scholarly journals rab5 GTPase Regulates Adenovirus Endocytosis

1999 ◽  
Vol 73 (11) ◽  
pp. 9664-9668 ◽  
Author(s):  
Tanja Rauma ◽  
Juha Tuukkanen ◽  
Jeffrey M. Bergelson ◽  
Gerene Denning ◽  
Timo Hautala
Keyword(s):  
Tyrosine Phosphorylation ◽  
Hela Cells ◽  
Intracellular Signaling ◽  
Virus Entry ◽  
Dominant Negative ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

ABSTRACT Adenovirus interaction with αv integrins is important for virus entry. We have examined the effects of adenovirus attachment on intracellular signaling in HeLa cells, with an emphasis on pathways known to be activated following integrin interaction with other ligands. We found no evidence for [Ca2+]c-mediated signaling or for tyrosine phosphorylation of pp125FAK, p130CAS, and paxillin. However, adenovirus attachment is known to activate phosphatidylinositol-3 kinase, which in turn may regulate endocytosis via rab5 GTPase. We found that adenovirus uptake was increased by overexpression of wild-type rab5 and decreased by dominant-negative rab5. These results indicate a role for rab5 in adenovirus entry.

Erythropoietin Induces the Tyrosine Phosphorylation of GAB1 and Its Association With SHC, SHP2, SHIP, and Phosphatidylinositol 3-Kinase

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2578-2585 ◽  
Author(s):  
Carinne Lecoq-Lafon ◽  
Frédérique Verdier ◽  
Serge Fichelson ◽  
Stany Chrétien ◽  
Sylvie Gisselbrecht ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Direct Consequence ◽  
Receptor Activation ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitors ◽  
Epo Receptor ◽  
Gm Csf ◽  
Macrophage Colony ◽  
Phosphatidylinositol 3

Abstract Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells. The 180- and 70-kD proteins have been previously shown to be IRS2 and the Epo receptor. In this report, we show that the 116-kD protein is the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics and Epo dose-response experiments showed that GAB1 tyrosine phosphorylation was a direct consequence of Epo receptor activation. After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase, the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1 was also associated with the molecular adapter GRB2 in unstimulated cells, and this association dramatically increased after Epo stimulation. Thus, GAB1 could be a scaffold protein able to couple the Epo receptor activation with the stimulation of several intracellular signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1 in UT-7 cells, indicating that this molecule participates in the signal transduction of several cytokine receptors.

Erythropoietin Induces the Tyrosine Phosphorylation of GAB1 and Its Association With SHC, SHP2, SHIP, and Phosphatidylinositol 3-Kinase

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2578-2585 ◽  
Author(s):  
Carinne Lecoq-Lafon ◽  
Frédérique Verdier ◽  
Serge Fichelson ◽  
Stany Chrétien ◽  
Sylvie Gisselbrecht ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Direct Consequence ◽  
Receptor Activation ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitors ◽  
Epo Receptor ◽  
Gm Csf ◽  
Macrophage Colony ◽  
Phosphatidylinositol 3

Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells. The 180- and 70-kD proteins have been previously shown to be IRS2 and the Epo receptor. In this report, we show that the 116-kD protein is the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics and Epo dose-response experiments showed that GAB1 tyrosine phosphorylation was a direct consequence of Epo receptor activation. After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase, the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1 was also associated with the molecular adapter GRB2 in unstimulated cells, and this association dramatically increased after Epo stimulation. Thus, GAB1 could be a scaffold protein able to couple the Epo receptor activation with the stimulation of several intracellular signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1 in UT-7 cells, indicating that this molecule participates in the signal transduction of several cytokine receptors.

scholarly journals Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase–dependent Activation of Phospholipase C-γ1

2005 ◽  
Vol 16 (7) ◽  
pp. 3236-3246 ◽  
Author(s):  
Zhongjian Xie ◽  
Patrick A. Singleton ◽  
Lilly Y.W. Bourguignon ◽  
Daniel D. Bikle
Keyword(s):  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Dominant Negative ◽  
Keratinocyte Differentiation ◽  
Regulatory Subunit ◽  
Alternative Mechanism ◽  
Phosphatidylinositol 3 Kinase ◽  
Differentiation Markers ◽  
Human Keratinocyte ◽  
Phosphatidylinositol 3

We have previously demonstrated that phospholipase C (PLC)-γ1 is required for calcium-induced human keratinocyte differentiation. In the present study, we investigated whether the activation of PLC-γ1 by nonreceptor kinases such as src and fyn plays a role in mediating this process. Our results showed that the combination of dominant negative src and fyn blocked calcium-stimulated PLC-γ1 activity and human keratinocyte differentiation, whereas each separately has little effect. However, unlike the activation of PLC-γ1 by epidermal growth factor, calcium-induced activation of PLC-γ1 was not a result of direct tyrosine phosphorylation. Therefore, we examined an alternative mechanism, in particular phosphatidylinositol 3,4,5-triphosphate (PIP3) formed as a product of phosphatidylinositol 3-kinase (PI3K) activity. PIP3 binds to and activates PLC-γ1. The combination of dominant negative src and fyn blocked calcium-induced tyrosine phosphorylation of the regulatory subunit of PI3K, p85α, and the activity of the catalytic subunit of PI3K. PI3K inhibitors blocked calcium activation of PLC-γ1 as well as the induction of keratinocyte differentiation markers involucrin and transglutaminase. These data indicate that calcium activates PLC-γ1 via increased PIP3 formation mediated by c-src– and fyn-activated PI3K. This activation is required for calcium-induced human keratinocyte differentiation.

scholarly journals Phosphatidylinositol 3-Kinase Activity Is Required for Epidermal Growth Factor to Suppress Proteolysis

2002 ◽  
Vol 13 (4) ◽  
pp. 903-909
Author(s):  
Harold A. Franch ◽  
Xiaonan Wang ◽  
Sira Sooparb ◽  
Nikia S. Brown ◽  
Jie Du
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Intracellular Signaling ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Protein Breakdown ◽  
Phosphatidylinositol 3 Kinase ◽  
Class 1 ◽  
Epidermal Growth ◽  
Phosphatidylinositol 3

ABSTRACT. Suppression of protein breakdown occurs commonly in cell growth, but the pathways responsible for controlling proteolysis are poorly understood. Protein breakdown in NRK-52E renal epithelial cells treated with epidermal growth factor (EGF) and intracellular signaling inhibitors or dominant negative signaling molecules contained in an adenoviral vector were measured. The tyrosine kinase inhibitor, herbimycin A, eliminated the suppression of proteolysis induced by EGF. In contrast, the Src inhibitor, PP1, had no effect. Expression of dominant negative H-RasY57 blocked the ability of EGF to stimulate downstream targets of Ras and also reduced the ability of EGF to suppress proteolysis. Inhibiting MEK did not influence the ability of EGF to suppress proteolysis, but the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY249002, stimulated basal proteolysis and completely eliminated the proteolytic response to EGF. Use of an adenovirus that expresses a dominant negative p85 subunit of class 1 PI 3-kinase completely blocked the ability of EGF to suppress proteolysis, whereas use of an adenovirus expressing a K227E constitutively active p110 subunit reproduced the reduction in protein breakdown. It was concluded that EGF suppresses proteolysis by a mechanism that involves Ras and class 1 PI 3-kinase.

Peptide antisera to human colony-stimulating factor 1 receptor detect ligand-induced conformational changes and a binding site for phosphatidylinositol 3-kinase

1991 ◽  
Vol 11 (5) ◽  
pp. 2489-2495
Author(s):  
J R Downing ◽  
S A Shurtleff ◽  
C J Sherr
Keyword(s):  
Binding Site ◽  
Tyrosine Phosphorylation ◽  
Stable Complex ◽  
Colony Stimulating Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Vitro Assay ◽  
Juxtamembrane Region ◽  
Stimulating Factor ◽  
Phosphatidylinositol 3

A peptide antiserum (anti-A) directed to the intracellular, juxtamembrane region (residues 552 to 574) of the human colony-stimulating factor 1 receptor (CSF-1R) precipitated only ligand-activated, native receptors from solution but bound to unstimulated forms after their denaturation. Two peptide antisera (anti-KI1 and -KI2), directed to residues 679 to 700 and 701 to 721, respectively, in the CSF-1R kinase insert (KI) domain and including mapped sites of ligand-induced phosphorylation at Tyr-699 and Tyr-708, bound at least 80% of the receptor molecules expressed in either CSF-1-stimulated or unstimulated cells. Immune complexes formed with anti-KI1, anti-A, or a peptide antiserum to the CSF-1R carboxyl terminus (anti-C-ter) coprecipitated CSF-1R complexed to a phosphatidylinositol 3-kinase (PtdIns 3-K) from CSF-1-stimulated cells, whereas anti-KI2 serum did not. In an in vitro assay, binding of CSF-1R to PtdIns 3-K required receptor tyrosine phosphorylation but not CSF-1R-mediated phosphorylation of the lipid kinase, and the association was specifically blocked by anti-KI2 or antibodies to phosphotyrosine. Neither anti-KI1, anti-A, nor anti-C-ter serum inhibited binding. We conclude that (i) only a minority of ligand-activated receptors form a stable complex with PtdIns 3-K in vivo, (ii) efficient binding of the lipid kinase requires receptor tyrosine phosphorylation within the CSF-1R KI domain, and (iii) a region within the KI domain defined by residues 701 to 721 at least partially overlaps the PtdIns 3-K binding site.

scholarly journals Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

1993 ◽  
Vol 13 (10) ◽  
pp. 6012-6023 ◽  
Author(s):  
R Cafferkey ◽  
P R Young ◽  
M M McLaughlin ◽  
D J Bergsma ◽  
Y Koltin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Dna Sequence ◽  
Leucine Zipper ◽  
Sequence Similarity ◽  
Missense Mutations ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Haploid Strain ◽  
Diploid Cells ◽  
Phosphatidylinositol 3

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

scholarly journals Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver

2000 ◽  
Vol 279 (2) ◽  
pp. E266-E274 ◽  
Author(s):  
Paul G. Drake ◽  
Alejandro Balbis ◽  
Jiong Wu ◽  
John J. M. Bergeron ◽  
Barry I. Posner
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Rat Liver ◽  
Intracellular Signaling ◽  
Kinase Activity ◽  
Glycogen Synthase ◽  
Metabolic Effects ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK β-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.

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