The Pleckstrin Homology (PH) Domain of the Arf Exchange Factor Brag2 Is an Allosteric Binding Site

Jian, Xiaoying; Gruschus, James M.; Sztul, Elizabeth; Randazzo, Paul A.

doi:10.1074/jbc.m112.368084

Cited by 37 publications

(62 citation statements)

References 38 publications

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“…For example Syt1p has a Sec7‐homology guanine exchange factor (GEF) domain, and related GEFs in other species have a classical PH domain. The non‐significant hit in Syt1p (E‐values: SMART = 0.002, Pfam not found (>10), SuperFamily = 0.05) has been accepted as PH‐like, because the GEF domain homology strongly enhances the likelihood of a PH‐like domain existing 43, 44…”

Section: Profile‐sequence Tools Currently Used To Define Domainsmentioning

confidence: 99%

Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains

Fidler¹,

Murphy²,

Courtis³

et al. 2016

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Advances in membrane cell biology are hampered by the relatively high proportion of proteins with no known function. Such proteins are largely or entirely devoid of structurally significant domain annotations. Structural bioinformaticians have developed profile‐profile tools such as HHsearch (online version called HHpred), which can detect remote homologies that are missed by tools used to annotate databases. Here we have applied HHsearch to study a single structural fold in a single model organism as proof of principle. In the entire clan of protein domains sharing the pleckstrin homology domain fold in yeast, systematic application of HHsearch accurately identified known PH‐like domains. It also predicted 16 new domains in 13 yeast proteins many of which are implicated in intracellular traffic. One of these was Vps13p, where we confirmed the functional importance of the predicted PH‐like domain. Even though such predictions require considerable work to be corroborated, they are useful first steps. HHsearch should be applied more widely, particularly across entire proteomes of model organisms, to significantly improve database annotations.

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Section: Profile‐sequence Tools Currently Used To Define Domainsmentioning

confidence: 99%

Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains

Fidler¹,

Murphy²,

Courtis³

et al. 2016

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“…29 It also contacts Arf in a manner that positions the GTPase and the canonical PIP 2 -binding site in register to interact with membranes. Remarkably, although BRAG2 Sec7PH is highly active in solution on Arf1 and Arf6, its activity is further increased by more than 2 orders of magnitude by membranes (Table 1), 29,46 approaching the theoretical maximal value of about 10 8 M ¡1 s ¡1 , corresponding to a rate which is limited only by the diffusion of the proteins. 63 Arf-GTP did not modify the activity of BRAG2, indicating that the PH domain is not involved in a feedback mechanism.…”

Section: Regulation Of Ph Domain-containing Arfgefs By Membranesmentioning

confidence: 99%

“…54,55 Whereas the activities and specificities of these 3 subfamilies differ broadly in solution, all are highly potent at activating both Arf1 and Arf6 in the presence of membranes, including EFA6 which is not active on Arf1 in solution. 29,46,49,[56][57][58] These stark differences highlight the importance to assess specificity using membranes and myristoylated Arf GTPases, as this is often the starting point for building models of the biology of the proteins being studied. k cat /K m toward Arf1 in solution and on membranes determined using a range of GEF concentrations are given in Table 1 for Sec-PH constructs of representative members of the cytohesin (ref.…”

Section: Regulation Of Ph Domain-containing Arfgefs By Membranesmentioning

confidence: 99%

“…29,36 Unlike in cytohesins, this extended PH domain is not autoinhibitory. 29,46 The crystal structure of a human BRAG2 construct comprising the Sec7 and PH domains in complex with Arf showed that the PH domain forms extensive intramolecular interactions with the Sec7 domain. 29 It also contacts Arf in a manner that positions the GTPase and the canonical PIP 2 -binding site in register to interact with membranes.…”

Section: Regulation Of Ph Domain-containing Arfgefs By Membranesmentioning

confidence: 99%

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Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

2016

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Arf GTPases assemble protein complexes on membranes to carry out major functions in cellular traffic. An essential step is their activation by guanine nucleotide exchange factors (GEFs), whose Sec7 domain stimulates GDP/GTP exchange. ArfGEFs form 2 major families: ArfGEFs with DCB, HUS and HDS domains (GBF1 and BIG1/BIG2 in humans), which act at the Golgi; and ArfGEFs with a Cterminal PH domain (cytohesin, EFA6 and BRAG), which function at the plasma membrane and endosomes. In addition, pathogenic bacteria encode an ArfGEF with a unique membrane-binding domain. Here we review the allosteric regulation of Arf GTPases and their GEFs at the membrane interface. Membranes contribute several regulatory layers: at the GTPase level, where activation by GTP is coupled to membrane recruitment by a built-in structural device; at the Sec7 domain, which manipulates this device to ensure that Arf-GTP is attached to membranes; and at the level of noncatalytic ArfGEF domains, which form direct or GTPase-mediated interactions with membranes that enable a spectacular diversity of regulatory regimes. Notably, we show here that membranes increase the efficiency of a large ArfGEF (human BIG1) by 32-fold by interacting directly with its Nterminal DCB and HUS domains. The diversity of allosteric regulatory regimes suggests that ArfGEFs can function in cascades and circuits to modulate the shape, amplitude and duration of Arf signals in cells. Because Arf-like GTPases feature autoinhibitory elements similar to those of Arf GTPases, we propose that their activation also requires allosteric interactions of these elements with membranes or other proteins.

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“…Therefore, we overexpressed following constructs in COS7 cells: WTBrag2a-GFP, Brag2a-DSec-GFP lacking the catalytic domain Sec7, which is essential for the activation of GTPases of the Arf family, Brag2a-DPH-GFP lacking the pleckstrin homology domain, which is required for the activation of the enzymatic activity of Brag2 by binding PI(4,5)P2 on membranes [26,45], or EGFP control vector and assessed b1-integrin endocytosis in a pulse-chased endocytosis assay by FACS in GFP ? cells.…”

Section: Brag2 Promotes Endocytosis Of B1-integrins and Recycling Of mentioning

confidence: 99%

Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis

et al. 2014

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b1-Integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated Brag2-silencing reduced EC angiogenic sprouting and migration. Brag2-siRNA transfection differentially affected a5b1-and aVb3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and adhesion on b1-integrin ligands (fibronectin and collagen), while reducing the adhesion on the aVb3-integrin ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of a5b1-integrin, while reducing surface expression of aVb3-integrin. Mechanistically, Brag2-mediated aVb3-integrin-recycling and b1-integrin endocytosis and specifically of the active/matrix-bound a5b1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via b1-integrin endocytosis. Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, b1-integrin endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating b1-integrin internalization and link for the first time the process of b1-integrin endocytosis with angiogenesis.

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The Pleckstrin Homology (PH) Domain of the Arf Exchange Factor Brag2 Is an Allosteric Binding Site

Cited by 37 publications

References 38 publications

Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains

Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis

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