Nuclear localization of phosphatidylinositol 4-kinase β

2002 ◽  
Vol 115 (8) ◽  
pp. 1769-1775 ◽  
Author(s):  
Petra de Graaf ◽  
Elsa E. Klapisz ◽  
Thomas K. F. Schulz ◽  
Alfons F. M. Cremers ◽  
Arie J. Verkleij ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Kinase Activity ◽  
Insoluble Fraction ◽  
Type Ii ◽  
3T3 Cells ◽  
Type Iii ◽  
Pore Complexes ◽  
Phosphatidylinositol 4 ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Whereas most phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity is localized in the cytoplasm, PtdIns 4-kinase activity has also been detected in membranedepleted nuclei of rat liver and mouse NIH 3T3 cells. Here we have characterized the PtdIns 4-kinase that is present in nuclei from NIH 3T3 cells. Both type II and type III PtdIns 4-kinase activity were observed in the detergent-insoluble fraction of NIH 3T3 cells. Dissection of this fraction into cytoplasmic actin filaments and nuclear lamina-pore complexes revealed that the actin filament fraction contains solely type II PtdIns 4-kinase,whereas lamina-pore complexes contain type III PtdIns 4-kinase activity. Using specific antibodies, the nuclear PtdIns 4-kinase was identified as PtdIns 4-kinase β. Inhibition of nuclear export by leptomycin B resulted in an accumulation of PtdIns 4-kinase β in the nucleus. These data demonstrate that PtdIns 4-kinase β is present in the nuclei of NIH 3T3 fibroblasts,suggesting a specific function for this kinase in nuclear processes.

scholarly journals Recombinant Keratinocyte Growth Factor 1 in Tobacco Potentially Promotes Wound Healing in Diabetic Rats

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Zhi-Guo Feng ◽  
Shi-Feng Pang ◽  
Ding-Jiong Guo ◽  
Yue-Tao Yang ◽  
Bin Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Keratinocyte Growth Factor ◽  
Diabetic Rats ◽  
Diabetic Rat ◽  
Type Ii ◽  
3T3 Cells ◽  
Tobacco Plants ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Keratinocyte growth factor 1 (KGF1) is a growth factor that promotes epidermal cell proliferation, migration, differentiation, and wound repair. It is expressed at low levels in a form of inclusion body inE. coli.In order to increase its expression and activity, we produced tobacco plants expressing KGF1 viaAgrobacterium-mediatedtransformation using apotato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the KGF1 gene fused with a green florescence protein. The recombinant plasmid was introduced into leaf cells ofNicotiana benthamiana(a wild Australian tobacco) viaAgrobacterium-mediatedagroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the KGF1 gene was correctly translated into the tobacco plants. The recombinant KGF1 was purified from plant tissues by heparin affinity chromatography, and cell proliferation in NIH/3T3 cells was stimulated by the purified KGF1. The purified KGF1 was also applied to the wounds of type-II diabetic rats. KGF1 had accumulated to levels as high as 530 μg/g fresh weight in the leaves of agroinfected plants. We show that plant-derived KGF1 can promote the proliferation of NIH/3T3 cells and have significant effects on the type-II diabetic rat. The present findings indicated that KGF1 from tobacco maintains its biological activity, implying prospective industrial production in a plant bioreactor.

Elevated phosphocholine concentration in ras-transformed NIH 3T3 cells arises from increased choline kinase activity, not from phosphatidylcholine breakdown

1989 ◽  
Vol 9 (1) ◽  
pp. 325-328
Author(s):  
I G Macara
Keyword(s):  
Phospholipase C ◽  
Kinase Activity ◽  
Transformed Cells ◽  
Choline Kinase ◽  
3T3 Cells ◽  
Cellular Concentration ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

The cellular concentration of phosphocholine has been reported to be significantly elevated in Ha-ras-transformed NIH 3T3 cells, but not in v-sis transformants (J. C. Lacal, J. Moscat, and S. A. Aaronson, Nature [London] 330:269-271, 1987). It was suggested that the phosphocholine arises from constitutive hydrolysis of phosphatidylcholine by phospholipase C, an activity that would also account for the elevated 1,2-diacylglycerol found in ras-transformed cells. I have demonstrated that the increased phosphocholine arises through the induction of choline kinase activity. No increased breakdown of phosphatidylcholine was observed in ras-transformed cells. The elevation in diacylglycerol is therefore unlikely to be a consequence of phosphatidylinositol or phosphatidylcholine turnover.

scholarly journals Heme Induces Ubiquitination and Degradation of the Transcription Factor Bach1

2007 ◽  
Vol 27 (19) ◽  
pp. 6962-6971 ◽  
Author(s):  
Yukari Zenke-Kawasaki ◽  
Yoshihiro Dohi ◽  
Yasutake Katoh ◽  
Tsuyoshi Ikura ◽  
Masae Ikura ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Regulatory Protein ◽  
Heme Oxygenase 1 ◽  
3T3 Cells ◽  
Embryonic Fibroblasts ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
Nih 3T3 ◽  
Murine Erythroleukemia Cells ◽  
Nih 3T3 Cells

ABSTRACT The transcription repressor Bach1 is a sensor and effector of heme that regulates the expression of heme oxygenase 1 and globin genes. Heme binds to Bach1, inhibiting its DNA binding activity and inducing its nuclear export. We found that hemin further induced the degradation of endogenous Bach1 in NIH 3T3 cells, murine embryonic fibroblasts, and murine erythroleukemia cells. In contrast, succinylacetone, an inhibitor of heme synthesis, caused accumulation of Bach1 in murine embryonic fibroblasts, indicating that physiological levels of heme regulated the Bach1 turnover. Polyubiquitination and rapid degradation of overexpressed Bach1 were induced by hemin treatment. HOIL-1, an ubiquitin-protein ligase which recognizes heme-bound, oxidized iron regulatory protein 2, was found to bind with Bach1 when both were overexpressed in NIH 3T3 cells. HOIL-1 stimulated the polyubiquitination of Bach1 in a purified in vitro ubiquitination system depending on the intact heme binding motifs of Bach1. Expression of dominant-negative HOIL-1 in murine erythroleukemia cells resulted in higher stability of endogenous Bach1, raising the possibility that the heme-regulated degradation involved HOIL-1 in murine erythroleukemia cells. These results suggest that heme within a cell regulates the polyubiquitination and degradation of Bach1.

Mechanisms of action in NIH-3T3 cells of genistein, an inhibitor of EGF receptor tyrosine kinase activity

1990 ◽  
Vol 39 (1) ◽  
pp. 187-193 ◽  
Author(s):  
Claude Linassier ◽  
Michel Pierre ◽  
Jean-Bernard Le Pecq ◽  
Josiane Pierre
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Mechanisms Of Action ◽  
3T3 Cells ◽  
Tyrosine Kinase Activity ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals mos gene transforming efficiencies correlate with oocyte maturation and cytostatic factor activities.

1991 ◽  
Vol 11 (2) ◽  
pp. 604-610 ◽  
Author(s):  
N Yew ◽  
M Oskarsson ◽  
I Daar ◽  
D G Blair ◽  
G F Vande Woude
Keyword(s):  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Kinase Activity ◽  
Vertebrate Species ◽  
3T3 Cells ◽  
Transforming Activity ◽  
Cytostatic Factor ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The mos proto-oncogenes from different vertebrate species transform mouse NIH 3T3 cells with markedly different efficiencies. v-mos, mouse (c-mosmu), and chicken (c-mosch) mos transform NIH 3T3 cells 10- to 100-fold more efficiently than do human (c-moshu) and Xenopus (c-mosxc) mos. The mos genes with the highest transforming activity efficiently induce maturation in Xenopus oocytes and mimic cytostatic factor (CSF) by causing mitotic cleavage arrest in embryos. Chimeric v-mos/c-moshu proteins that had high transforming efficiencies in NIH 3T3 cells were also effective in the induction of oocyte maturation and CSF cleavage arrest. We measured the in vitro autophosphorylation activities of the different mos proteins and found that the levels of kinase activity of v-mos, c-mosmu, and c-mosch were much higher than that of c-mosxc. These data indicate that mos gene transforming efficiency and the ability to induce oocyte maturation or mimic CSF activity are correlated with in vitro autophosphorylation activity and suggest that the mos protein plays a similar role in transformed cells and normal oocytes.

scholarly journals Elevated phosphocholine concentration in ras-transformed NIH 3T3 cells arises from increased choline kinase activity, not from phosphatidylcholine breakdown.

1989 ◽  
Vol 9 (1) ◽  
pp. 325-328 ◽  
Author(s):  
I G Macara
Keyword(s):  
Phospholipase C ◽  
Kinase Activity ◽  
Transformed Cells ◽  
Choline Kinase ◽  
3T3 Cells ◽  
Cellular Concentration ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

The cellular concentration of phosphocholine has been reported to be significantly elevated in Ha-ras-transformed NIH 3T3 cells, but not in v-sis transformants (J. C. Lacal, J. Moscat, and S. A. Aaronson, Nature [London] 330:269-271, 1987). It was suggested that the phosphocholine arises from constitutive hydrolysis of phosphatidylcholine by phospholipase C, an activity that would also account for the elevated 1,2-diacylglycerol found in ras-transformed cells. I have demonstrated that the increased phosphocholine arises through the induction of choline kinase activity. No increased breakdown of phosphatidylcholine was observed in ras-transformed cells. The elevation in diacylglycerol is therefore unlikely to be a consequence of phosphatidylinositol or phosphatidylcholine turnover.

scholarly journals Identification of Raf-1 Ser621 kinase activity from NIH 3T3 cells as AMP-activated protein kinase

FEBS Letters ◽  
1997 ◽  
Vol 403 (3) ◽  
pp. 254-258 ◽  
Author(s):  
Amy B Sprenkle ◽  
Stephen P Davies ◽  
David Carling ◽  
D.Grahame Hardie ◽  
Thomas W Sturgill
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
3T3 Cells ◽  
Amp Activated Protein Kinase ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals Phosphatidylinositol 3-kinase binds to α-actinin through the p85 subunit

1994 ◽  
Vol 302 (2) ◽  
pp. 551-557 ◽  
Author(s):  
F Shibasaki ◽  
K Fukami ◽  
Y Fukui ◽  
T Takenawa
Keyword(s):  
Signal Transduction ◽  
Kinase Activity ◽  
Cytoskeletal Reorganization ◽  
Phosphatidylinositol 3 Kinase ◽  
3T3 Cells ◽  
Rich Region ◽  
Phosphatidylinositol 4 ◽  
Nih 3T3 ◽  
Proline Rich Region ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to play an important role in the signal transduction of cell growth. It is also suggested that it is involved in cytoskeletal reorganization. We have found that alpha-actinin copurifies with PI 3-kinase from bovine thymus. The antibody against PI 3-kinase 85 kDa subunit (p85) also co-immunoprecipitates alpha-actinin from lysates of NIH/3T3 cells. In addition, anti-alpha-actinin antibody coprecipitates PI 3-kinase activity. This coprecipitation was observed even after depolymerization of actin fibres, suggesting that PI 3-kinase binds directly to alpha-actinin. As alpha-actinin is a phosphatidylinositol 4,5-bisphosphate (PI4,5P2)-binding protein, binding experiments using various constructs of truncated p85 were carried out in the presence or absence of PI4,5P2. In the absence of PI4,5P2, chicken gizzard alpha-actinin binds only to the whole p85 construct, but it binds to the proline-rich region of p85 fragments in the presence of PI4,5P2. This binding is enhanced with increased concentrations of Pi4,5P2 up to 10 microM, whereas phosphatidylinositol and phosphatidylinositol 4-phosphate were not good activators of alpha-actinin binding. These results suggest that PI 3-kinase binds to alpha-actinin and regulates cytoskeletal reorganization.

scholarly journals Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

1994 ◽  
Vol 14 (9) ◽  
pp. 5961-5974 ◽  
Author(s):  
Beth B. Koenig ◽  
Jonathan S. Cook ◽  
Dana Hance Wolsing ◽  
Jerry Ting ◽  
Jay P. Tiesman ◽  
...  
Keyword(s):  
Transforming Growth Factor Beta ◽  
Binding Sites ◽  
Transforming Growth Factor ◽  
Type I ◽  
Type Ii ◽  
Tgf Beta ◽  
3T3 Cells ◽  
Related Factors ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs.

mos gene transforming efficiencies correlate with oocyte maturation and cytostatic factor activities

1991 ◽  
Vol 11 (2) ◽  
pp. 604-610
Author(s):  
N Yew ◽  
M Oskarsson ◽  
I Daar ◽  
D G Blair ◽  
G F Vande Woude
Keyword(s):  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Kinase Activity ◽  
Vertebrate Species ◽  
3T3 Cells ◽  
Transforming Activity ◽  
Cytostatic Factor ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The mos proto-oncogenes from different vertebrate species transform mouse NIH 3T3 cells with markedly different efficiencies. v-mos, mouse (c-mosmu), and chicken (c-mosch) mos transform NIH 3T3 cells 10- to 100-fold more efficiently than do human (c-moshu) and Xenopus (c-mosxc) mos. The mos genes with the highest transforming activity efficiently induce maturation in Xenopus oocytes and mimic cytostatic factor (CSF) by causing mitotic cleavage arrest in embryos. Chimeric v-mos/c-moshu proteins that had high transforming efficiencies in NIH 3T3 cells were also effective in the induction of oocyte maturation and CSF cleavage arrest. We measured the in vitro autophosphorylation activities of the different mos proteins and found that the levels of kinase activity of v-mos, c-mosmu, and c-mosch were much higher than that of c-mosxc. These data indicate that mos gene transforming efficiency and the ability to induce oocyte maturation or mimic CSF activity are correlated with in vitro autophosphorylation activity and suggest that the mos protein plays a similar role in transformed cells and normal oocytes.

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