Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity

Ananthanarayanan, Bharath; Ni, Qing Zhe; Zhang, Jin

doi:10.1073/pnas.0502889102

Cited by 95 publications

(107 citation statements)

References 45 publications

(58 reference statements)

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“…Another restriction of this type of probe is in their sensitivity. Moreover, Ananthanarayanan et al (2005) have observed a significant timelag between the increase in the level of PtdIns(3,4,5)P 3 and activation of Akt monitored by a type 1 probe. The first probe belonging to the second type of probe was reported by Calleja et al (2003).…”

Section: Discussionmentioning

confidence: 99%

“…Two types of FRET-based monitors of Akt activity have been reported previously (Calleja et al, 2003;Sasaki et al, 2003;Ananthanarayanan et al, 2005;Kunkel et al, 2005): 1) measurement of phosphorylation of Akt substrate (Sasaki et al, 2003;Kunkel et al, 2005) and 2) measurement of the conformational change of Akt (Calleja et al, 2003). Probes belonging to the first type detect the balance between the activities of Akt and antagonizing phosphatases.…”

Section: Discussionmentioning

confidence: 99%

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Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

Yoshizaki

Mochizuki

Gotoh

et al. 2007

MBoC

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We studied the spatiotemporal regulation of Akt (also called protein kinase B), phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P 2 ], and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] by using probes based on the principle of fluorescence resonance energy transfer. On epidermal growth factor (EGF) stimulation, the amount of PtdIns(3,4,5)P 3 was increased diffusely in the plasma membrane, whereas that of PtdIns(3,4)P 2 was increased more in the nascent lamellipodia than in the plasma membrane of the central region. The distribution and time course of Akt activation were similar to that of increased PtdIns(3,4)P 2 levels, which were most prominent in the nascent lamellipodia. Moreover, we found that upon EGF stimulation 3-phosphoinositide-dependent protein kinase-1 (PDK1) was also recruited to nascent lamellipodia in an Akt-dependent manner. Because PDK1 is known to activate Ral GTPase and because Ral is required for EGF-induced lamellipodial protrusion, we speculated that the PDK1-Akt complex may be indispensable for the induction of lamellipodia. In agreement with this idea, EGF-induced lamellipodia formation was promoted by the overexpression of Akt and inhibited by an Akt inhibitor or a Ral-binding domain of Sec5. These results identified the Akt-PDK1 complex as an upstream positive regulator of Ral GTPase in the induction of lamellipodial protrusion. INTRODUCTIONClass I phosphoinositide 3-kinase (PI3K) is a key mediator of intracellular signaling pathways that regulate actin cytoskeletal reorganization and polarized cell migration. Activated PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ] to generate phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ], which, in turn, activates a variety of pleckstrin homology (PH) domain-containing proteins such as Akt (also called protein kinase B) (Saltiel and Pessin, 2002;Sulis and Parsons, 2003). PtdIns(3,4,5)P 3 is dephosphorylated to yield PtdIns(4,5)P 2 by a tumor suppressor protein, phosphatase and tensin homolog deleted in chromosome 10 (PTEN), which is frequently mutated or deleted in various human cancers (Sulis and Parsons, 2003). The resulting PTEN deficiency causes accumulation of PtdIns(3,4,5)P 3 in cells, and, as a consequence, an increase in cell motility in fibroblasts (Liliental et al., 2000;Higuchi et al., 2001;Hafizi et al., 2005). Another dephosphorylated derivative of PtdIns (3,4,5)P 3 is PtdIns(3,4)P 2 produced by phosphoinositide 5-phosphatases, including Src homology 2-containing inositol-5-phosphatase (SHIP). This PtdIns(3,4)P 2 also binds to various PH domain-containing proteins, which overlap significantly to those bound to PtdIns(3,4,5)P 3 (Lemmon and Ferguson, 2000;Maffucci and Falasca, 2001).Among many PH domain-containing proteins, a serinethreonine kinase, Akt, has been studied most extensively. Akt has been implicated in the control of diverse cellular functions, including glucose metabolism, gene transcription, cell proliferation, and apoptosis (Brazil et al., 2004;Fayard et al., 2005)...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

Yoshizaki

Mochizuki

Gotoh

et al. 2007

MBoC

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“…If, however, there is not a large enough ligand binding-induced conformational change in the binding protein to induce a FRET change, one must come up with other strategies to induce such a change. One such strategy, which has been implemented for a PIP 3 biosensor, indicator for phosphoinositides based on Akt (InPAkt), is to incorporate a pseudoligand to the biosensor that binds to the binding domain in the absence of natural ligand binding but is displaced upon natural ligand binding, thereby generating a conformational change (29). In principle, this modular design can be extended to other ligands using corresponding ligand-binding domains and engineered pseudoligands.…”

Section: Modular Designmentioning

confidence: 99%

“…And for this same reason, they can be directed to subcellular regions via specific amino acid targeting sequences that are added to the biosensor. Targeting of biosensors to specific locations is important for studying the spatial regulation of signaling dynamics, and, as will be discussed in later sections, represents one proven strategy for the coimaging of FRET-based biosensors (29,31).…”

Section: Genetic Encodabilitymentioning

confidence: 99%

“…Currently, there are FRET-based second messenger biosensors to detect cellular fluctuations in Ca 11 (27,28), PIP 3 (29,30), 3 0 ,5 0 -cyclic adenosine monophosphate (cAMP) (31-33), 3 0 ,5 0 -cyclic guanosine monophosphate (cGMP) (34), nitric oxide (35), and more. Many of these biosensors rely on a ligandinduced conformational change in the binding protein in order to alter the distance and/or orientation between the two FPs that flank the second messenger binding domain (Fig.…”

Section: Modular Designmentioning

confidence: 99%

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Dynamic visualization of signal transduction in living cells: From second messengers to kinases

Herbst

Zhang

2009

IUBMB Life

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SummaryThe study of signal transduction, or the highly regulated series of biochemical events which allow a cell to convert a given stimulus into a functional response, has seen a paradigm shift with a recent explosion in the number of genetically encoded FRET-based biosensors capable of detecting spatial and temporal regulation of various signaling events in living cells. The two classes of biosensors discussed, namely kinase activity and second messenger biosensors, utilize two fluorescent proteins (FP) suitable for FRET and convert a signaling event of interest into a conformational change in the biosensor that can be measured as a change in FRET between the two FPs. Individually, these biosensors have been used to elucidate many complex signal transduction mechanisms in various biological systems. However, it has become increasingly clear that it is often more desirable to study multiple signaling events simultaneously, allowing for precise correlation of the temporal profiles of multiple signaling molecules without the complication of cell to cell variability. With the design of spectrally distinct biosensors and new coimaging strategies, simultaneous imaging of multiple signaling events is not only possible, but has aided in mapping the intricate network of cellular signal transduction cascades. Furthermore, as aberrant signal transduction involving second messengers and kinases is implicated in numerous disease states, it is hopeful that these FRET-based biosensors and coimaging strategies can help to unravel the molecular links between altered signal transduction and certain disease states.2009 IUBMB IUBMB Life, 61(9): 902-908, 2009

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Visualization of Cellular Phosphoinositide Pools with GFP‐Fused Protein‐Domains

Balla

Várnai

2009

CP Cell Biology

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This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present in only tiny amounts as compared to structural lipids, but they are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane recruitment and activity of many membrane protein signaling complexes in specific membrane compartments, and they have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single‐cell level. The only available technique in live cell applications is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules, when fused to fluorescent proteins, can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular localization, and it has rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. This unit summarizes the design and practical use of these constructs and also reviews important considerations for interpretation of the data obtained by this technique. Curr. Protoc. Cell Biol. 42:24.4.1‐24.4.27. © 2009 by John Wiley & Sons, Inc.

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Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity

Cited by 95 publications

References 45 publications

Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

Dynamic visualization of signal transduction in living cells: From second messengers to kinases

Visualization of Cellular Phosphoinositide Pools with GFP‐Fused Protein‐Domains

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