidylinositol 3'-kinase (p85) and Src homology 2 domain-containing phosphatase-2 (SHP2) were then determined in hindlimb muscle and liver. Additionally, protein tyrosine phosphatase (PTPase) activities towards immobilized phosphorylated insulin receptor or phosphorylated IRS-1 of membrane (MF) and cytosolic fractions (CF) of these tissues were measured. Ramipril treatment increased IRS-1-protein content in muscle by 31+/-9% (P<0.05). No effects were observed on IRS-1 content in liver or on insulin binding or protein expression of p85 or SHP2 in both tissues. Ramipril treatment also increased dephosphorylation of insulin receptor by muscle CF (22.0+/-1.0%/60 min compared to 16.8+/-1.5%/60 min; P<0.05), and of IRS-1 by liver MF (37.2+/-1.7%/7.5 min compared to 33.8+/-1.7%/7.5 min; P<0.05) and CF (36.8+/-1.0%/7.5 min compared to 33.2+/-1.0%/7.5 min; P<0.05). We conclude that the observed effects of ACE-inhibition by ramipril on the protein expression of IRS-1 and on PTPase activity might contribute to its effect on insulin sensitivity.

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Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

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Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

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Medulloblastoma (MB) is the most common malignant brain tumor in childhood and represents the main cause of cancer-related death in this age group. The phosphoinositide 3-kinase (PI3K) pathway has been shown to play an important role in the regulation of medulloblastoma cell survival and proliferation, but the molecular mechanisms and downstream effectors underlying PI3K signaling still remain elusive. The impact of RNA interference (RNAi)-mediated silencing of PI3K isoforms p110α and p110δ on global gene expression was investigated by DNA microarray analysis in medulloblastoma cell lines. A subset of genes with selectively altered expression upon p110α silencing in comparison to silencing of the closely related p110δ isoform was revealed. Among these genes, the leukemia inhibitory factor receptor α (LIFR α) was validated as a novel p110α target in medulloblastoma. A network involving c-Myc and miR-125b was shown to be involved in the control of LIFRα expression downstream of p110α. Targeting the LIFRα by RNAi, or by using neutralizing reagents impaired medulloblastoma cell proliferation in vitro and induced a tumor volume reduction in vivo. An analysis of primary tumors revealed that LIFRα and p110α expression were elevated in the sonic hedgehog (SHH) subgroup of medulloblastoma, indicating its clinical relevance. Together, these data reveal a novel molecular signaling network, in which PI3K isoform p110α controls the expression of LIFRα via c-Myc and miR-125b to promote MB cell proliferation.

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Intravital imaging has revealed that T cells change their migratory behavior during physiological activation inside lymphoid tissue. Yet, it remains less well investigated how the intrinsic migratory capacity of activated T cells is regulated by chemokine receptor levels or other regulatory elements. Here, we used an adjuvant-driven inflammation model to examine how motility patterns corresponded with CCR7, CXCR4, and CXCR5 expression levels on ovalbumin-specific DO11.10 CD4(+) T cells in draining lymph nodes. We found that while CCR7 and CXCR4 surface levels remained essentially unaltered during the first 48-72 h after activation of CD4(+) T cells, their in vitro chemokinetic and directed migratory capacity to the respective ligands, CCL19, CCL21, and CXCL12, was substantially reduced during this time window. Activated T cells recovered from this temporary decrease in motility on day 6 post immunization, coinciding with increased migration to the CXCR5 ligand CXCL13. The transiently impaired CD4(+) T cell motility pattern correlated with increased LFA-1 expression and augmented phosphorylation of the microtubule regulator Stathmin on day 3 post immunization, yet neither microtubule destabilization nor integrin blocking could reverse TCR-imprinted unresponsiveness. Furthermore, protein kinase C (PKC) inhibition did not restore chemotactic activity, ruling out PKC-mediated receptor desensitization as mechanism for reduced migration in activated T cells. Thus, we identify a cell-intrinsic, chemokine receptor level-uncoupled decrease in motility in CD4(+) T cells shortly after activation, coinciding with clonal expansion. The transiently reduced ability to react to chemokinetic and chemotactic stimuli may contribute to the sequestering of activated CD4(+) T cells in reactive peripheral lymph nodes, allowing for integration of costimulatory signals required for full activation.