scholarly journals Protein Kinase B Activation and Lamellipodium Formation Are Independent Phosphoinositide 3-Kinase-Mediated Events Differentially Regulated by Endogenous Ras

1998 ◽  
Vol 18 (4) ◽  
pp. 1802-1811 ◽  
Author(s):  
David H. J. van Weering ◽  
Johan de Rooij ◽  
Barbara Marte ◽  
Julian Downward ◽  
Johannes L. Bos ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Protein Kinase ◽  
Egf Receptor ◽  
Protein Kinase B ◽  
Ectopic Expression ◽  
Differential Sensitivity ◽  
Phosphorylated Proteins ◽  
Phosphoinositide 3 Kinase

ABSTRACT Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events.

scholarly journals Domain Swapping Used To Investigate the Mechanism of Protein Kinase B Regulation by 3-Phosphoinositide-Dependent Protein Kinase 1 and Ser473 Kinase

1999 ◽  
Vol 19 (7) ◽  
pp. 5061-5072 ◽  
Author(s):  
Mirjana Andjelković ◽  
Sauveur-Michel Maira ◽  
Peter Cron ◽  
Peter J. Parker ◽  
Brian A. Hemmings
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase B ◽  
Second Messengers ◽  
Activation Mechanism ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Phosphoinositide 3 Kinase ◽  
Regulatory Sites

ABSTRACT Protein kinase B (PKB or Akt), a downstream effector of phosphoinositide 3-kinase (PI 3-kinase), has been implicated in insulin signaling and cell survival. PKB is regulated by phosphorylation on Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and on Ser473 by an unidentified kinase. We have used chimeric molecules of PKB to define different steps in the activation mechanism. A chimera which allows inducible membrane translocation by lipid second messengers that activate in vivo protein kinase C and not PKB was created. Following membrane attachment, the PKB fusion protein was rapidly activated and phosphorylated at the two key regulatory sites, Ser473 and Thr308, in the absence of further cell stimulation. This finding indicated that both PDK1 and the Ser473 kinase may be localized at the membrane of unstimulated cells, which was confirmed for PDK1 by immunofluorescence studies. Significantly, PI 3-kinase inhibitors prevent the phosphorylation of both regulatory sites of the membrane-targeted PKB chimera. Furthermore, we show that PKB activated at the membrane was rapidly dephosphorylated following inhibition of PI 3-kinase, with Ser473 being a better substrate for protein phosphatase. Overall, the results demonstrate that PKB is stringently regulated by signaling pathways that control both phosphorylation/activation and dephosphorylation/inactivation of this pivotal protein kinase.

Cross-talk between receptors with intrinsic tyrosine kinase activity and α1b-adrenoceptors

2000 ◽  
Vol 350 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Luz DEL CARMEN MEDINA ◽  
José VÁZQUEZ-PRADO ◽  
J. Adolfo GARCÍA-SÁINZ
Keyword(s):  
Protein Kinase C ◽  
Growth Factors ◽  
Growth Factor ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Functional Coupling ◽  
Kinase C ◽  
Epidermal Growth ◽  
Phosphoinositide 3 Kinase ◽  
And Function

The effect of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) on the phosphorylation and function of α1b-adrenoceptors transfected into Rat-1 fibroblasts was studied. EGF and PDGF increased the phosphorylation of these adrenoceptors. The effect of EGF was blocked by tyrphostin AG1478 and that of PDGF was blocked by tyrphostin AG1296, inhibitors of the intrinsic tyrosine kinase activities of the receptors for these growth factors. Wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the α1b-adrenoceptor phosphorylation induced by EGF but not that induced by PDGF. Inhibition of protein kinase C blocked the adrenoceptor phosphorylation induced by EGF and PDGF. The ability of noradrenaline to increase [35S]guanosine 5´-[γ-thio]triphosphate ([35S]GTP[S]) binding in membrane preparations was used as an index of the functional coupling of the α1b-adrenoceptors and G-proteins. Noradrenaline-stimulated [35S]GTP[S] binding was markedly decreased in membranes from cells pretreated with EGF or PDGF. Our data indicate that: (i) activation of EGF and PDGF receptors induces phosphorylation of α1b-adrenoceptors, (ii) phosphatidylinositol 3-kinase is involved in the EGF response, but does not seem to play a major role in the action of PDGF, (iii) protein kinase C mediates this action of both growth factors and (iv) the phosphorylation of α1b-adrenoceptors induced by EGF and PDGF is associated with adrenoceptor desensitization.

The life of a cell: apoptosis regulation by the PI3K/PKB pathway

2008 ◽  
Vol 415 (3) ◽  
pp. 333-344 ◽  
Author(s):  
Vincent Duronio
Keyword(s):  
Growth Factors ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Protein Kinase B ◽  
Cell Type ◽  
A Cell ◽  
Phosphoinositide 3 Kinase ◽  
The Many ◽  
Apoptosis Regulation ◽  
Inositol Ring

The activation of PI3K (phosphoinositide 3-kinase) family members is a universal event in response to virtually all cytokines, growth factors and hormones. As a result of formation of PtdIns with an added phosphate at the 3 position of the inositol ring, activation of the protein kinases PDK1 (phosphoinositide-dependent kinase 1) and PKB (protein kinase B)/Akt occurs. The PI3K/PKB pathway impinges upon a remarkable array of intracellular events that influence either directly or indirectly whether or not a cell will undergo apoptosis. In this review, the many ways in which PI3K/PKB can control these processes are summarized. Not all of the events described will necessarily play a role in any one cell type, but a subset of these events is probably essential for the survival of every cell.

scholarly journals Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism

2004 ◽  
Vol 380 (3) ◽  
pp. 795-804 ◽  
Author(s):  
Troy A. HORNBERGER ◽  
Rudy STUPPARD ◽  
Kevin E. CONLEY ◽  
Mark J. FEDELE ◽  
Marta L. FIOROTTO ◽  
...  
Keyword(s):  
Growth Factors ◽  
Protein Kinase ◽  
Mechanical Load ◽  
Ex Vivo ◽  
Protein Kinase B ◽  
Mtor Pathway ◽  
Central Component ◽  
Translational Efficiency ◽  
Mechanical Stimuli ◽  
Phosphoinositide 3 Kinase

In response to growth factors, mTOR (mammalian target of rapamycin) has been identified as a central component of the signalling pathways that control the translational machinery and cell growth. Signalling through mTOR has also been shown to be necessary for the mechanical load-induced growth of cardiac and skeletal muscles. Although the mechanisms involved for mechanically induced activation of mTOR are not known, it has been suggested that activation of PI3K (phosphoinositide 3-kinase) and protein kinase B (Akt), via the release of locally acting growth factors, underlies this process. In the present study, we show that mechanically stimulating (passive stretch) the skeletal muscle ex vivo results in the activation of mTOR-dependent signalling events. The activation of mTOR-dependent signalling events was necessary for an increase in translational efficiency, demonstrating the physiological significance of this pathway. Using pharmacological inhibitors, we show that activation of mTOR-dependent signalling occurs through a PI3K-independent pathway. Consistent with these results, mechanically induced signalling through mTOR was not disrupted in muscles from Akt1−/− mice. In addition, ex vivo co-incubation experiments, along with in vitro conditioned-media experiments, demonstrate that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for the activation of the mTOR pathway. Taken together, our results demonstrate that mechanical stimuli can activate the mTOR pathway independent of PI3K/Akt1 and locally acting growth factors. Thus mechanical stimuli and growth factors provide distinct inputs through which mTOR co-ordinates an increase in the translational efficiency.

scholarly journals Protein kinase B/Akt: a nexus of growth factor and cytokine signaling in determining muscle mass

2007 ◽  
Vol 103 (1) ◽  
pp. 378-387 ◽  
Author(s):  
Robert A. Frost ◽  
Charles H. Lang
Keyword(s):  
Skeletal Muscle ◽  
Growth Factors ◽  
Growth Factor ◽  
Protein Kinase ◽  
Muscle Mass ◽  
Muscle Protein ◽  
Contractile Activity ◽  
Protein Kinase B ◽  
Muscle Size ◽  
Cytokine Signaling

Although the boundaries of skeletal muscle size are fundamentally determined by genetics, this dynamic tissue also demonstrates great plasticity in response to environmental and hormonal factors. Recent work indicates that contractile activity, nutrients, growth factors, and cytokines all contribute to determining muscle mass. Muscle responds not only to endocrine hormones but also to the autocrine production of growth factors and cytokines. Skeletal muscle synthesizes anabolic growth factors such as insulin-like growth factor (IGF)-I and potentially inhibitory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and myostatin. These self-regulating inputs in turn influence muscle metabolism, including the use of nutrients such as glucose and amino acids. These changes are principally achieved by altering the activity of the protein kinase known as protein kinase B or Akt. Akt plays a central role in integrating anabolic and catabolic responses by transducing growth factor and cytokine signals via changes in the phosphorylation of its numerous substrates. Activation of Akt stimulates muscle hypertrophy and antagonizes the loss of muscle protein. Here we review the many signals that funnel through Akt to alter muscle mass.

scholarly journals c-erbB2–induced Disruption of Matrix Adhesion and Morphogenesis Reveals a Novel Role for Protein Kinase B as a Negative Regulator of α2β1 Integrin Function

2002 ◽  
Vol 13 (8) ◽  
pp. 2894-2908 ◽  
Author(s):  
Lachmi E. Lindberg ◽  
Shahram Hedjazifar ◽  
Dan Baeckström
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Growth Factor Receptor ◽  
Mammary Epithelial ◽  
Negative Regulator ◽  
Epithelial Cell Line ◽  
Adhesive Properties ◽  
Direct Role ◽  
Phosphoinositide 3 Kinase

Overexpression of the growth factor receptor subunit c-erbB2, leading to its ligand-independent homodimerization and activation, has been implicated in the pathogenesis of mammary carcinoma. Here, we have examined the effects of c-erbB2 on the adhesive properties of a mammary epithelial cell line, HB2/tnz34, in which c-erbB2 homodimerization can be induced by means of a transfected hybrid “trk-neu” construct. trk-neu consists of the extracellular domain of the trkA nerve growth factor (NGF) receptor fused to the transmembrane and cytoplasmic domains of c-erbB2, allowing NGF-induced c-erbB2 homodimer signaling. Both spreading and adhesion on collagen surfaces were impaired on c-erbB2 activation in HB2/tnz34 cells. Antibody-mediated stimulation of α2β1 integrin function restored adhesion, suggesting a direct role for c-erbB2 in integrin inactivation. Using pharmacological inhibitors and transient transfections, we identified signaling pathways required for suppression of integrin function by c-erbB2. Among these was the MEK-ERK pathway, previously implicated in integrin inactivation. However, we could also show that downstream of phosphoinositide-3-kinase (PI3K), protein kinase B (PKB) acted as a previously unknown, potent inhibitor of integrin function and mediator of the disruptive effects of c-erbB2 on adhesion and morphogenesis. The integrin-linked kinase, previously identified as a PKB coactivator, was also found to be required for integrin inactivation by c-erbB2. In addition, the PI3K-dependent mTOR/S6 kinase pathway was shown to mediate c-erbB2–induced inhibition of adhesion (but not spreading) independently of PKB. Overexpression of MEK1 or PKB suppressed adhesion without requirement for c-erbB2 activation, suggesting that these two pathways partake in integrin inhibition by targeting common downstream effectors. These results demonstrate a major novel role for PI3K and PKB in regulation of integrin function.

scholarly journals p70 S6 Kinase Is Regulated by Protein Kinase Cζ and Participates in a Phosphoinositide 3-Kinase-Regulated Signalling Complex

1999 ◽  
Vol 19 (4) ◽  
pp. 2921-2928 ◽  
Author(s):  
Angela Romanelli ◽  
Kathleen A. Martin ◽  
Alex Toker ◽  
John Blenis
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Independent Manner ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Dependent Signal ◽  
Catalytic Loop ◽  
Phosphoinositide 3 Kinase ◽  
P70s6k Activation

ABSTRACT p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCζ as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCζ mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCζ mutant (myristoylated PKCζ [myr-PKCζ]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCζ. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCζ in vivo in a growth factor-independent manner, while PDK-1 and PKCζ can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.

Effect of multiple phosphorylation events on the transcription factors FKHR, FKHRLI and AFX

2002 ◽  
Vol 30 (4) ◽  
pp. 391-397 ◽  
Author(s):  
Y. L. Woods ◽  
G. Rena
Keyword(s):  
Transcription Factors ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Critical Role ◽  
Chromosome X ◽  
Kinase Cascade ◽  
Phosphoinositide 3 Kinase ◽  
Fused Gene ◽  
Pkb Phosphorylation

The insulin-stimulated phosphoinositide 3-kinase (PI 3-kinase)/3-phosphoinositide-dependent kinase-1 (PDK1)/protein kinase B (PKB) kinase cascade is believed to play a critical role in metabolic control and cell survival, largely mediated through PKB phosphorylation of many proteins. Recent findings demonstrate that the transcription factors FKHR (forkhead in rhabdomyosarcoma), AFX (ALL1 fused gene from chromosome X) and FKHRL1 (FKHR-like 1; termed FKHR isoforms) are phosphorylated by PKB in cells, leading to their exit from the nucleus. These exciting results suggest that FKHR isoforms may be critical effectors of PI 3-kinase/PDK1/PKB signalling in vivo.

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