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.

Functional consequence of targeting protein kinase B/Akt to GLUT4 vesicles

2002 ◽  
Vol 115 (14) ◽  
pp. 2857-2866 ◽  
Author(s):  
Pierre-Henri Ducluzeau ◽  
Laura M. Fletcher ◽  
Gavin I. Welsh ◽  
Jeremy M. Tavaré
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Glucose Transporter ◽  
Protein Kinase B ◽  
Protein A ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Functional Consequence ◽  
Insulin Dependent ◽  
Inactive Protein

We have investigated the role of protein kinase B (Akt) in the insulin-stimulated translocation of vesicles containing the insulin-responsive isoform of glucose transporter (GLUT4) to the plasma membrane of adipocytes. Previous reports have suggested that protein kinase B can bind to intracellular GLUT4 vesicles in an insulin-dependent manner, but the functional consequence of this translocation is not known. In this study we have artificially targeted constitutively active and kinase-inactive mutants of protein kinase B to intracellular GLUT4 vesicles by fusing them with the N-terminus of GLUT4 itself. We examined the effect of these mutants on the insulin-dependent translocation of the insulin-responsive amino peptidase IRAP(a bona fide GLUT4-vesicle-resident protein). A kinase-inactive protein kinase B targeted to GLUT4 vesicles was an extremely effective dominant-negative inhibitor of insulin-stimulated IRAP translocation to the plasma membrane. By contrast, a kinase-inactive protein kinase B expressed in the cytoplasm did not have an effect. The results suggest that protein kinase B has an important functional role at, or in the vicinity of, GLUT4 vesicles in the insulin-dependent translocation of those vesicles to the plasma membrane of adipocytes.

scholarly journals Protein kinase D inhibits plasma membrane Na+/H+exchanger activity

1999 ◽  
Vol 277 (6) ◽  
pp. C1202-C1209 ◽  
Author(s):  
Robert S. Haworth ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt ◽  
Metin Avkiran
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Wild Type ◽  
Ph Indicator

The regulation of plasma membrane Na+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux ( J H), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased J H (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J Hwas already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637–815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

scholarly journals A Cell Number Counting Factor Regulates Akt/Protein Kinase B To Regulate Dictyostelium discoideum Group Size

2004 ◽  
Vol 3 (5) ◽  
pp. 1176-1184 ◽  
Author(s):  
Tong Gao ◽  
David Knecht ◽  
Lei Tang ◽  
R. Diane Hatton ◽  
Richard H. Gomer
Keyword(s):  
Protein Kinase ◽  
Dictyostelium Discoideum ◽  
Group Size ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Protein Kinase B ◽  
Cell Number ◽  
Pleckstrin Homology Domain ◽  
Wild Type ◽  
High Concentration

ABSTRACT Little is known about how individual cells can organize themselves to form structures of a given size. During development, Dictyostelium discoideum aggregates in dendritic streams and forms groups of ∼20,000 cells. D. discoideum regulates group size by secreting and simultaneously sensing a multiprotein complex called counting factor (CF). If there are too many cells in a stream, the associated high concentration of CF will decrease cell-cell adhesion and increase cell motility, causing aggregation streams to break up. The pulses of cyclic AMP (cAMP) that mediate aggregation cause a transient translocation of Akt/protein kinase B (Akt/PKB) to the leading edge of the plasma membrane and a concomitant activation of the kinase activity, which in turn stimulates motility. We found that countin− cells (which lack bioactive CF) and wild-type cells starved in the presence of anticountin antibodies (which block CF activity) showed a decreased level of cAMP-stimulated Akt/PKB membrane translocation and kinase activity compared to parental wild-type cells. Recombinant countin has the bioactivity of CF, and a 1-min treatment of cells with recombinant countin potentiated Akt/PKB translocation to membranes and Akt/PKB activity. Western blotting of total cell lysates indicated that countin does not affect the total level of Akt/PKB. Fluorescence microscopy of cells expressing an Akt/PKB pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion protein indicated that recombinant countin and anti-countin antibodies do not obviously alter the distribution of Akt/PKB PH-GFP when it translocates to the membrane. Our data indicate that CF increases motility by potentiating the cAMP-stimulated activation and translocation of Akt/PKB.

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 Analysis of the role of protein kinase B (cAKT) in insulin-dependent induction of glucokinase and sterol regulatory element-binding protein 1 (SREBP1) mRNAs in hepatocytes

2003 ◽  
Vol 376 (3) ◽  
pp. 697-705 ◽  
Author(s):  
Pascale G. RIBAUX ◽  
Patrick B. IYNEDJIAN
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Binding Protein ◽  
Regulatory Element ◽  
Necessary Condition ◽  
Insulin Effect ◽  
Element Binding Protein ◽  
Insulin Dependent ◽  
Sterol Regulatory Element ◽  
Stimulation Of

Previous work showed that acute stimulation of a conditionally active protein kinase B (PKB or cAKT) was sufficient to elicit insulin-like induction of GCK (glucokinase) and SREBP1 (sterol regulatory element-binding protein 1) in hepatocytes [Iynedjian, Roth, Fleischmann and Gjinovci (2000) Biochem. J. 351, 621–627; Fleischmann and Iynedjian (2000) Biochem. J. 349, 13–17]. The objective of the present study was to determine whether activation of PKB during insulin stimulation of hepatocytes was a necessary condition for the induction of the two genes. Activation of PKB by insulin was inhibited by pretreatment of the hepatocytes with C2 ceramide. This resulted in the inhibition of insulin-dependent increases in GCK and SREBP1 mRNAs. A triple mutant of PKB failed to interfere with insulin activation of PKB in hepatocytes even at high overexpression levels achieved after adenovirus transduction. A PKB–CaaX fusion protein, which can act as a dominant-negative inhibitor of PKB activation in other cells, was shown to be constitutively activated in hepatocytes and to trigger insulin-like induction of GCK and SREBP1. In addition, constitutive PKB–CaaX activity caused refractoriness of the hepatocytes to insulin signalling at an upstream step resulting in the inhibition of both extracellular-signal-regulated kinase 1/2 and endogenous PKB activation. The stimulation of gene expression by constitutively active PKB–CaaX and inhibition of the insulin effect by ceramide are compatible with a role for PKB in the insulin-dependent induction of GCK and SREBP1.

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.

Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B

2001 ◽  
Vol 356 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Mireille CORMONT ◽  
Nadine GAUTIER ◽  
Karine ILC ◽  
Yannick Le MARCHAND-BRUSTEL
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Active Form ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Glut4 Translocation ◽  
Phosphatidylinositol 3 Kinase ◽  
Isolated Adipocytes ◽  
Phosphatidylinositol 3

The small GTPase Rab4 has been shown to participate in the subcellular distribution of GLUT4 under both basal and insulin-stimulated conditions in adipocytes. In the present work, we have characterized the effect of Rab4 ΔCT, a prenylation-deficient and thus cytosolic form of Rab4, in this process. We show that the expression of Rab4 ΔCT in freshly isolated adipocytes inhibits insulin-induced GLUT4 translocation, but only when this protein is in its GTP-bound active form. Further, it not only blocks the effect of insulin, but also that of a hyperosmotic shock, but does not interfere with the effect of zinc ions on GLUT4 translocation. Rab4 ΔCT was then shown to prevent GLUT4 translocation induced by the expression of an active form of phosphatidylinositol 3-kinase or of protein kinase B, without altering the activities of the enzymes. Our results are consistent with a role of Rab4 ΔCT acting as a dominant negative protein towards Rab4, possibly by binding to Rab4 effectors.

scholarly journals Vitamin D3 Activates Phosphatidylinositol-3-Kinase/Protein Kinase B via Insulin-Like Growth Factor-1 to Improve Testicular Function in Diabetic Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Yanyan He ◽  
Yang Liu ◽  
Qing-Zhu Wang ◽  
Feng Guo ◽  
Fengjuan Huang ◽  
...  
Keyword(s):  
Vitamin D ◽  
Growth Factor ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Diabetic Rats ◽  
Testicular Function ◽  
Insulin Like Growth Factor ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Objective. In diabetes mellitus, vitamin D3 deficiency affects sex hormone levels and male fertility; however, the mechanism leading to the disorder is unclear. This research was designed to investigate the mechanism of vitamin D3 deficiency and hypogonadism in diabetic rats. Our aim was to assess serum vitamin D3 levels and the relationship among vitamin D3, insulin-like growth factor-1 (IGF-1), and testicular function. Materials and Methods. Rats with streptozotocin-induced diabetes were randomly divided into four groups and treated with different doses of vitamin D3: no vitamin D3, low (0.025 μg/kg/day), high (0.1 μg/kg/day), and high (0.1 μg/kg/day) with JB-1 (the insulin-like growth factor-1 receptor inhibitor group, 100 μg/kg/day). The groups were compared with wild-type rats, which function as the control group. Various parameters such as vitamin D3 and IGF-1 were compared between the experimental and wild-type groups, and their correlations were determined. Results. Twelve weeks of vitamin D3 supplementation improved the testosterone levels, as shown by the increase in the level of serum IGF-1 in diabetic rats. Phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT), which was a downstream of the signaling pathway of IGF-1, was significantly increased after vitamin D3 treatment. Conclusions. The study shows that vitamin D3 may promote the expression of testosterone and improve testicular function in diabetic rats by activating PI3K/AKT via IGF-1.

scholarly journals Protein Kinase B Gene Homologue pkbR1 Performs One of Its Roles at First Finger Stage of Dictyostelium

2011 ◽  
Vol 10 (4) ◽  
pp. 512-520 ◽  
Author(s):  
Hiroshi Ochiai ◽  
Kosuke Takeda ◽  
Masashi Fukuzawa ◽  
Atsushi Kato ◽  
Shigeharu Takiya ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Stalk Cell ◽  
Null Mutant ◽  
Wild Type ◽  
Cell Region ◽  
Content Type ◽  
Mutant Forms ◽  
Second Wave ◽  
Gene Homologue

ABSTRACT Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB , which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form nonviable stalk cells. These results suggest that the mutant has defects in a system that changes the physiological dynamics in the prestalk cell region of a finger. We suggest that the arrest of its development is due to the loss of the second wave of expression of a protein kinase A catalytic subunit gene ( pkaC ) only in the prestalk region of the pkbR1 null mutant.

scholarly journals A Mutation Linked with Bartter's Syndrome Locks Kir 1.1a (Romk1) Channels in a Closed State

1999 ◽  
Vol 114 (5) ◽  
pp. 685-700 ◽  
Author(s):  
Thomas P. Flagg ◽  
Margaret Tate ◽  
Jean Merot ◽  
Paul A. Welling
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
K Channel ◽  
Bartter’S Syndrome ◽  
Wild Type ◽  
Bartter's Syndrome ◽  
Closed State ◽  
Negative Effect

Mutations in the inward rectifying renal K+ channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332–391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP–wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332–351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.

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