scholarly journals Cytosolic Protein Tyrosine Phosphatase-ε Is a Negative Regulator of Insulin Signaling in Skeletal Muscle

Endocrinology ◽  
2007 ◽  
Vol 149 (2) ◽  
pp. 605-614 ◽  
Author(s):  
Shlomit Aga-Mizrachi ◽  
Tamar Brutman-Barazani ◽  
Avraham I. Jacob ◽  
Asia Bak ◽  
Ari Elson ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Rna Silencing ◽  
Glycogen Synthase ◽  
Protein Kinase B ◽  
Negative Regulator ◽  
Glycogen Synthase Kinase 3 ◽  
Wild Type ◽  
Stimulation Of

Whereas positive regulatory events triggered by insulin binding to insulin receptor (IR) have been well documented, the mechanism by which the activated IR is returned to the basal status is not completely understood. Recently studies focused on the involvement of protein tyrosine phosphatases (PTPs) and how they might influence IR signaling. In this study, we examined the possibility that cytosolic PTPε (cytPTPε) is involved in IR signaling. Studies were performed on L6 skeletal muscle cells. cytPTPε was overexpressed by using pBABE retroviral expression vectors. In addition, we inhibited cytPTPε by RNA silencing. We found that insulin induced rapid association of cytPTPε with IR. Interestingly, this association appeared to occur in the plasma membrane and on stimulation with insulin the two proteins internalized together. Moreover, it appeared that almost all internalized IR was associated with cytPTPε. We found that knockdown of cytPTPε by RNA silencing increased insulin-induced tyrosine phosphorylation of IR and IR substrate (IRS)-1 as well as phosphorylation of protein kinase B and glycogen synthase kinase-3 and insulin-induced stimulation of glucose uptake. Moreover, overexpression of wild-type cytPTPε reduced insulin-induced tyrosine phosphorylation of IR, IRS-1, and phosphorylation of protein kinase B and glycogen synthase kinase-3 and insulin-induced stimulation of glucose uptake. Finally, insulin-induced tyrosine phosphorylation of IR and IRS-1 was greater in skeletal muscle from mice lacking the cytPTPε gene than that from wild-type control animals. We conclude that cytPTPε serves as another major candidate negative regulator of IR signaling in skeletal muscle.

scholarly journals Cell-Extracellular Matrix Interactions Stimulate the AP-1 Transcription Factor in an Integrin-Linked Kinase- and Glycogen Synthase Kinase 3-Dependent Manner

1999 ◽  
Vol 19 (11) ◽  
pp. 7420-7427 ◽  
Author(s):  
Armelle A. Troussard ◽  
Clara Tan ◽  
T. Nathan Yoganathan ◽  
Shoukat Dedhar
Keyword(s):  
Extracellular Matrix ◽  
Protein Kinase ◽  
Glycogen Synthase ◽  
Negative Regulator ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Dependent Manner ◽  
Wild Type ◽  
Integrin Linked Kinase ◽  
Stimulation Of

ABSTRACT Integrin-mediated interactions of cells with components of the extracellular matrix regulate cell survival, cell proliferation, cell differentiation, and cell migration. Some of these physiological responses are regulated via activation of transcription factors such as activator protein 1 (AP-1). Integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase whose activity is rapidly and transiently stimulated by cell-fibronectin interactions as well as by insulin stimulation. ILK activates protein kinase B and inhibits the glycogen synthase kinase 3 (GSK-3) activity in a phosphatidylinositol-3-kinase (PI 3-kinase)-dependent manner. We now show that cell adhesion to fibronectin results in a rapid and transient stimulation of AP-1 activity. At the same time, the kinase activity of ILK is stimulated whereas that of GSK-3 is inhibited. This fibronectin-dependent activation of AP-1 activity is inhibited in a dose-dependent manner if the cells are transfected with wild-type GSK-3, and also by inhibitors of PI 3-kinase. Stable or transient overexpression of ILK results in a stimulation of AP-1 activity which is inhibited by cotransfection with wild-type GSK-3 and kinase-deficient ILK. Transient transfection of ILK in HEK-293 cells stimulates complex formation between an AP-1 consensus oligonucleotide and nuclear proteins containing c-jun. The formation of this complex is inhibited by cotransfection with active GSK-3 or kinase-deficient ILK, suggesting that ILK may regulate AP-1 activation by inhibiting GSK-3, which has previously been shown to be a negative regulator of AP-1. In the presence of serum, ILK has no effect on the phosphorylation of Ser-73 in the N-terminal transactivation domain of c-jun. These results demonstrate a novel signaling pathway for the adhesion-mediated stimulation of AP-1 transcriptional activity involving ILK and GSK-3 and the subsequent regulation of the c-jun–DNA interaction.

scholarly journals TcR and TcR-CD28 Engagement of Protein Kinase B (PKB/AKT) and Glycogen Synthase Kinase-3 (GSK-3) Operates Independently of Guanine Nucleotide Exchange Factor VAV-1

2006 ◽  
Vol 281 (43) ◽  
pp. 32385-32394 ◽  
Author(s):  
Joanne E. Wood ◽  
Helga Schneider ◽  
Christopher E. Rudd
Keyword(s):  
T Cells ◽  
Protein Kinase ◽  
Glycogen Synthase ◽  
Protein Kinase B ◽  
Guanine Nucleotide Exchange Factor ◽  
Glycogen Synthase Kinase 3 ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

TcRζ/CD3 and TcRζ/CD3-CD28 signaling requires the guanine nucleotide exchange factor (GEF) Vav-1 as well as the activation of phosphatidylinositol 3-kinase, protein kinase B (PKB/AKT), and its inactivation of glycogen synthase kinase-3 (GSK-3). Whether these two pathways are connected or operate independently of each other in T-cells has been unclear. Here, we report that anti-CD3 and anti-CD3/CD28 can induce PKB and GSK-3α phosphorylation in the Vav-1–/– Jurkat cell line J. Vav.1 and in primary CD4-positive Vav-1–/– T-cells. Reduced GSK-3α phosphorylation was observed in Vav-1,2,3–/– T-cells together with a complete loss of FOXO1 phosphorylation. Furthermore, PKB and GSK-3 phosphorylation was unperturbed in the presence of GEF-inactive Vav-1 that inhibited interleukin-2 gene activation and a form of Src homology 2 domain-containing lymphocytic protein of 76-kDa (SLP-76) that is defective in binding to Vav-1. The pathway also was intact under conditions of c-Jun N-terminal kinase (JNK) inhibition and disruption of the actin cytoskeleton by cytochalasin D. Both events are down-stream targets of Vav-1. Overall, our findings indicate that the TcR and TcR-CD28 driven PKB-GSK-3 pathway can operate independently of Vav-1 in T-cells.

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.

scholarly journals Glycogen Synthase Kinase 3 Phosphorylates Hypoxia-Inducible Factor 1α and Mediates Its Destabilization in a VHL-Independent Manner

2007 ◽  
Vol 27 (9) ◽  
pp. 3253-3265 ◽  
Author(s):  
Daniela Flügel ◽  
Agnes Görlach ◽  
Carine Michiels ◽  
Thomas Kietzmann
Keyword(s):  
Protein Kinase ◽  
Metabolic Diseases ◽  
Glycogen Synthase ◽  
Protein Kinase B ◽  
Hypoxia Inducible Factor ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Independent Manner ◽  
Von Hippel Lindau ◽  
Gsk 3Β

ABSTRACT Hypoxia-inducible transcription factor 1α (HIF-1α) is a key player in the response to hypoxia. Additionally, HIF-1α responds to growth factors and hormones which can act via protein kinase B (Akt). However, HIF-1α is not a direct substrate for this kinase. Therefore, we investigated whether the protein kinase B target glycogen synthase kinase 3 (GSK-3) may have an impact on HIF-1α. We found that the inhibition or depletion of GSK-3 induced HIF-1α whereas the overexpression of GSK-3β reduced HIF-1α. These effects were mediated via three amino acid residues in the oxygen-dependent degradation domain of HIF-1α. In addition, mutation analyses and experiments with von Hippel-Lindau (VHL)-defective cells indicated that GSK-3 mediates HIF-1α degradation in a VHL-independent manner. In line with these observations, the inhibition of the proteasome reversed the GSK-3 effects, indicating that GSK-3 may target HIF-1α to the proteasome by phosphorylation. Thus, the direct regulation of HIF-1α stability by GSK-3 may influence physiological processes or pathophysiological situations such as metabolic diseases or tumors.

Muscle-specific overexpression of wild type and R225Q mutant AMP-activated protein kinase γ3-subunit differentially regulates glycogen accumulation

2006 ◽  
Vol 291 (3) ◽  
pp. E557-E565 ◽  
Author(s):  
Haiyan Yu ◽  
Michael F. Hirshman ◽  
Nobuharu Fujii ◽  
Jason M. Pomerleau ◽  
Lauren E. Peter ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Glycogen Content ◽  
Specific Activity ◽  
Glycogen Synthase ◽  
Underlying Mechanism ◽  
Wild Type ◽  
Glycogen Accumulation ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a heterotrimeric complex that works as an energy sensor to integrate nutritional and hormonal signals. The naturally occurring R225Q mutation in the γ3-subunit in pigs is associated with abnormally high glycogen content in skeletal muscle. Becauses skeletal muscle accounts for most of the body's glucose uptake, and γ3 is specifically expressed in skeletal muscle, it is important to understand the underlying mechanism of this mutation in regulating glucose and glycogen metabolism. Using skeletal muscle-specific transgenic mice overexpressing wild type γ3 (WTγ3) and R225Q mutant γ3 (MUTγ3), we show that both WTγ3 and MUTγ3 mice have 1.5- to 2-fold increases in muscle glycogen content. In WTγ3 mice, increased glycogen content was associated with elevated total glycogen synthase activity and reduced glycogen phosphorylase activity, whereas alterations in activities of these enzymes could not explain elevated glycogen in MUTγ3 mice. Basal, 5-aminoimidazole- AICAR- and phenformin-stimulated AMPKα2 isoform-specific activities were decreased only in MUTγ3 mice. Basal rates of 2-DG glucose uptake were decreased in both WTγ3 and MUTγ3 mice. However, AICAR- and phenformin-stimulated 2-DG glucose uptake were blunted only in MUTγ3 mice. In conclusion, expression of either wild type or mutant γ3-subunit of AMPK results in increased glycogen concentrations in muscle, but the mechanisms underlying this alteration appear to be different. Furthermore, mutation of the γ3-subunit is associated with decreases in AMPKα2 isoform-specific activity and impairment in AICAR- and phenformin-stimulated skeletal muscle glucose uptake.

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