Human spectrin Src homology 3 domain binding protein 1 regulates macropinocytosis in NIH 3T3 cells

2000 ◽  
Vol 113 (21) ◽  
pp. 3805-3814 ◽  
Author(s):  
J. Xu ◽  
D. Ziemnicka ◽  
G.S. Merz ◽  
L. Kotula
Keyword(s):  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Binding Protein ◽  
Sh3 Domain ◽  
Water Soluble ◽  
Protein Markers ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Macropinocytosis is an endocytic process that occurs through non-clathrin coated vesicles larger than 0.2 microm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates with macropinosomes in NIH 3T3 fibroblasts. Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid alpha-spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.

scholarly journals The histone acetyltransferases CBP/p300 are degraded in NIH 3T3 cells by activation of Ras signalling pathway

2006 ◽  
Vol 398 (2) ◽  
pp. 215-224 ◽  
Author(s):  
Sara Sánchez-Molina ◽  
José Luis Oliva ◽  
Susana García-Vargas ◽  
Ester Valls ◽  
José M. Rojas ◽  
...  
Keyword(s):  
Binding Protein ◽  
Camp Response Element Binding ◽  
Signalling Pathway ◽  
3T3 Cells ◽  
Protein Levels ◽  
3T3 Fibroblasts ◽  
Differentiation And Proliferation ◽  
Nih 3T3 ◽  
Ras Signalling ◽  
Nih 3T3 Cells

The CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300 acetyltransferases function as transcriptional co-activators and play critical roles in cell differentiation and proliferation. Accumulating evidence shows that alterations of the CBP/p300 protein levels are linked to human tumours. In the present study, we show that the levels of the CBP/p300 co-activators are decreased dramatically by continuous PDGF (platelet-derived growth factor) and Ras signalling pathway activation in NIH 3T3 fibroblasts. This effect occurs by reducing the expression levels of the CBP/p300 genes. In addition, CBP and p300 are degraded by the 26 S proteasome pathway leading to an overall decrease in the levels of the CBP/p300 proteins. Furthermore, we provide evidence that Mdm2 (murine double minute 2), in the presence of active H-Ras or N-Ras, induces CBP/p300 degradation in NIH 3T3 cells. These findings support a novel mechanism for modulating other signalling transduction pathways that require these common co-activators.

Potent transforming activity of the small GTP-binding protein Rit in NIH 3T3 cells: evidence for a role of a p38γ-dependent signaling pathway

FEBS Letters ◽  
2001 ◽  
Vol 511 (1-3) ◽  
pp. 15-20 ◽  
Author(s):  
Kaoru Sakabe ◽  
Hidemi Teramoto ◽  
Muriel Zohar ◽  
Babak Behbahani ◽  
Hiroshi Miyazaki ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Binding Protein ◽  
3T3 Cells ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Dependent Signaling Pathway ◽  
Nih 3T3 Cells

Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain

1994 ◽  
Vol 14 (12) ◽  
pp. 7943-7952
Author(s):  
R R Mattingly ◽  
A Sorisky ◽  
M R Brann ◽  
I G Macara
Keyword(s):  
Muscarinic Receptor ◽  
Muscarinic Receptors ◽  
Cellular Transformation ◽  
Sh3 Domain ◽  
Gtpase Activating Protein ◽  
3T3 Cells ◽  
Focus Formation ◽  
Ras Gtpase ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected.

scholarly journals Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.

1994 ◽  
Vol 14 (12) ◽  
pp. 7943-7952 ◽  
Author(s):  
R R Mattingly ◽  
A Sorisky ◽  
M R Brann ◽  
I G Macara
Keyword(s):  
Muscarinic Receptor ◽  
Muscarinic Receptors ◽  
Cellular Transformation ◽  
Sh3 Domain ◽  
Gtpase Activating Protein ◽  
3T3 Cells ◽  
Focus Formation ◽  
Ras Gtpase ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected.

scholarly journals Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin

1994 ◽  
Vol 124 (5) ◽  
pp. 757-768 ◽  
Author(s):  
K Takenaga ◽  
Y Nakamura ◽  
S Sakiyama ◽  
Y Hasegawa ◽  
K Sato ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
S100 Protein ◽  
Calcium Binding Protein ◽  
Immunofluorescent Staining ◽  
Dependent Manner ◽  
3T3 Cells ◽  
Cell Extracts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The cDNA coding for mouse fibroblast tropomyosin isoform 2 (TM2) was placed into a bacterial expression vector to produce a fusion protein containing glutathione-S-transferase (GST) and TM2 (GST/TM2). Glutathione-Sepharose beads bearing GST/TM2 were incubated with [35S]methionine-labeled NIH 3T3 cell extracts and the materials bound to the fusion proteins were analyzed to identify proteins that interact with TM2. A protein of 10 kD was found to bind to GST/TM2, but not to GST. The binding of the 10-kD protein to GST/TM2 was dependent on the presence of Ca2+ and inhibited by molar excess of free TM2 in a competition assay. The 10-kD protein-binding site was mapped to the region spanning residues 39-107 on TM2 by using several COOH-terminal and NH2-terminal truncation mutants of TM2. The 10-kD protein was isolated from an extract of NIH 3T3 cells transformed by v-Ha-ras by affinity chromatography on a GST/TM2 truncation mutant followed by SDS-PAGE and electroelution. Partial amino acid sequence analysis of the purified 10-kD protein, two-dimensional polyacrylamide gel analysis and a binding experiment revealed that the 10-kD protein was identical to a calcium-binding protein derived from mRNA named pEL98 or 18A2 that is homologous to S100 protein. Immunoblot analysis of the distribution of the 10-kD protein in Triton-soluble and -insoluble fractions of NIH 3T3 cells revealed that some of the 10-kD protein was associated with the Triton-insoluble cytoskeletal residue in a Ca(2+)-dependent manner. Furthermore, immunofluorescent staining of NIH 3T3 cells showed that some of the 10-kD protein colocalized with nonmuscle TMs in microfilament bundles. These results suggest that some of the pEL98 protein interacts with microfilament-associated nonmuscle TMs in NIH 3T3 cells.

scholarly journals Fibroblast growth factor-1 stimulation of quiescent NIH 3T3 cells increases G/T mismatch-binding protein expression

1996 ◽  
Vol 319 (1) ◽  
pp. 9-12 ◽  
Author(s):  
Patrick J. DONOHUE ◽  
Sheau-Line Y. FENG ◽  
Gregory F ALBERTS ◽  
Yan GUO ◽  
Kimberly A PEIFLEY ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Binding Protein ◽  
Calf Serum ◽  
Mrna Levels ◽  
Fibroblast Growth ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Polypeptide growth factors promote cell-cycle progression in part by the transcriptional activation of a diverse group of specific genes. We have used an mRNA differential-display approach to identify several fibroblast growth factor (FGF)-1 (acidic FGF)-inducible genes in NIH 3T3 cells. Here we report that one of these genes, called FGF-regulated (FR)-3, is predicted to encode G/T mismatch-binding protein (GTBP), a component of the mammalian DNA mismatch correction system. The murine GTBP gene is transiently expressed after FGF-1 or calf serum treatment, with maximal mRNA levels detected at 12 and 18 h post-stimulation. FGF-1-stimulated NIH 3T3 cells also express an increased amount of GTBP as determined by immunoblot analysis. These results indicate that elevated levels of GTBP may be required during the DNA synthesis phase of the cell cycle for efficient G/T mismatch recognition and repair.

Interaction of antiflammin-1 with uteroglobin-binding protein induces phosphorylation of ERK1/2 in NIH 3T3 cells

Peptides ◽  
2007 ◽  
Vol 28 (11) ◽  
pp. 2137-2145 ◽  
Author(s):  
Chen Li ◽  
Jianzhong Han ◽  
Lian Li ◽  
Shaojie Yue ◽  
Jinfeng Li ◽  
...  
Keyword(s):  
Binding Protein ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals Structural and functional characterization of the mouse p8 gene: promotion of transcription by the CAAT-enhancer binding protein α (C/EBPα) and C/EBPβ trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter

1999 ◽  
Vol 343 (2) ◽  
pp. 377-383 ◽  
Author(s):  
Sophie VASSEUR ◽  
Gustavo Vidal MALLO ◽  
Andrés GARCIA-MONTERO ◽  
Emilia Mariana ORTIZ ◽  
Fritz FIEDLER ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Binding Protein ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Expression Vectors ◽  
3T3 Cells ◽  
Cis Acting ◽  
Enhancer Binding Protein ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Rat p8 mRNA was discovered because of its strong activation in pancreas during the acute phase of pancreatitis. We report here structural and functional data on the mouse p8 gene. The mouse p8 polypeptide is 80 amino acids long and shows 91% and 75% identity with its rat and human counterparts respectively. The p8 gene is organized into three exons interrupted by two introns. Promoter regions involved in the regulation of p8 gene expression in NIH 3T3 cells were analysed. Chloramphenicol acetyltransferase (CAT) reporter assays with progressive deletions of the 5′ flanking region of the mouse p8 gene revealed four silencer elements located from nucleotides -5000 to -1472, -1471 to -671, -670 to -473, and -239 to -117 respectively. One positive element was identified between nucleotides -117 and -72. We identified a CAAT-enhancer binding protein (C/EBP) cis-acting element at position -111. Site-directed mutagenesis of this consensus site decreased promoter activity to 5% of that of the wild-type. An electrophoretic mobility-shift assay, using an oligonucleotide probe corresponding to the C/EBP consensus and nuclear extracts of NIH 3T3 cells transfected with C/EBPα or C/EBPβ expression vectors, generated specific DNA-protein complexes that were supershifted with specific antibodies against C/EBPα and C/EBPβ. Co-transfection with C/EBPα or C/EBPβ expression vectors and the p-116/+36p8-CAT construct increased the reporter gene activity in a dose-dependent fashion. Surprisingly, overexpression of C/EBPα or C/EBPβ still increased the promoter activity of both pC/EBPmut-116/+36p8-CAT (which contains the C/EBP mutated site) and the p-71/+36-CAT construct (which does not contain the C/EBP site). Collectively, these results show that C/EBPα and C/EBPβ trans-acting factors can promote transcription of the mouse p8 gene (i) by direct binding to the C/EBP consensus site, and (ii) by enhancing the activity of other trans-acting factors interacting with the p8 promoter.

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