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At least 1,297 records · Page 72Linked to original sources
PubMed1993

Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.

Amino Acid Sequence↗
PubMed1995

Repression of platelet-derived growth factor beta-receptor expression by mitogenic growth factors and transforming oncogenes in murine 3T3 fibroblasts.

Platelet-derived growth factor BB (PDGF-BB) is an important extracellular factor for regulating the G0-S phase transition of murine BALB/c-3T3 fibroblasts. We have investigated the expression of the PDGF beta receptor (PDGF beta R) in these cells. We show that the state of growth arrest in G0, resulting from serum deprivation, is associated with increased expression of the PDGF beta R. When the growth-arrested fibroblasts are stimulated to reenter the cell cycle by the mitogenic action of serum or certain specific combinations of growth factors, PDGF beta R mRNA levels and cell surface PDGF-BB-binding sites are markedly downregualted. Oncogene-transformed 3T3 cell lines, which fail to undergo growth arrest following prolonged serum deprivation, express constitutively low levels of the PDGF beta R mRNA and possess greatly reduced numbers of cell surface PDGF receptors, as determined by PDGF-BB binding and Western blotting (immunoblotting). Nuclear runoff assays indicate the mechanism of repression of PDGF beta R expression to be, at least in large part, transcriptional. These data indicate that expression of the PDGF beta R is regulated in a growth state-dependent manner in fibroblasts and suggest that this may provide a means by which cells can modulate their responsiveness to the actions of PDGF.

3T3 Cells↗
PubMed1995

Characterization of fus1 of Schizosaccharomyces pombe: a developmentally controlled function needed for conjugation.

In Schizosaccharomyces pombe, the fus1 mutation blocks conjugation at a point after cell contact and agglutination. The cell walls separating the mating partners are not degraded, which prevents cytoplasmic fusion. In order to investigate the molecular mechanism of conjugation, we cloned the fus1 gene and found that it is capable of encoding a 1,372-amino-acid protein with no significant similarities to other known proteins. Expression of the fus1 gene is regulated by the developmental state of the cells. Transcription is induced by nitrogen starvation and requires a pheromone signal in both P and M cell types. Consequently, mutants defective in the pheromone response pathway fail to induce fus1 expression. The ste11 gene, which encodes a transcription factor controlling expression of many genes involved in sexual differentiation, is also required for transcription of fus1. Furthermore, deletion of two potential Ste11 recognition sites in the fus1 promoter region abolished transcription, and expression could be restored when we inserted a different Ste11 site from the mat1-P promoter. Since this element was inverted relative to the fus1 element, we conclude that activation of transcription by Ste11 is independent of orientation. Although the fus1 mutant has a phenotype very similar to that of Saccharomyces cerevisiae fus1 mutants, the two proteins appear to have different roles in the process of cell fusion. Budding yeast Fus1 is a typical membrane protein and contains an SH3 domain. Fission yeast Fus1 has no features of a membrane protein, yet it appears to localize to the projection tip. A characteristic proline-rich potential SH3 binding site may mediate interaction with other proteins.

Amino Acid Sequence↗
PubMed2000

A role for the Hsp40 Ydj1 in repression of basal steroid receptor activity in yeast.

In addition to its roles in translocation of preproteins across membranes, Ydj1 facilitates the maturation of Hsp90 substrates, including mammalian steroid receptors, which activate transcription in yeast in a hormone-dependent manner. To better understand Ydj1's function, we have constructed and analyzed an array of Ydj1 mutants in vivo. Both the glucocorticoid receptor and the estrogen receptor exhibited elevated activity in the absence of hormone in all ydj1 mutant strains, indicating a strict requirement for Ydj1 activity in hormonal control. Glucocorticoid receptor containing a mutation in the carboxy-terminal transcriptional activation domain, AF-2, retained elevated basal activity, while mutation of the amino-terminal transactivation domain, AF-1, eliminated the elevated basal activity observed in ydj1 mutant strains. This result indicates that the source of activity is AF-1, which is normally repressed by the carboxy-terminal hormone binding domain in the absence of hormone. Chimeric proteins containing the hormone binding domain of the estrogen or glucocorticoid receptor fused to heterologous activation and DNA binding domains also exhibited elevated activity in the absence of hormone. Thus, Ydj1 mutants appear to increase basal receptor activity by altering the ability of the hormone binding domain of the receptor to repress nearby activation domains. We propose that Ydj1 functions to present steroid receptors to the Hsp90 pathway for folding and hormonal control. In the presence of Ydj1 mutants that fail to bind substrate efficiently, some receptor escapes the Hsp90 pathway, resulting in constitutive activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

The role of protein tyrosine kinases and protein tyrosine phosphatases in T cell antigen receptor signal transduction.

Engagement of the T cell antigen receptor (TCR) by peptide antigen bound to the major histocompatibility complex (MHC) molecules initiates a biochemical cascade involving protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Recent biochemical and genetic evidence has implicated at least three cytoplasmic protein tyrosine kinases (PTKs), Lck, Fyn, and ZAP-70, that are involved in the initiation of TCR signal transduction. In addition, genetic evidence has demonstrated the requirement of the transmembrane PTPase, CD45, for TCR function. Activation of T cells through the TCR represents an alteration in the dynamic equilibrium between PTKs and PTPases. The TCR is a multi-subunit complex composed of at least six different gene products. Dissection of the TCR utilizing chimeric receptors and TCR mutants has demonstrated that the multi-subunit receptor is composed of at least two signal transducing modules, the CD3 and the zeta chain subunits. These two modules have in common peptide sequences within their cytoplasmic domains termed antigen recognition activation motifs (ARAMs) that are responsible for transducing signaling events. Moreover, the ARAM sequence is also found in subunits associated with a variety of other hematopoietic cell antigen receptors and is likely to form the basis for interactions with effector molecules within the signaling cascades of these receptors. Here we review the mechanism by which the ARAM sequences interact with PTKs and the cascades of PTKs and PTPases that are involved in mediating TCR function.

Amino Acid Sequence↗
PubMed2001

Effect of dietary K intake on apical small-conductance K channel in CCD: role of protein tyrosine kinase.

We have used Western blot to examine the expression of cSrc protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP)-1D in the renal cortex, and the patch-clamp technique to determine the role of PTK in mediating the effect of dietary K intake on the small-conductance K (SK) channel in the cortical collecting duct (CCD). When rats were on a K-deficient (KD) diet for 1, 3, 5, and 7 days, the expression of cSrc increased by 40, 90, 140, and 135%, respectively. In contrast, the expression of cSrc in the renal cortex from rats on a high-K (HK) diet for 1, 2, and 3 days decreased by 40, 60, and 75%, respectively. However, the protein level of PTP-1D was not significantly changed by dietary K intake. The addition of 1 microM herbimycin A increased NP(o), a product of channel number (N) and open probability (P(o)) in the CCD from rats on a normal diet or on a KD diet. The increase in NP(o) was 0.30 (normal), 0.45 (1-day KD), 0.65 (3-day KD), 1.55 (5-day KD), and 1.85 (7-day KD), respectively. Treatment of the CCD with herbimycin A from rats on a KD diet increased NP(o) per patch from the control value (0.7) to 1.4 (1-day KD), 1.6 (3-day KD), 2.6 (5-day KD), and 3.5 (7-day KD), respectively. In contrast, HK intake for as short as 1 day abolished the effect of herbimycin A. Furthermore, the expression of ROMK channels in the renal cortex was the same between rats on a KD diet or on a HK diet. Moreover, treatment with herbimycin A did not further increase NP(o) in the CCDs from rats on a HK diet. We conclude that dietary K intake plays a key role in regulating the activity of the SK channels and that PTK is involved in mediating the effect of the K intake on channel activity in the CCD.

Animals↗
PubMed2004

Sensitization to the lysosomal cell death pathway upon immortalization and transformation.

Tumorigenesis is associated with several changes that alter the cellular susceptibility to programmed cell death. Here, we show that immortalization and transformation sensitize cells in particular to the cysteine cathepsin-mediated lysosomal death pathway. Spontaneous immortalization increased the susceptibility of wild-type murine embryonic fibroblasts (MEFs) to tumor necrosis factor (TNF)-mediated cytotoxicity >1000-fold, whereas immortalized MEFs deficient for lysosomal cysteine protease cathepsin B (CathB) retained the resistant phenotype of primary cells. This effect was specific for cysteine cathepsins, because also lack of cathepsin L (a lysosomal cysteine protease), but not that of cathepsin D (a lysosomal aspartyl protease) or caspase-3 (the major executioner protease in classic apoptosis) inhibited the immortalization-associated sensitization of MEFs to TNF. Oncogene-driven transformation of immortalized MEFs was associated with a dramatic increase in cathepsin expression and additional sensitization to the cysteine cathepsin-mediated death pathway. Importantly, exogenous expression of CathB partially reversed the resistant phenotype of immortalized CathB-deficient MEFs, and the inhibition of CathB activity by pharmacological inhibitors or RNA interference attenuated TNF-induced cytotoxicity in immortalized and transformed wild-type cells. Thus, tumorigenesis-associated changes in lysosomes may counteract cancer progression and enhance therapeutic responses by sensitizing cells to programmed cell death.

Animals↗
PubMed2003

The c-SRC1 gene visualized by in situ hybridization on Xenopus laevis chromosomes.

Fluorescent in situ hybridization followed by tyramide signal amplification was used to map the site of the c-SRC1 gene on XENOPUS LAEVIS chromosomes. Positive results were obtained with a cDNA probe of about 1 kb. The c-SRC1 gene is located in the subcentromeric region in the long arm of one of the acrocentric chromosomes of the G category (classified according to Graf and Kobel, 1991). The c-SRC1 gene and the XENOPUS major histocompatibility complex (MHC) 1b locus, which consists of 20 tandemly arranged gene copies, are situated on different chromosomes of the G category.

Animals↗
PubMed2000

Tyrosine kinase signalling in breast cancer.

Cells are continuously exposed to diverse stimuli ranging from soluble endocrine and paracrine factors to signalling molecules on neighbouring cells. Receptors of the tyrosine kinase family play an important role in the integration and interpretation of these external stimuli, allowing a cell to respond appropriately to its environment. The activation of receptor tyrosine kinases (RTKs) is tightly controlled, allowing a normal cell to correctly integrate its external environment with internal signal transduction pathways. In contrast, due to numerous molecular alterations arising during the course of malignancy, a tumour is characterized by an abnormal response to its environment, which allows cancer cells to evade the normal mechanisms controlling cellular proliferation. Alterations in the expression of various RTKs, in their activation, and in the signalling molecules lying downstream of the receptors play important roles in the development of cancer. This topic is the major focus of the thematic review section of this issue of Breast Cancer Research.

Breast Neoplasms↗
PubMed2000

Tyrosine kinase signalling in breast cancer: modulation of tyrosine kinase signalling in human breast cancer through altered expression of signalling intermediates.

The past decade has seen the definition of key signalling pathways downstream of receptor tyrosine kinases (RTKs) in terms of their components and the protein-protein interactions that facilitate signal transduction. Given the strong evidence that links signalling by certain families of RTKs to the progression of breast cancer, it is not surprising that the expression profile of key downstream signalling intermediates in this disease has also come under scrutiny, particularly because some exhibit transforming potential or amplify mitogenic signalling pathways when they are overexpressed. Reflecting the diverse cellular processes regulated by RTKs, it is now clear that altered expression of such signalling proteins in breast cancer may influence not only cellular proliferation (eg Grb2) but also the invasive properties of the cancer cells (eg EMS1/cortactin).

Adaptor Proteins, Signal Transducing↗
PubMed2006

Molecular epidemiologic evidence for diabetogenic effects of dioxin exposure in U.S. Air force veterans of the Vietnam war.

BACKGROUND: One of the outcomes positively associated with dioxin exposure in humans is type 2 diabetes. OBJECTIVES: This study was conducted in order to find the molecular biological evidence for the diabetogenic action of dioxin in adipose samples from Vietnam veterans. METHODS: We obtained 313 adipose tissue samples both from Vietnam veterans who were exposed to dioxin (Operation Ranch Hand) and from comparison veterans who served in Southeast Asia with no record of dioxin exposure. We conducted quantitative reverse-transcribed polymerase chain reaction studies on selected marker mRNAs from these samples. RESULTS: We found the most sensitive and reliable molecular indicator of dioxin-induced diabetes to be the ratio of mRNA of glucose transporter 4 (GLUT4) and nuclear transcription factor kappa B (NFkappaB), a marker of inflammation. This ratio showed significant correlations to serum dioxin residues and to fasting glucose among those in the Ranch Hand group and, surprisingly, even in the comparison group, who have low levels of dioxin comparable to the general public. Such a correlation in the comparison group was particularly significant among those with known risk factors such as obesity and family history of diabetes. CONCLUSIONS: These results show that the GLUT4:NFkappaB ratio is a reliable marker for the diabetogenic action of dioxin, particularly at very low exposure levels that are not much higher than those found in the general public, implying a need to address current exposure levels.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1998

TPA induces a block of differentiation and increases the susceptibility to neoplastic transformation of a rat thyroid epithelial cell line.

The PC Cl 3 cell line is a well-characterized epithelial cell line of rat thyroid origin. This cell line retains in vitro the typical markers of thyroid differentiation: thyroglobulin (TG) synthesis and secretion, iodide uptake, thyroperoxidase (TPO) expression, and dependency on TSH for growth. Although the differentiated phenotype of thyroid cells has been relatively well described, the molecular mechanisms that regulate both differentiation and neoplastic transformation of thyroid cells still need to be investigated in detail. Protein kinase C (PKC), the target of tetradecanoylphorbol acetate (TPA), regulates growth and differentiation of several cell types. Here we show that treatment of PC Cl 3 cells with TPA induces an acute block of thyroid differentiation. TPA-treated PC Cl 3 cells are unable to trap iodide and the expression levels of thyroglobulin, TSH receptor, and TPO genes are drastically reduced by TPA treatment. This differentiation block is not caused by a reduced expression of one of the master genes of thyroid differentiation, the thyroid transcription factor 1 (TTF-1). TPA-treated PC Cl 3 cells display an increased growth rate indicating that, in addition to the differentiation block, TPA also significantly affects the growth regulation of thyroid cells. Finally, TPA treatment dramatically increases the number of transformation foci induced in PC Cl 3 cells by retroviruses carrying v-Ki-ras, v-Ha-ras, and v-mos oncogenes. These findings support the notion that the PKC pathway can influence proliferation, differentiation, and neoplastic transformation of thyroid cells in culture.

Animals↗
PubMed1999

cSrc is a major cytosolic tyrosine kinase in vascular tissue.

We are interested in identifying, in vascular tissue, nonreceptor tyrosine kinases that may be responsible for the contractile actions of G-protein-coupled agonists such as angiotensin II. By using a series of chromatographic steps, including ion exchange, hydrophobic, and affinity chromatography, we have isolated a major fraction of tyrosine kinase activity from the cytosolic fraction of porcine aorta tissue. According to (i) its immunologic cross-reactivity with the monoclonal anti-cSrc antibody, m327, and with the N-terminally directed monoclonal cSrc2-17 antibody, (ii) its inhibition by the C-terminal cSrc kinase, CSK, and (iii) its specificity for phosphorylating tyrosine 15 in the cdc2(6-20) peptide kinase substrate, we conclude that the kinase we have isolated represents porcine cSrc. A substantial proportion of the enzyme (>70%) was recovered in the cytoplasmic fraction from aorta tissue. The profile of inhibition of the human and porcine cSrc enzymes by a spectrum of tyrosine kinase inhibitors (PP1 >> AG82 > AG490 approximately/= genistein > AG10) was compared with the profile of inhibition of angiotensin II mediated contraction in a porcine coronary vascular preparation (AG10 >> genistein > or = AG82 > or = AG490; PP1 inactive). The different inhibitory profiles indicated that cSrc does not represent the vascular tyrosine kinase responsible for the contractile actions of angiotensin II. We suggest, nonetheless, that cSrc plays a key role for other actions of angiotensin II in intact vascular tissue, such as the regulation of mitogen-activated protein kinase activity and gene transcription.

Amino Acid Sequence↗
PubMed2001

[Tumor markers for colorectal cancer].

CEA and CA19-9 are the two most common tumor markers for colorectal cancer that are currently utilized clinically. The positive rate of CEA is 40-60% and that of CA19-9 is 30-50%. Simultaneous use of the two markers is useful in evaluating the therapeutic effect and monitoring the recurrence of advanced colorectal cancer. Surgical specimens may also provide useful information for the appropriate treatment of patients. Using surgically resected lymph nodes, we examined micrometastasis to assess the spread of the cancer cells and the malignant potential of colorectal cancer. Immunohistochemical analysis using anti-cytokeratin antibody revealed no significant impact of micrometastasis on patient prognosis, while RT-PCR assay using CEA as a genetic marker suggested a positive value in predicting a rapid recurrence. Among various molecular markers, we found that CDC25B phosphatase was a powerful prognostic factor for colorectal cancer. Diagnosis of the existence and malignant potential of cancer cells, together with serum tumor marker levels, may help to construct a more useful system for the better treatment of colorectal cancer.

Antigens, Neoplasm↗
PubMed1992

Multilineage phenotypes of interleukin-3-dependent progenitor cells.

Interleukin-3 (IL-3)-dependent murine FDCP-mix cells have multilineage differentiation capacity; they are nonleukemic, have a normal karyotype, and are nonimmortalized. These cells coexpress on their cell surface the "early" B-lineage marker B220/CD45R and the myeloid marker Mac-1/iC3b receptor (CR3), transcribe germline T-cell receptor gamma genes, and express the macrophage lineage growth factor receptor gene c-fms as a predominant 8.4-kb transcript. They do not detectably express at the stable mRNA or protein level other lymphoid precursor cell genes including CD2, TdT, lambda 5, and BP1. Induction of granulocyte/macrophage differentiation in these cells closes down expression of the lymphoid genes and activates stable expression of genes specific to the myeloid lineage, including myeloperoxidase. Expression of the c-fms gene at the mRNA level is upregulated and the dominant stable transcript is now in the 4.1-kb form typical of the macrophage lineage. These data provide a plausible explanation for the coexpression of lymphoid and myeloid lineage markers on human leukemic cells of stem cell or progenitor cell origin and have implications for the programming of lineage potential in normal multipotential hematopoiteic progenitor cells.

Animals↗