scholarly journals Regulation of Apoptosis by the Ft1 Protein, a New Modulator of Protein Kinase B/Akt

2004 ◽  
Vol 24 (4) ◽  
pp. 1493-1504 ◽  
Author(s):  
Ingrid Remy ◽  
Stephen W. Michnick
Keyword(s):  
Protein Kinase ◽  
Fas Ligand ◽  
Fluorescent Protein ◽  
Protein Kinase B ◽  
Protein A ◽  
Cell Types ◽  
Wide Distribution ◽  
Upstream Kinase ◽  
Protein Fragment Complementation Assay ◽  
Green Fluorescent

ABSTRACT The serine/threonine kinase protein kinase B (PKB)/Akt plays a central role in many cellular processes, including cell growth, glucose metabolism, and apoptosis. However, the identification and validation of novel regulators or effectors is key to future advances in understanding the multiple functions of PKB. Here we report the identification of a novel PKB binding protein, called Ft1, from a cDNA library screen using a green fluorescent protein-based protein-fragment complementation assay. We show that the Ft1 protein interacts directly with PKB, enhancing the phosphorylation of both of its regulatory sites by promoting its interaction with the upstream kinase PDK1. Further, the modulation of PKB activity by Ft1 has a strong effect on the apoptosis susceptibility of T lymphocytes treated with glucocorticoids. We demonstrate that this phenomenon occurs via a PDK1/PKB/GSK3/NF-ATc signaling cascade that controls the production of the proapoptotic hormone Fas ligand. The wide distribution of Ft1 in adult tissues suggests that it could be a general regulator of PKB activity in the control of differentiation, proliferation, and apoptosis in many cell types.

scholarly journals Protein Kinase B/Akt Participates in GLUT4 Translocation by Insulin in L6 Myoblasts

1999 ◽  
Vol 19 (6) ◽  
pp. 4008-4018 ◽  
Author(s):  
Qinghua Wang ◽  
Romel Somwar ◽  
Philip J. Bilan ◽  
Zhi Liu ◽  
Jing Jin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Protein Kinase B ◽  
Protein A ◽  
Dominant Negative ◽  
Wild Type ◽  
Gag Protein ◽  
Membrane Ruffling ◽  
Insulin Dependent

ABSTRACT L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protein kinase B alpha (PKBα)/Akt1 in the insulin-induced translocation of GLUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescent labeling of the myc epitope in nonpermeabilized cells. Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Δp85α). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A constitutively active PKBα, created by fusion of a viral Gag protein at its N terminus (GagPKB), increased the cell surface density of GLUT4myc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKBα/Akt1 construct with the mutations K179A (substitution of alanine for the lysine at position 179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent activation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cotransfected BAD, demonstrating inhibition of the endogenous PKB/Akt. Under the same conditions, AAA-PKB almost entirely blocked the insulin-dependent increase in surface GLUT4myc. PKBα with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insulin-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation. AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKBα/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.

Interaction of the Repressors Nrg1 and Nrg2 With the Snf1 Protein Kinase in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 563-572 ◽  
Author(s):  
Valmik K Vyas ◽  
Sergei Kuchin ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Hybrid System ◽  
Fluorescent Protein ◽  
Zinc Finger Protein ◽  
Glucose Repression ◽  
Snf1 Kinase ◽  
Cell Extracts ◽  
Green Fluorescent ◽  
Two Hybrid

Abstract The Snf1 protein kinase is essential for the transcription of glucose-repressed genes in Saccharomyces cerevisiae. We identified Nrg2 as a protein that interacts with Snf1 in the two-hybrid system. Nrg2 is a C2H2 zinc-finger protein that is homologous to Nrg1, a repressor of the glucose- and Snf1-regulated STA1 (glucoamylase) gene. Snf1 also interacts with Nrg1 in the two-hybrid system and co-immunoprecipitates with both Nrg1 and Nrg2 from cell extracts. A LexA fusion to Nrg2 represses transcription from a promoter containing LexA binding sites, indicating that Nrg2 also functions as a repressor. An Nrg1 fusion to green fluorescent protein is localized to the nucleus, and this localization is not regulated by carbon source. Finally, we show that VP16 fusions to Nrg1 and Nrg2 allow low-level expression of SUC2 in glucose-grown cells, and we present evidence that Nrg1 and Nrg2 contribute to glucose repression of the DOG2 gene. These results suggest that Nrg1 and Nrg2 are direct or indirect targets of the Snf1 kinase and function in glucose repression of a subset of Snf1-regulated genes.

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

scholarly journals Live-Cell Fluorescence Imaging Reveals the Dynamics of Protein Kinase CK2 Individual Subunits

2003 ◽  
Vol 23 (3) ◽  
pp. 975-987 ◽  
Author(s):  
Odile Filhol ◽  
Arsenio Nueda ◽  
Véronique Martel ◽  
Delphine Gerber-Scokaert ◽  
Maria José Benitez ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Protein Kinase Ck2 ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Trafficking ◽  
Green Fluorescent ◽  
Nucleocytoplasmic Distribution ◽  
Kinase Ck2

ABSTRACT Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (α or α′) and two regulatory (β) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2α or GFP-CK2β revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2β, CK2α can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2α is dramatically changed by its association with CK2β, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.

scholarly journals RACK1 Regulates Integrin-mediated Adhesion, Protrusion, and Chemotactic Cell Migration via Its Src-binding Site

2003 ◽  
Vol 14 (2) ◽  
pp. 658-669 ◽  
Author(s):  
Elisabeth A. Cox ◽  
David Bennin ◽  
Ashley T. Doan ◽  
Timothy O'Toole ◽  
Anna Huttenlocher
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Site ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Expression Cloning ◽  
Chemotactic Migration ◽  
Cell Protrusion ◽  
Kinase C ◽  
Green Fluorescent

Mammalian cDNA expression cloning was used to identify novel regulators of integrin-mediated cell-substratum adhesions. Using a focal adhesion morphology screen, we identified a cDNA with homology to a receptor for activated protein kinase C (RACK1) that induced a loss of central focal adhesions and stress fibers in CHO-K1 cells. The identified cDNA was a C-terminal truncated form of RACK1 that had one of the putative protein kinase C binding sites but lacked the region proposed to bind the β integrin cytoplasmic domain and the tyrosine kinase Src. To investigate the role of RACK1 during cell spreading and migration, we tagged RACK1, a C-terminal truncated RACK1 and a point mutant that does not bind Src (RACK Y246F) with green fluorescent protein and expressed them in CHO-K1 cells. We found that RACK1 regulates the organization of focal adhesions and that it localizes to a subset of nascent focal complexes in areas of protrusion that contain paxillin but not vinculin. We also found that RACK1 regulates cell protrusion and chemotactic migration through its Src binding site. Together, these findings suggest that RACK1 regulates adhesion, protrusion, and chemotactic migration through its interaction with Src.

Green Fluorescent Protein: A Tool for Gene Expression and Cell Biology in Mycobacteria

2003 ◽  
pp. 245-260
Author(s):  
Laura E. Via ◽  
Subramanian Dhandayuthapani ◽  
Dusanka Deretic ◽  
V. Deretic
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein A ◽  
Green Fluorescent

A new recombinant rabies virus expressing a green fluorescent protein: A novel and fast approach to quantify virus neutralizing antibodies

Biologicals ◽  
2019 ◽  
Vol 59 ◽  
pp. 56-61 ◽  
Author(s):  
Shuyun Qin ◽  
Dmitriy Volokhov ◽  
Elvira Rodionova ◽  
Christoph Wirblich ◽  
Matthias J. Schnell ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Protein A ◽  
Fast Approach ◽  
Green Fluorescent

scholarly journals Multiple Adhesin-Like Functions of Xanthomonas oryzae pv. oryzae Are Involved in Promoting Leaf Attachment, Entry, and Virulence on Rice

2009 ◽  
Vol 22 (1) ◽  
pp. 73-85 ◽  
Author(s):  
Amit Das ◽  
Nandini Rangaraj ◽  
Ramesh V. Sonti
Keyword(s):  
Bacterial Adhesion ◽  
Bacterial Blight ◽  
Fluorescent Protein ◽  
Protein A ◽  
Disease Process ◽  
Xanthomonas Oryzae ◽  
Type Iv ◽  
Genes Encoding ◽  
Green Fluorescent

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. We have used enhanced green fluorescent protein-tagged X. oryzae pv. oryzae cells in conjunction with confocal microscopy to monitor the role of several adhesin-like functions in bacterial adhesion to leaf surface and early stages of leaf entry. Mutations in genes encoding either the Xanthomonas adhesin-like protein A (XadA) or its paralog, Xanthomonas adhesin-like protein B (XadB), as well as the X. oryzae pv. oryzae homolog of Yersinia autotransporter-like protein H (YapH), exhibit deficiencies in leaf attachment or entry. A mutation in the X. oryzae pv. oryzae pilQ gene, which is predicted to encode the type IV pilus secretin, appears to have no effect on leaf attachment or entry. The xadA– mutant is deficient in the ability to cause disease following surface inoculation while the XadB, YapH, and PilQ functions are less important than XadA for this process. The xadA– and xadB– mutants have no effect on virulence following wound inoculation whereas the yapH– and pilQ– mutants are always virulence deficient following wound inoculation. Overall, these results indicate that multiple adhesin-like functions are involved in promoting virulence of X. oryzae pv. oryzae, with preferential involvement of individual functions at different stages of the disease process.

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