Interaction of BIG2, a brefeldin A-inhibited guanine nucleotide-exchange protein, with exocyst protein Exo70

Xu, Kai‐Feng; Shen, X. Y.; Li, Hewang; Pacheco–Rodriguez, Gustavo; Moss, Joel; Vaughan, Martha

doi:10.1073/pnas.0409871102

Cited by 48 publications

(35 citation statements)

References 25 publications

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“…1A,B). The protrusions resemble those previously described in Exo70-overexpressing HepG2 and NRK cells Xu et al, 2005). Consistent with findings in these cell types, YFP-Exo70 exhibited predominantly plasma membrane staining in PC12 cells (Fig.…”

Section: Exo70 Is Essential For Neurite Outgrowth and Leads To Membrasupporting

confidence: 78%

An NGF-induced Exo70-TC10 complex locally antagonises Cdc42-mediated activation of N-WASP to modulate neurite outgrowth

Pommereit

Wouters

2007

Journal of Cell Science

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NGF-induced differentiation of PC12 cells is mediated by actin-polymerisation-driven membrane protrusion, involving GTPase signalling pathways that activate actin nucleation promoting factors such as the neural Wiskott-Aldrich syndrome protein (N-WASP). Expression of the exocyst subunit Exo70 in PC12 cells and neurons leads to the generation of numerous membrane protrusions, an effect that is strongly potentiated upon NGF-induced differentiation. Förster resonance energy transfer (FRET) imaging by fluorescence lifetime microscopy (FLIM) reveals an NGF-induced interaction of activated TC10 with Exo70. Expression of dominant-negative mutants and siRNA-mediated knockdown implicates N-WASP in NGF-induced Exo70-TC10-mediated membrane protrusion. However, FRET imaging of N-WASP activation levels of cells expressing Exo70 and/or constitutively active TC10 reveals that this complex locally antagonises the NGF-induced activation of N-WASP in membrane protrusions. Experiments involving siRNA-mediated knockdown of Cdc42 and overexpression of constitutively active Cdc42 confirm that the Exo70-TC10 complex mainly targets the NGF-induced Cdc42-dependent activation of N-WASP. Our results show that Exo70 is responsible for the correct targeting of the Exo70-TC10 complex to sites of membrane protrusion. The functional uncoupling between both pathways represents a novel regulatory mechanism that enables switching between morphologically distinct – Cdc42- or TC10-dominated – forms of cellular membrane outgrowth.

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Section: Exo70 Is Essential For Neurite Outgrowth and Leads To Membrasupporting

confidence: 78%

An NGF-induced Exo70-TC10 complex locally antagonises Cdc42-mediated activation of N-WASP to modulate neurite outgrowth

Pommereit

Wouters

2007

Journal of Cell Science

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“…The absence of nuclear accumulation of BIG1 in cells after depolymerization of microtubules by nocodazole is consistent with their involvement in the delivery of BIG1 for nuclear import. BIG1 was present, along with BIG2 and exocyst proteins, in microtubules purified from HepG2 cells by taxol polymerization (31). It seems most probable that the interactions of BIG1 and BIG2 with microtubules are independent and quite different, both structurally and functionally.…”

Section: Discussionmentioning

confidence: 90%

Effect of protein kinase A on accumulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) in HepG2 cell nuclei

Citterio

Jones

Pacheco–Rodriguez

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Brefeldin A-inhibited guanine nucleotide-exchange proteins, BIG1 and BIG2, are activators of ADP-ribosylation factor GTPases that are essential for regulating vesicular traffic among intracellular organelles. Biochemical analyses and immunofluorescence microscopy demonstrated BIG1 in nuclei as well as membranes and cytosol of serum-starved HepG2 cells. Within 20 min after addition of 8-Br-cAMP, BIG1 accumulated in nuclei, and this effect was blocked by protein kinase A (PKA) inhibitors H-89 and PKI, suggesting a dependence on PKA-catalyzed phosphorylation. BIG2 localization was not altered by cAMP, nor did BIG2 small interfering RNA influence nuclear accumulation of BIG1 induced by cAMP. Mutant BIG1 (S883A) in which Ala replaced Ser-883, a putative PKA phosphorylation site, did not move to the nucleus with cAMP addition, whereas replacement with Asp (S883D) resulted in nuclear accumulation of BIG1 without or with cAMP exposure, consistent with the mechanistic importance of a negative charge at that site. Mutation (712KPK714) of the nuclear localization signal inhibited BIG1 accumulation in nuclei, and PKA-catalyzed phosphorylation of S883, although necessary, was not sufficient for nuclear accumulation, as shown by the double mutation S883D͞ nuclear localization signal. A role for microtubules in cAMP-induced translocation of BIG1 is inferred from its inhibition by nocodazole. Thus, two more critical elements of BIG1 molecular structure were identified, as well as the potential function of microtubules in a novel PKA effect on BIG1 translocation.ADP-ribosylation factor ͉ protein trafficking ͉ A kinase-anchoring protein M ammalian ADP-ribosylation factors (ARFs) comprise six 20-kDa GTP-binding proteins with essential regulatory roles in the formation of membrane trafficking vesicles. These vesicles bud from a donor membrane and fuse with a target membrane to deliver cargo molecules (1-3). Conversion of ARF from a cytosolic GDP-bound inactive state to a GTP-bound active form that is tightly membrane-associated is accelerated by guanine nucleotide-exchange factors (GEFs) (4). All known ARF GEFs contain a Sec7 domain of Ϸ200 aa that catalyzes ARF activation (5-7). The large-molecular-weight BIG1, BIG2, and GBF1, which localize in Golgi regions, are, with different sensitivities, inhibited by the fungal fatty acid metabolite brefeldin A (8-10). The Ϸ200-kDa BIG1 and Ϸ190-kDa BIG2 were first purified together from bovine brain cytosol on the basis of their BFA-inhibited GEF activities (11) and appeared to exist as parts of the same macromolecular complex(es) in HepG2 cells (12). When these cells were growing in medium with serum, BIG1 was primarily cytosolic and Golgi-associated. After overnight incubation without serum, a large fraction of endogenous BIG1 was found in the nuclei localized with nucleoporin p62 at the nuclear envelope (probably in transit between nucleus and cytoplasm), as well as in the nuclear matrix and nucleoli (13).The cAMP-dependent protein kinase A (PKA) is a tetrameric protein comprising two ca...

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“…Very interestingly, the exocyst has also been proposed to use microtubules tracks to target secretory vesicles. Exocyst has been shown to associate and colocalize with microtubules (Vega and Hsu, 2001;Xu et al, 2005). In neuronal cells, this association promotes neurite outgrowth (Vega and Hsu, 2001).…”

Section: Discussionmentioning

confidence: 99%

Direct interaction between exocyst and Wave complexes promotes cell protrusions and motility

Biondini

Sadou-Dubourgnoux

Paul‐Gilloteaux

et al. 2016

Journal of Cell Science

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Coordination between membrane trafficking and actin polymerization is fundamental in cell migration, but a dynamic view of the underlying molecular mechanisms is still missing. The Rac1 GTPase controls actin polymerization at protrusions by interacting with its effector, the Wave regulatory complex (WRC). The exocyst complex, which functions in polarized exocytosis, has been involved in the regulation of cell motility. Here, we show a physical and functional connection between exocyst and WRC. Purified components of exocyst and WRC directly associate in vitro, and interactions interfaces are identified. The exocyst-WRC interaction is confirmed in cells by co-immunoprecipitation and is shown to occur independently of the Arp2/3 complex. Disruption of the exocyst-WRC interaction leads to impaired migration. By using time-lapse microscopy coupled to image correlation analysis, we visualized the trafficking of the WRC towards the front of the cell in nascent protrusions. The exocyst is necessary for WRC recruitment at the leading edge and for resulting cell edge movements. This direct link between the exocyst and WRC provides a new mechanistic insight into the spatio-temporal regulation of cell migration.

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Interaction of BIG2, a brefeldin A-inhibited guanine nucleotide-exchange protein, with exocyst protein Exo70

Cited by 48 publications

References 25 publications

An NGF-induced Exo70-TC10 complex locally antagonises Cdc42-mediated activation of N-WASP to modulate neurite outgrowth

An NGF-induced Exo70-TC10 complex locally antagonises Cdc42-mediated activation of N-WASP to modulate neurite outgrowth

Effect of protein kinase A on accumulation of brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) in HepG2 cell nuclei

Direct interaction between exocyst and Wave complexes promotes cell protrusions and motility

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