Search PubMed⌕ Search

Biomedical subjects

F McCormick

Publications and source records attributed to F McCormick.

At least 37 records · Page 2Linked to original sources
PubMed2000

Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling.

We have examined the role of the mouse Diaphanous-related formin (DRF) Rho GTPase binding proteins, mDia1 and mDia2, in cell regulation. The DRFs are required for cytokinesis, stress fiber formation, and transcriptional activation of the serum response factor (SRF). 'Activated' mDia1 and mDia2 variants, lacking their GTPase binding domains, cooperated with Rho-kinase or ROCK to form stress fibers but independently activated SRF. Src tyrosine kinase associated and co-localized with the DRFs in endosomes and in mid-bodies of dividing cells. Inhibition of Src also blocked cytokinesis, SRF induction by activated DRFs, and cooperative stress fiber formation with active ROCK. Our results show that the DRF proteins couple Rho and Src during signaling and the regulation of actin dynamics.

3T3 Cells↗
PubMed2000

Loss of p14ARF in tumor cells facilitates replication of the adenovirus mutant dl1520 (ONYX-015).

The adenovirus mutant dl1520 (ONYX-015) does not express the E1B-55K protein that binds and inactivates p53. This virus replicates in tumor cells with mutant p53, but not in normal cells with functional p53. Although intra-tumoral injection of dl1520 shows promising responses in patients with solid tumors, previous in vitro studies have not established a close correlation between p53 status and dl1520 replication. Here we identify loss of p14ARF as a mechanism that allows dl1520 replication in tumor cells retaining wild-type p53. We demonstrate that the re-introduction of p14ARF into tumor cells with wild-type p53 suppresses replication of dl1520 in a p53-dependent manner. Our study supports the therapeutic use of dl1520 in tumors with lesions within the p53 pathway other than mutation of p53.

Adenoviridae↗
PubMed1999

Phosphoinositide 3-OH kinase (PI3K) and PKB/Akt delay the onset of p53-mediated, transcriptionally dependent apoptosis.

The phosphoinositide 3-OH kinase (PI3K)-PKB/Akt signaling pathway has been shown to mediate both Ras- and cytokine-induced protection from apoptosis. In addition, apoptosis induced by the p53 tumor suppressor protein can be inhibited by Ras- and cytokine-mediated signaling pathways. It was therefore of interest to determine if the PI3K-PKB/Akt signaling pathway was capable of conferring protection from apoptosis induced by p53. We demonstrate in this report that constitutively active PI3K and PKB/Akt are capable of significantly delaying the onset of p53-mediated apoptosis. This was manifested as a delay in the kinetics of DNA degradation and cell death as well as a profound attenuation in the accumulation of cells with a sub-G(1) DNA content. Moreover, we found that this effect is mediated in the absence of changes in expression of Bcl-2, Bcl-Xl, and the pro-apoptotic protein Bax. Our results provide the first direct and unambiguous link between p53-mediated apoptosis and the PI3K-PKB/Akt signaling pathway.

Animals↗
PubMed1999

Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells.

Mutations in the adenomatous polyposis coli (APC) tumour-suppressor gene occur in most human colon cancers. Loss of functional APC protein results in the accumulation of beta-catenin. Mutant forms of beta-catenin have been discovered in colon cancers that retain wild-type APC genes, and also in melanomas, medulloblastomas, prostate cancer and gastric and hepatocellular carcinomas. The accumulation of beta-catenin activates genes that are responsive to transcription factors of the TCF/LEF family, with which beta-catenin interacts. Here we show that beta-catenin activates transcription from the cyclin D1 promoter, and that sequences within the promoter that are related to consensus TCF/LEF-binding sites are necessary for activation. The oncoprotein p21ras further activates transcription of the cyclin D1 gene, through sites within the promoter that bind the transcriptional regulators Ets or CREB. Cells expressing mutant beta-catenin produce high levels of cyclin D1 messenger RNA and protein constitutively. Furthermore, expression of a dominant-negative form of TCF in colon-cancer cells strongly inhibits expression of cyclin D1 without affecting expression of cyclin D2, cyclin E, or cyclin-dependent kinases 2, 4 or 6. This dominant-negative TCF causes cells to arrest in the G1 phase of the cell cycle; this phenotype can be rescued by expression of cyclin D1 under the cytomegalovirus promoter. Abnormal levels of beta-catenin may therefore contribute to neoplastic transformation by causing accumulation of cyclin D1.

Binding Sites↗
PubMed1999

Early medieval cattle remains from a Scandinavian settlement in Dublin: genetic analysis and comparison with extant breeds.

A panel of cattle bones excavated from the 1000-year-old Viking Fishamble Street site in Dublin was assessed for the presence of surviving mitochondrial DNA (mtDNA). Eleven of these bones gave amplifiable mtDNA and a portion of the hypervariable control region was determined for each specimen. A comparative analysis was performed with control region sequences from five extant Nordic and Irish cattle breeds. The medieval population displayed similar levels of mtDNA diversity to modern European breeds. However, a number of novel mtDNA haplotypes were also detected in these bone samples. In addition, the presence of a putative ancestral sequence at high frequency in the medieval population supports an early post-domestication expansion of cattle in Europe.

Animals↗
PubMed1999

A frequent activated smoothened mutation in sporadic basal cell carcinomas.

Basal-cell carcinomas (BCCs) are the most common cancer in Caucasians. It has been reported that the patched gene is inactivated in 30-40% sporadic BCCs and 20% sporadic medulloblastomas via loss of heterozygosity and nonsense mutations. Recently, two activating smoothened mutations have been found in the sporadic basal cell carcinomas. One, at base pair 1604 (G-to-T transversion) of exon 9, changes codon 535 from tryptophan to leucine, and the other, at base pair 1685 (G-to-A transition) of exon 10, changes codon 562 from arginine to glutamine (Xie et al., 1998). In our study, 1604G-->T was found in 20 out of 97 (20.6%) sporadic BCCs. The high prevalence indicates that 1604G is the mutation hot spot in our tumor samples. This mutation was detected in all three histological subtypes of BCCs, suggesting that smoothened mutation is an early event during the development of the tumor. Our finding of a high smoothened mutation rate, together with high frequent patched gene mutations reported recently, indicates that activation of the hedgehog signal transduction pathway is the most common and early event in the development of sporadic BCCs. Additionally, to determine whether smoothened, like patched, is also involved in the carcinogenesis of medulloblastomas, we screened medulloblastoma samples for these two mutations by restriction analysis. We have found the 1604G-->T mutation in 1 out of 21 medulloblastomas. This result confirmed smoothened gene involvement in the carcinogenesis of medulloblastoma.

Basal Cell Carcinoma↗
PubMed1999

Expression of the c-erbB-3/HER-3 and c-erbB-4/HER-4 growth factor receptors and their ligands, neuregulin-1 alpha, neuregulin-1 beta, and betacellulin, in normal endometrium and endometrial cancer.

The objective of this study was to determine the immunohistochemical expression of the c-erbB-3 and c-erbB-4 growth factor receptors and their principal ligands, the neuregulins and betacellulin, in normal endometrium and determine whether there was evidence of under- or overexpression in endometrial adenocarcinoma. Immunohistochemistry was performed using well-characterized antibodies against each of the five proteins analyzed on formalin-fixed, paraffin-embedded archival material. Forty-three normal endometrial samples (16 proliferative, 19 secretory, and 8 hyperplastic) and 41 endometrial adenocarcinoma cases were analyzed. There was variable expression of the growth factor receptors and the ligands in the two principal phases of the menstrual cycle as well as in endometrial adenocarcinoma. In normal endometrium, the c-erbB-3 receptor was weakly expressed in both phases. The c-erbB-4 receptor and all of the ligands examined, neuregulin alpha, neuregulin beta, and betacellulin, were expressed at significantly higher levels in the secretory as compared with the proliferative phase of the menstrual cycle, suggesting a role for these proteins in endometrial maturation. In endometrial adenocarcinoma, overexpression of c-erbB-3, c-erbB-4, and betacellulin with underexpression of neuregulin a as compared with normal controls was observed. Neuregulin beta expression was not found to be significantly different in the two groups. These results suggest that signaling through the c-erbB-3 and c-erbB-4 receptors and the ligands neuregulin alpha, neuregulin beta, and betacellulin are important in endometrial carcinogenesis.

Amino Acid Sequence↗
PubMed1999

Signalling networks that cause cancer.

Cancer is caused by the stepwise accumulation of mutations that affect growth control, differentiation and survival. The view that mutations affect discrete signalling pathways, each contributing to a specific aspect of the full malignant phenotype, has proved to be too simplistic. We now know that oncogenes and tumour suppressors depend on one another for their selective advantage, and that they affect multiple pathways that intersect and overlap. The interactive nature of each genetic change has important implications for cancer therapy and for the stepwise model of carcinogenesis.

Adenomatous Polyposis Coli Protein↗
PubMed1998

Going for the GAP.

Explore the source record for details and available documents.

Amino Acid Substitution↗
PubMed1998

Akt activation by growth factors is a multiple-step process: the role of the PH domain.

The protein kinase encoded by the Akt proto-oncogene is activated by phospholipid binding, membrane translocation and phosphorylation. To address the relative roles of these mechanisms of Akt activation, we have employed a combination of genetic and pharmacological approaches. Transient transfection of NIH3T3 cells with wild-type Akt, pleckstrin homology (PH) domain mutants, generated on the basis of a PH domain structural model, and phosphorylation site Akt mutants provided evidence for a model of Akt activation consisting of three sequential steps: (1) a PH domain-dependent, growth factor-independent step, marked by constitutive phosphorylation of threonine 450 (T450) and perhaps serine 124 (S124), that renders the protein responsive to subsequent activation events; (2) a growth factor-induced, PI3-K-dependent membrane-translocation step; and (3) a PI3-K-dependent step, characterized by phosphorylation at T308 and S473, that occurs in the cell membrane and is required for activation. When forced to translocate to the membrane, wild-type Akt and PH domain Akt mutants that are defective in the first step become constitutively active, suggesting that the purpose of this step is to prepare the protein for membrane translocation. Both growth factor stimulation and forced membrane translocation, however, failed to activate a T308A mutant. This, combined with the finding that T308D/S473D double mutant is constitutively active, suggests that the purpose of the three-step process of Akt activation is the phosphorylation of the protein at T308 and S473. The proposed model provides a framework for a comprehensive understanding of the temporal and spatial requirements for Akt activation by growth factors.

3T3 Cells↗
PubMed1998

Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B.

Protein kinase B (PKB) is activated in response to phosphoinositide 3-kinases and their lipid products phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3] and PtdIns(3,4)P2 in the signaling pathways used by a wide variety of growth factors, antigens, and inflammatory stimuli. PKB is a direct target of these lipids, but this regulation is complex. The lipids can bind to the pleckstrin homologous domain of PKB, causing its translocation to the membrane, and also enable upstream, Thr308-directed kinases to phosphorylate and activate PKB. Four isoforms of these PKB kinases were purified from sheep brain. They bound PtdIns(3,4,5)P3 and associated with lipid vesicles containing it. These kinases contain an NH2-terminal catalytic domain and a COOH-terminal pleckstrin homologous domain, and their heterologous expression augments receptor activation of PKB, which suggests they are the primary signal transducers that enable PtdIns(3,4,5)P3 or PtdIns- (3,4)P2 to activate PKB and hence to control signaling pathways regulating cell survival, glucose uptake, and glycogen metabolism.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1997

Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B.

Protein kinase B (PKB) is a proto-oncogene that is activated in signaling pathways initiated by phosphoinositide 3-kinase. Chromatographic separation of brain cytosol revealed a kinase activity that phosphorylated and activated PKB only in the presence of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Phosphorylation occurred exclusively on threonine-308, a residue implicated in activation of PKB in vivo. PtdIns(3,4,5)P3 was determined to have a dual role: Its binding to the pleckstrin homology domain of PKB was required to allow phosphorylation by the upstream kinase and it directly activated the upstream kinase.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1997

Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-related kinase-dependent and Ca2+-dependent signal pathways in Rat-1 cells.

Stimulation of Rat-1 cells with lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results in a biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide or sodium orthovanadate had little effect on the early peak of ERK1 activity but potentiated the sustained phase. Cycloheximide also potentiated ERK1 activation in Rat-1 cells expressing DeltaRaf-1:ER, an estradiol-regulated form of the oncogenic, human Raf-1. Since cycloheximide did not potentiate MEK activity but abrogated the expression of mitogen-activated protein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, we speculated that the level of MKP-1 expression may be an important regulator of ERK1 activity in Rat-1 cells. Inhibition of LPA-stimulated MEK and ERK activation with PD98059 and pertussis toxin, a selective inhibitor of Gi-protein-coupled signaling pathways, reduced LPA-stimulated MKP-1 expression by only 50%, suggesting the presence of additional MEK- and ERK-independent pathways for MKP-1 expression. Specific activation of the MEK/ERK pathway by DeltaRaf-1:ER had little or no effect on MKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1 expression in Rat-1 cells. Activation of PKC played little part in growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression was blocked by buffering [Ca2+]i, leading to a potentiation of the sustained phase of ERK1 activation without potentiating MEK activity. In Rat-1DeltaRaf-1:ER cells, we observed a strong synergy of MKP-1 expression when cells were stimulated with estradiol in the presence of ionomycin, phorbol 12-myristate 13-acetate, or okadaic acid under conditions where these agents did not synergize for ERK activation. These results suggest that activation of the Raf/MEK/ERK pathway is insufficient to induce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute the response seen with growth factors. In this way, ERK-dependent and -independent signals may regulate MKP-1 expression, the magnitude of sustained ERK1 activity, and therefore gene expression.

Animals↗
PubMed1997

Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro.

The c-Raf-1 protein kinase plays a critical role in intracellular signaling downstream from many tyrosine kinase and G-protein-linked receptors. c-Raf-1 binds to the proto-oncogene Ras in a GTP-dependent manner, but the exact mechanism of activation of c-Raf-1 by Ras is still unclear. We have established a system to study the activation of c-Raf-1 in vitro. This involves mixing membranes from cells expressing oncogenic H-RasG12V, with cytosol from cells expressing epitope-tagged full-length wild-type c-Raf-1. This results in a fraction of the c-Raf-1 binding to the membranes and a concomitant 10- to 20-fold increase in specific activity. Ras was the only component in these membranes required for activation, as purified recombinant farnesylated K-Ras.GTP, but not non-farnesylated K-Ras.GTP or farnesylated K-Ras.GDP, was able to activate c-Raf-1 to the same degree as intact H-RasG12V membranes. The most potent activation occurred under conditions in which phosphorylation was prohibited. Under phosphorylation-permissive conditions, activation of c-Raf-1 by Ras was substantially inhibited. Consistent with the results from other groups, we find that the activation of c-Raf-1 by Src in vivo occurs concomitant with tyrosine phosphorylation on c-Raf-1, and in vitro, activation of c-Raf-1 by Src requires the presence of ATP. Therefore we propose that activation of c-Raf-1 by Ras or by Src occurs through different mechanisms.

Animals↗
PubMed1997

Differential regulation of extracellular signal-regulated protein kinase 1 and Jun N-terminal kinase 1 by Ca2+ and protein kinase C in endothelin-stimulated Rat-1 cells.

The extracellular signal-regulated protein kinase (ERK) and Jun N-terminal kinase (JNK) signalling cascades transduce signals from the cell cytoplasm to the nucleus, where they regulate gene expression. The activation of ERK1 by lysophosphatidic acid (LPA) and endothelin 1 (Et-1) was compared in Rat-1 cells. Both stimulated DNA synthesis to a similar degree but, in contrast with LPA, Et-1 did not stimulate sustained ERK1 activation, a signal that is thought to be important for the proliferation of fibroblasts. Et-1, but not LPA, was able to activate JNK1; pharmacological analysis revealed that the same EtA receptor mediates DNA synthesis, ERK1 and JNK1 activation. However, activation of JNK1 required higher concentrations of Et-1 than was required for stimulation of ERK1 or DNA synthesis. Signalling to ERK1 and JNK1 was partly inhibited by pertussis toxin, suggesting that both pathways are regulated in part by Gi or G0 proteins. Activation of JNK1 by Et-1 lagged behind ERK1 activation but was not dependent on it because PD98059, an inhibitor of mitogen-activated protein kinase (or ERK) kinase, was without effect on JNK1 activation. In contrast with recent studies, activation of protein kinase C (PKC) or Ca2+ fluxes inhibited activation of JNK1 but not ERK1; furthermore inhibition of PKC or sequestration of Ca2+ potentiated JNK1 activation by Et-1 but not by anisomycin, and again had little effect on ERK1 activation. These results demonstrate that the same G-protein-coupled receptor can activate both the ERK and JNK signal pathways but the two kinase cascades seem to be separate, parallel pathways that are differentially regulated by PKC and Ca2+. The results are discussed in terms of the role of ERK and JNK in proliferative signalling.

Animals↗