The Brain Exocyst Complex Interacts with RalA in a GTP-dependent Manner

Brymora, Adam; Valova, Valentina A.; Larsen, Martin R.; Roufogalis, Basil D.; Robinson, Phillip J.

doi:10.1074/jbc.c100320200

Cited by 131 publications

(55 citation statements)

References 36 publications

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“…In addition, the anti-sec8 antibody did not immunoprecipitate other exocyst subunits in the absence of sec8 subunit (lane 8). These immunoprecipitation results are consistent with previous in vitro protein binding and yeast two-hybrid studies, which showed that each exocyst subunit can interact with multiple exocyst subunits (4,8,16). The omission of a single exocyst subunit, therefore, would not affect the assembly of other seven subunits into the complex in vitro.…”

Section: E F H and I)supporting

confidence: 80%

“…Temperature-sensitive mutations in any of its eight subunits sec3, sec5, sec6, sec8, sec10, sec15, exo70, and exo84, resulted in defective secretion and polarized growth with concomitant accumulation of intracellular secretory vesicles under non-permissive temperature (1,4). In mammals, the exocyst complex is also large complex similar to that found in yeast (5)(6)(7)(8)(9). The role of this complex in mammals and other multicellular organisms has been mostly studied through perturbation of its function in whole organisms and tissue culture cells.…”

mentioning

confidence: 83%

“…Thus, the multiple, and likely regulated, association of exocyst with various cellular structures may play a role in coordinating protein synthesis, vesicle trafficking and cytoskeletal organization to promote cell growth and cell differentiation. Furthermore, some potential exocyst regulators discovered to date include members of the small GTPase family such as Rho, Ral, Rab, and TC10 which are downstream messengers of many signaling pathways (8,19,(27)(28)(29)(30)(31). The establishment of in vitro exocyst reconstitution assay and the availability of exocyst subunit-specific monoclonal antibodies should allow us to further investigate the regulation of exocyst function in response to various intracellular and extracellular signals during development.…”

Section: Discussionmentioning

confidence: 99%

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The Mammalian Exocyst, a Complex Required for Exocytosis, Inhibits Tubulin Polymerization

Wang

Liu

Adamson

et al. 2004

Journal of Biological Chemistry

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The exocyst is a 734-kDa complex essential for development. Perturbation of its function results in early embryonic lethality. Extensive investigation has revealed that this complex participates in multiple biological processes, including protein synthesis and vesicle/ protein targeting to the plasma membrane. In this article we report that the exocyst may also play a role in modulating microtubule dynamics. Using monoclonal antibodies, we observed that endogenous exocyst subunits co-localized with microtubules and mitotic spindles in normal rat kidney cells. To test for a functional relationship between the exocyst complex and microtubules, we established an in vitro exocyst reconstitution assay and studied exocyst effect on microtubule dynamics. We found that the exocyst complex reconstituted from eight recombinant exocyst subunits inhibited tubulin polymerization in vitro. Deletion of exocyst subunit sec5, sec6, sec15, or exo70 diminished its tubulin polymerization inhibition activity. Surprisingly, exocyst subunit exo70 itself was also capable of inhibiting tubulin polymerization, although exocyst complex with exo70 deletion did not lose its activity completely. Overexpression of exo70 in NRK cells resulted in microtubule network disruption and the formation of filopodia-like plasma membrane protrusions. The formation of these membrane protrusions was greatly hampered by stabilizing microtubules with taxol. Overexpression of exo84, an exocyst subunit that did not show tubulin polymerization inhibition activity, did not cause this phenotype. Results shown in this article, along with a previous report that localized microtubule instability induces plasma membrane addition, implicates a novel role for the exocyst in modulating microtubule dynamics underlying exocytosis.

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Section: E F H and I)supporting

confidence: 80%

mentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

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The Mammalian Exocyst, a Complex Required for Exocytosis, Inhibits Tubulin Polymerization

Wang

Liu

Adamson

et al. 2004

Journal of Biological Chemistry

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“…Our question arising here is whether RalA acts upstream or downstream of Rheb in the nutrient-sensing system, because RalA may exert its effect on diverse processes, such as cytoskeletal reorganization and membrane dynamics (19 -22), which potentially influence the nutrient uptake process. In fact, a Ral effector, the exocyst complex, is involved in vesicle transport (20,39,40); Exo70, an exocyst component, plays a role in targeting of the glucose transporter 4 to the plasma membrane (41,42). Therefore, we first FIGURE 2.…”

Section: Resultsmentioning

confidence: 99%

RalA Functions as an Indispensable Signal Mediator for the Nutrient-sensing System

Maehama

Tanaka

Nishina

et al. 2008

Journal of Biological Chemistry

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Cells sense nutrients present in the extracellular environment and modulate the activities of intracellular signaling systems in response to nutrient availability. This study demonstrates that RalA and its activator RalGDS participate in nutrient sensing and are indispensable for activation of mammalian target of rapamycin complex 1 (mTORC1) induced by extracellular nutrients. Knockdown of RalA or RalGDS abolished amino acidand glucose-induced mTORC1 activation, as judged by phosphorylation of S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. The amount of GTP-bound RalA increased in response to increased amino acid availability. In addition, RalA knockdown suppressed Rheb-induced S6 kinase phosphorylation, and the constitutively active form of RalA induced mTORC1 activation in the absence of Rheb. These results collectively suggest that RalGDS and RalA act downstream of Rheb and that RalA activation is a crucial step in nutrient-induced mTORC1 activation.Nutrients such as amino acids and carbohydrates are available in the extracellular space and are vital for cell homeostasis. Cells are able to detect extracellular nutrients and modulate their intracellular signaling systems in response to changes in nutrient levels. Recent studies have shown that mammalian target of rapamycin complex 1 (mTORC1) 2 functions as a critical intracellular component of the nutrient-sensing system and governs many aspects of cellular responses to changing nutrient levels (1-5). Signaling cascades initiated by growth factors and leading to mTORC1 activation have been extensively characterized. Growth factor stimulation results primarily in the phosphorylation of TSC2, a GTPase-activating protein (GAP) for Rheb GTPase, in a phosphoinositide 3-kinase-and protein kinase B (PKB)-dependent manner, followed by inactivation of TSC2 GAP activity (6 -9). It has been proposed that the decreased GAP activity allows Rheb to stay in an "active" GTPbound form, leading to mTORC1 activation. In contrast to the mechanism of the growth factor-initiated mTORC1 activation, little is known about the mechanism(s) by which mTORC1 activity is regulated by amino acid availability (10 -16). Recent studies have shown that calmodulin and hVps34 play a role in the amino acid-sensing system (12) and that Rag GTPases escort mTORC1 to sites where they can activate the kinase (17, 18). However, most of the amino acid-sensing signaling machinery, including the amino acid sensors themselves, is unknown, and the mechanism(s) underlying the nutrient-sensing process remains to be identified.RalA and RalB GTPases belong to the Ras superfamily and are known to participate in multiple cellular processes through binding to diverse effector proteins. RalA effectors described to date include Ral-binding protein 1, Sec5, Exo84, filamin, and ZO-1-associated nucleic acid-binding protein (19 -22); these interactions are indicative of crucial roles for RalA in cell migration, membrane dynamics, and transcriptional regulation. RalB is highly homologous to R...

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“…Samples (30 l) were taken as indicated, quenched by adding SDS-PAGE loading buffer, and analyzed by Tris/ Tricine-PAGE (16). In-gel trypsin digestion and analysis was performed as described previously (17). Far-UV CD Spectropolarimetry-CD spectra were recorded on a Jasco J-720 spectropolarimeter equipped with a Neslab RTE-111 temperature controller.…”

Section: Protein Expression and Purification-humanmentioning

confidence: 99%

Dimerization of CtIP, a BRCA1- and CtBP-interacting Protein, Is Mediated by an N-terminal Coiled-coil Motif

Dubin

Stokes

Sum

et al. 2004

Journal of Biological Chemistry

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The Brain Exocyst Complex Interacts with RalA in a GTP-dependent Manner

Abstract: Ral is a small

Cited by 131 publications

References 36 publications

The Mammalian Exocyst, a Complex Required for Exocytosis, Inhibits Tubulin Polymerization

The Mammalian Exocyst, a Complex Required for Exocytosis, Inhibits Tubulin Polymerization

RalA Functions as an Indispensable Signal Mediator for the Nutrient-sensing System

Dimerization of CtIP, a BRCA1- and CtBP-interacting Protein, Is Mediated by an N-terminal Coiled-coil Motif

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