scholarly journals Inhibition of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Hampers the Vasopressin-dependent Stimulation of Myogenic Differentiation

2019 ◽  
Vol 20 (17) ◽  
pp. 4188
Author(s):  
Silvia Sorrentino ◽  
Alessandra Barbiera ◽  
Gabriella Proietti ◽  
Gigliola Sica ◽  
Sergio Adamo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Protein Kinase B ◽  
Myogenic Differentiation ◽  
Neuromuscular Disorders ◽  
Neuromuscular Diseases ◽  
Molecular Techniques ◽  
Physiological Factor ◽  
Pi3k Inhibitor Ly294002

Arginine-vasopressin (AVP) promotes muscle differentiation, hypertrophy, and regeneration through the combined activation of the calcineurin and Calcium/Calmodulin-dependent Protein Kinase (CaMK) pathways. The AVP system is impaired in several neuromuscular diseases, suggesting that AVP may act as a physiological factor in skeletal muscle. Since the Phosphoinositide 3-kinases/Protein Kinase B/mammalian Target Of Rapamycin (PI3K/Akt/mTOR) signaling plays a significant role in regulating muscle mass, we evaluated its role in the AVP myogenic effect. In L6 cells AKT1 expression was knocked down, and the AVP-dependent expression of mTOR and Forkhead box O3 (FoxO) was analyzed by Western blotting. The effect of the PI3K inhibitor LY294002 was evaluated by cellular and molecular techniques. Akt knockdown hampered the AVP-dependent mTOR expression while increased the levels of FoxO transcription factor. LY294002 treatment inhibited the AVP-dependent expression of Myocyte Enhancer Factor-2 (MEF2) and myogenin and prevented the nuclear translocation of MEF2. LY294002 also repressed the AVP-dependent nuclear export of histone deacetylase 4 (HDAC4) interfering with the formation of multifactorial complexes on the myogenin promoter. We demonstrate that the PI3K/Akt pathway is essential for the full myogenic effect of AVP and that, by targeting this pathway, one may highlight novel strategies to counteract muscle wasting in aging or neuromuscular disorders.

scholarly journals Dual Phosphorylation of Btk by Akt/Protein Kinase B Provides Docking for 14-3-3ζ, Regulates Shuttling, and Attenuates both Tonic and Induced Signaling in B Cells

2013 ◽  
Vol 33 (16) ◽  
pp. 3214-3226 ◽  
Author(s):  
Dara K. Mohammad ◽  
Beston F. Nore ◽  
Alamdar Hussain ◽  
Manuela O. Gustafsson ◽  
Abdalla J. Mohamed ◽  
...  
Keyword(s):  
Protein Kinase ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Nuclear Translocation ◽  
Kinase Inhibitor ◽  
Protein Kinase B ◽  
Negative Regulator ◽  
Pleckstrin Homology Domain ◽  
Loss Of Function ◽  
Interaction Sites

Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in theBtkgene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

Higenamine Induces Glioma Cell Death by Modulating Nuclear Factor-κB Nuclear Translocation, Phosphoinositide-3-Kinase/Protein Kinase B Signaling and Caspase Cascade

2020 ◽  
Vol 19 (3) ◽  
pp. 317-325
Author(s):  
Chao Geng ◽  
Shaowu Ou
Keyword(s):  
Protein Kinase ◽  
Nuclear Translocation ◽  
Protein Kinase B ◽  
Glioma Cells ◽  
B Cell Lymphoma ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
Nuclear Factor ◽  
Aspartic Proteases ◽  
Phosphoinositide 3 Kinase

We have investigated the effectiveness of higenamine in the treatment of malignant glioma, and explored its possible mechanism in C6 glioma cells. The efficacy of higenamine on viability of cells, apoptosis, cell cycle arrest, DNA fragmentation, and biochemical markers was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, enzyme-linked immunosorbent assay, and Western blotting. The biochemical markers investigated included the effect of higenamine on the expression of phosphoinositide-3-kinase/protein kinase B, B-cell lymphoma 2, BCL2- associated X protein, cysteine-aspartic proteases-3 and -9 proteins. The translocation of nuclear factor-kappa B from the nucleus was also analyzed. Results revealed that higenamine induced cytotoxic and antiproliferative effects on the C6 glioma cells. Higenamine led to cell arrest at G2/M phase of cell cycle and lowered cell count at S-phase. The maximum extent of DNA fragmentation was observed after 72 h exposure of higenamine. Nuclear translocation of nuclear factorkappa B was attenuated after higenamine treatment in the C6 glioma cells. The results also revealed that higenamine significantly modulated the phosphoinositide-3-kinase/protein kinase B signaling cascade. Also, higenamine elevated the cysteine-aspartic proteases-3 and -9 and BCL2-associated X protein, and downregulated B-cell lymphoma 2 expression in the C6 glioma cells. Overall, the investigation suggests higenamine modulation of phosphoinositide-3-kinase/protein kinase B signaling pathway, nuclear factor-kappa B nuclear translocation, and caspase cascade in the C6 glioma cells.

scholarly journals Regulation of the Intracellular Localization of Foxo3a by Stress-Activated Protein Kinase Signaling Pathways in Skeletal Muscle Cells

2009 ◽  
Vol 30 (2) ◽  
pp. 470-480 ◽  
Author(s):  
Stephan Clavel ◽  
Sandrine Siffroi-Fernandez ◽  
Anne Sophie Coldefy ◽  
Kim Boulukos ◽  
Didier F. Pisani ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Muscle Atrophy ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
C2c12 Myotubes

ABSTRACT Muscle atrophy is a debilitating process associated with many chronic wasting diseases, like cancer, diabetes, sepsis, and renal failure. Rapid loss of muscle mass occurs mainly through the activation of protein breakdown by the ubiquitin proteasome pathway. Foxo3a transcription factor is critical for muscle atrophy, since it activates the expression of ubiquitin ligase Atrogin-1. In several models of atrophy, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway induces nuclear import of Foxo3a through an Akt-dependent process. This study aimed to identify signaling pathways involved in the control of Foxo3a nuclear translocation in muscle cells. We observed that after nuclear import of Foxo3a by PI3K/Akt pathway inhibition, activation of stress-activated protein kinase (SAPK) pathways induced nuclear export of Foxo3a through CRM1. This mechanism involved the c-Jun NH2-terminal kinase (JNK) signaling pathway and was independent of Akt. Likewise, we showed that inhibition of p38 induced a massive nuclear relocalization of Foxo3a. Our results thus suggest that SAPKs are involved in the control of Foxo3a nucleocytoplasmic translocation in C2C12 cells. Moreover, activation of SAPKs decreases the expression of Atrogin-1, and stable C2C12 myotubes, in which the p38 pathway is constitutively activated, present partial protection against atrophy.

scholarly journals Phosphoinositide-Dependent Phosphorylation of PDK1 Regulates Nuclear Translocation

2005 ◽  
Vol 25 (6) ◽  
pp. 2347-2363 ◽  
Author(s):  
Michael P. Scheid ◽  
Michael Parsons ◽  
James R. Woodgett
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Pleckstrin Homology Domain ◽  
Subcellular Trafficking ◽  
Kinase C ◽  
Ribosomal S6 Kinase ◽  
Nuclear Shuttling

ABSTRACT 3-Phosphoinositide-dependent kinase 1 (PDK1) phosphorylates the activation loop of a number of protein serine/threonine kinases of the AGC kinase superfamily, including protein kinase B (PKB; also called Akt), serum and glucocorticoid-induced kinase, protein kinase C isoforms, and the p70 ribosomal S6 kinase. PDK1 contains a carboxyl-terminal pleckstrin homology domain, which targets phosphoinositide lipids at the plasma membrane and is central to the activation of PKB. However, PDK1 subcellular trafficking to other compartments is not well understood. We monitored the posttranslational modifications of PDK1 following insulin-like growth factor 1 stimulation. PDK1 underwent rapid and transient phosphorylation on S396, which was dependent upon plasma membrane localization. Phosphorylation of S396 was necessary for nuclear shuttling of PDK1, possibly through its influence on an adjacent nuclear export sequence. Thus, mitogen-stimulated phosphorylation of PDK1 provides a means for directed PDK1 subcellular trafficking, with potential implications for PDK1 signaling.

scholarly journals Inhibition of Nuclear Import by Protein Kinase B (Akt) Regulates the Subcellular Distribution and Activity of the Forkhead Transcription Factor AFX

2001 ◽  
Vol 21 (10) ◽  
pp. 3534-3546 ◽  
Author(s):  
Amy M. Brownawell ◽  
Geert J. P. L. Kops ◽  
Ian G. Macara ◽  
Boudewijn M. T. Burgering
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Transcriptional Activity ◽  
Protein Kinase B ◽  
Phosphorylation Site ◽  
Nuclear Accumulation ◽  
Forkhead Transcription Factor ◽  
Akt Phosphorylation

ABSTRACT AFX belongs to a subfamily of Forkhead transcription factors that are phosphorylated by protein kinase B (PKB), also known as Akt. Phosphorylation inhibits the transcriptional activity of AFX and changes the steady-state localization of the protein from the nucleus to the cytoplasm. Our goal was threefold: to identify the cellular compartment in which PKB phosphorylates AFX, to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and to elucidate the mechanism by which phosphorylation alters the localization of AFX. We show that phosphorylation of AFX by PKB occurs in the nucleus. In addition, nuclear export mediated by the export receptor, Crm1, is required for the inhibition of AFX transcriptional activity. Both phosphorylated and unphosphorylated AFX, however, bind Crm1 and can be exported from the nucleus. These results suggest that export is unregulated and that phosphorylation by PKB is not required for the nuclear export of AFX. We show that AFX enters the nucleus by an active, Ran-dependent mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical nuclear localization signal (NLS). S193, contained within this atypical NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a negative charge at S193 by mutating the residue to glutamate reduces nuclear accumulation. PKB-mediated phosphorylation of AFX, therefore, attenuates the import of the transcription factor, which shifts the localization of the protein from the nucleus to the cytoplasm and results in the inhibition of AFX transcriptional activity.

scholarly journals 5′ Phospholipid Phosphatase SHIP-2 Causes Protein Kinase B Inactivation and Cell Cycle Arrest in Glioblastoma Cells

2000 ◽  
Vol 20 (18) ◽  
pp. 6860-6871 ◽  
Author(s):  
Vanessa Taylor ◽  
Michelle Wong ◽  
Christian Brandts ◽  
Linda Reilly ◽  
Nicholas M. Dean ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Cycle Arrest ◽  
Protein Kinase B ◽  
Pten Expression ◽  
Glioblastoma Cells ◽  
Pi3k Inhibitor Ly294002 ◽  
Cycle Arrest ◽  
Phosphoinositide 3 Kinase ◽  
The Stability

ABSTRACT The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)–protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3,4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G1 in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27KIP1. Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

scholarly journals Regulation of nuclear translocation of Forkhead transcription factor AFX by protein kinase B

1999 ◽  
Vol 96 (21) ◽  
pp. 11836-11841 ◽  
Author(s):  
H. Takaishi ◽  
H. Konishi ◽  
H. Matsuzaki ◽  
Y. Ono ◽  
Y. Shirai ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Nuclear Translocation ◽  
Protein Kinase B ◽  
Forkhead Transcription Factor
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