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

Yoshizaki, Hisayoshi; Mochizuki, Naoki; Gotoh, Yukiko; Matsuda, Minoru

doi:10.1091/mbc.e06-05-0467

Cited by 59 publications

(67 citation statements)

References 63 publications

(105 reference statements)

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“…We show here that in addition to its previously identified PDK1 association domain in its N terminus, RalGDS also has an independent site in the middle of the protein that complexes with Akt. This scaffold model is consistent with recent fluorescence resonance energy transfer data that showed that RalGDS, PDK1, and Akt associate together in lamellipodia, where Ral and Akt are both activated upon EGF stimulation (36). Moreover, an analogous scaffold function has been ascribed to MAKAPa, which preferentially promotes PDK1 activation of p90RSK in the cytoplasm but not of Akt in the plasma membrane (21).…”

Section: Discussionsupporting

confidence: 89%

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RalGDS Couples Growth Factor Signaling to Akt Activation

Hao

Wong

Feig

2008

Molecular and Cellular Biology

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The Akt kinase is a key regulator of cell proliferation and survival. It is activated in part by PDK1-induced phosphorylation. Here we show that RalGDS, a Ras effector protein that activates Ral GTPases, has a second function that promotes Akt phosphorylation by PDK1 by bringing these two kinases together. In support of this conclusion is our finding that suppression of RalGDS expression in cells inhibits both epidermal growth factor and insulin-induced phosphorylation of Akt. Moreover, while PDK1 complexes with N-GDS, Akt complexes with the central region of RalGDS through an intermediary, JIP1. The biological significance of this newly discovered RalGDS function is highlighted by the observation that an N-terminally deleted mutant of RalGDS that retains the ability to activate Ral proteins but loses the ability to activate Akt also fails to promote cell proliferation. Thus, RalGDS forms a nexus that transduces growth factor signaling to both Ral GTPase and Akt-mediated signaling cascades.The Akt kinase is a key mediator of the actions of many growth factors and hormones (10). Its effects on cells include regulation of metabolism, gene transcription, cell proliferation, angiogenesis, and apoptosis. Loss or gain of Akt activity contributes to a variety of diseases, including diabetes and cancer. Akt influences these processes by phosphorylating a broad spectrum of substrates, including MDM2, FOXO transcription factors, p27 kip1 , Bad, and GSK3. The three Akt isoforms, Akt1 to -3, belong to the AGC family of kinases, all of which are activated by the PDK1 kinase (for review, see reference 20). The accepted mode of Akt activation involves ligand-induced activation of phosphatidylinositol 3-kinase (PI3-kinase), which leads to the production of PIP2 and PIP3. These phospholipids interact with the PH domain of Akt to target the protein to PDK1 in the plasma membrane, where PDK1 phosphorylates Akt on threonine 308 (2). How specific PDK1 substrates are chosen for activation in response to specific stimuli is poorly understood. A secondary activation event occurs through phosphorylation of Akt at serine 473. Multiple kinases have been reported to be responsible for this event, the most recent of which is the rictor/mTor complex (29).RalGDS is one of a family of guanine nucleotide exchange factors (GEFs) that activate Ral-GTPases in cells by promoting their switch from GDP-bound to GTP-bound states. A subset of these GEFs, including RalGDS, Rlf, Rgl, and Rgl3, are downstream effectors of Ras proteins, as active Ras binds to their C termini and targets them to Ral in cells (25). Recent studies on human cells and in knockout mice have highlighted the importance of this effector pathway in the Ras-mediated oncogenesis. For example, Ral exchange factors can replace Ras in assays designed to test the minimal number of genes that are required to transform primary human cells to a tumorigenic state (14, 26), while a RalGDS knockout suppresses tumor formation in a Ras-mediated tumor model (13).Activation of RalGDS in cells also ...

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Section: Discussionsupporting

confidence: 89%

“…Instead, the N terminus of PDK1 associates with the noncatalytic N terminus of RalGDS, which relieves the latter's inhibitory effect on the GEF's catalytic domain. Moreover, PDK1 has been shown to promote lamellipodia formation in response to epidermal growth factor (EGF) by activating the Ral signaling cascade at the site of membrane protrusions (36).…”

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confidence: 99%

RalGDS Couples Growth Factor Signaling to Akt Activation

Hao

Wong

Feig

2008

Molecular and Cellular Biology

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“…negative controls and quantitative analysis (Yoshizaki et al, 2007). Fö rster (or fluorescence) resonance energy transfer (FRET) is a process by which a fluorophore (donor) in an excited state nonradiatively transfers its energy to a neighboring fluorophore (acceptor), thereby causing the acceptor to emit fluorescence at its characteristic wavelength.…”

Section: Introductionmentioning

confidence: 99%

“…Again, such a gradient was not observed in cells expressing the Digda-C67/132S mutant (Supplemental Figure S3B). For the comparison, we also examined the distribution of products of phosphatidylinositol 3-kinase (PI3-kinase), PIP 3 , and PI(3,4)P 2 , in migrating MDCK cells by using the Pippi-type FRET biosensors Pippi-PIP 3 and -PI(3,4)P 2 , having the PH domains of GRP and TAPP1, respectively (Aoki et al, 2007;Yoshizaki et al, 2007). As reported previously (Parent et al, 1998;Servant et al, 2000), both PtdInss were enriched in the front with an increasing gradient toward the leading edge ( Figure 3, C and D).…”

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confidence: 99%

Rapid Turnover Rate of Phosphoinositides at the Front of Migrating MDCK Cells

Nishioka

Aoki

Hikake

et al. 2008

MBoC

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Phosphoinositides (PtdInss) play key roles in cell polarization and motility. With a series of biosensors based on Fö rster resonance energy transfer, we examined the distribution and metabolism of PtdInss and diacylglycerol (DAG) in stochastically migrating Madin-Darby canine kidney (MDCK) cells. The concentrations of phosphatidylinositol (4,5)-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), phosphatidylinositol (3,4)-bisphosphate, and DAG were higher at the plasma membrane in the front of the cell than at the plasma membrane of the rear of the cell. The difference in the concentrations of PtdInss was estimated to be less than twofold between the front and rear of the migrating MDCK cells. To decode the spatial activities of PtdIns metabolic enzymes from the obtained concentration maps of PtdInss, we developed a one-dimensional reaction diffusion model of PtdIns metabolism. In this model, the activities of phosphatidylinositol monophosphate 5-kinase, phosphatidylinositol 3-kinase, phospholipase C, and PIP 3 5-phosphatases were higher at the plasma membrane of the front than at the plasma membrane of the rear of the cell. This result suggests that, although the difference in the steady-state level of PtdInss is less than twofold, PtdInss were more rapidly turned over at the front than the rear of the migrating MDCK cells. INTRODUCTIONCell migration is an important event during early development, inflammatory responses to infection, and wound healing, and it is an important pathological event during tumor invasion and metastasis. Despite morphological and functional differences, different migratory cells share a conserved set of polarity signals. Kinases for phosphoinositides (PtdInss), Rho GTPases, and the actin and microtubule cytoskeletons play key roles in signaling polarity in cells ranging from Dictyostelium discoideum (Charest and Firtel, 2006) to neurons (Luo, 2000;Aoki et al., 2007) and neutrophils (Van Keymeulen et al., 2006;Wong et al., 2006).PtdInss are a family of phospholipids containing myoinositol as their head group (reviewed in Takenawa and Itoh, 2001). Despite a relatively low abundance in biological membranes, PtdInss have been reported to regulate a myriad of cellular processes. Among them, phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ] is the major PtdInss at the inner leaflet of the plasma membrane. On growth factor stimulation, intracellular second messengers such as inositol (1,4,5)-trisphosphate (IP 3 ), diacylglycerol (DAG), and phosphatidylinositol (3,4,5)-triphosphate (PIP 3 ) are generated from PI(4,5)P 2 .The discovery that the pleckstrin homology (PH) domain in the signaling proteins recognizes specific phosphoinositides revealed that stimulated proteins translocate to specific regions of the membrane to participate in signaling events via interaction with these lipids (reviewed in Di Paolo and De Camilli, 2006). A series of experiments indicated that the PH domains from different proteins recognize different PtdInss and led to the development of a novel t...

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“…5). Next, accumulation of mEGFP-Rab5 on the phagosome membrane was revealed with mRFP as a reference of entirely cytosolic protein 11 (Fig. 2b, f).…”

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confidence: 99%

Imaging of Rab5 activity identifies essential regulators for phagosome maturation

Kitano

Nakaya

Nakamura³

et al. 2008

Nature

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131

161

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Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response 1,2 . Rab5 is known as a key regulator of the early endocytic pathway 3 and we have recently shown that Rab5 is also implicated in apoptotic cell engulfment 4 ; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominantnegative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.To make Rab5 activity visible in living cells, we developed a genetically encoded fluorescence resonance energy transfer (FRET) probe, designated Raichu-Rab5. The probe comprised a modified yellow fluorescent protein (YFP) called Venus, the amino-terminal Rab5-binding domain of EEA1, a modified cyan fluorescent protein (CFP) called SECFP, and Rab5 (Fig. 1a). In this probe design, an increase in Rab5-GTP results in an increase in FRET, which can be represented by the 525 nm/475 nm emission ratio. Characterization of Raichu-Rab5 was conducted similarly to that of other Raichu probes reported previously 5,6 . In comparison with the wild-type Rab5 probe, Raichu-Rab5-Q79L-which lacks GTPase activity-had an increased FRET efficiency, whereas Raichu-Rab5-S34N-which shows a reduced affinity for guanine nucleotides-had a decreased FRET efficiency, as expected ( Supplementary Fig. 1a). The GTP loading of the Rab5 probes correlated well with that of the authentic Rab5 proteins ( Supplementary Fig. 1b), and the GTP loadings obtained here were similar to those reported previously 7 . Next, we examined the sensitivity of Raichu-Rab5 to guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) (Supplementary Fig. 1c). Rab5 GEFs such as Rabex-5, Rin1 and Gapex-5 induced a dose-dependent increase in the FRET efficiency of Raichu-Rab5. In contrast, the expression of Rab5 GAPs such as RabGAP-5 and RN-tre decreased the FRET efficiency in a dose-dependent manner. These results indicate that Raichu-Rab5 is capable of monitoring the balance between GEF and GA...

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

Cited by 59 publications

References 63 publications

RalGDS Couples Growth Factor Signaling to Akt Activation

RalGDS Couples Growth Factor Signaling to Akt Activation

Rapid Turnover Rate of Phosphoinositides at the Front of Migrating MDCK Cells

Imaging of Rab5 activity identifies essential regulators for phagosome maturation

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