A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.

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Capacity of simian immunodeficiency virus strain mac Nef for high-affinity Src homology 3 (SH3) binding revealed by ligand-tailored SH3 domains

Journal of General Virology ◽

10.1099/0022-1317-83-12-3147 ◽

2002 ◽

Vol 83 (12) ◽

pp. 3147-3152 ◽

Cited By ~ 9

Author(s):

Marita Hiipakka ◽

Kalle Saksela

Keyword(s):

Simian Immunodeficiency Virus ◽

Binding Motif ◽

Sh3 Domains ◽

Src Homology 3 ◽

High Affinity ◽

Loop Region ◽

Immunodeficiency Virus ◽

Src Homology ◽

Hiv 1

The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef. Substitution of six amino acids in the RT loop region of Hck-SH3 with the sequence E/DGWWG resulted in SH3 domains that bound in vitro to SIVmac Nef much better than the natural Hck- or Fyn-SH3 domains. These novel SH3 domains also efficiently associated with SIVmac Nef when co-expressed in 293T cells and displayed a strikingly differential specificity when compared with SH3 domains similarly targeted for binding to human immunodeficiency virus type 1 (HIV-1) Nef. Thus, SIVmac Nef is competent for high-affinity SH3 binding, but its natural SH3 protein partners are likely to be different from those of HIV-1 Nef.

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Identification of a Candidate Human Spectrin Src Homology 3 Domain-binding Protein Suggests a General Mechanism of Association of Tyrosine Kinases with the Spectrin-based Membrane Skeleton

Journal of Biological Chemistry ◽

10.1074/jbc.273.22.13681 ◽

1998 ◽

Vol 273 (22) ◽

pp. 13681-13692 ◽

Cited By ~ 63

Author(s):

Dorota Ziemnicka-Kotula ◽

Jiliu Xu ◽

Hong Gu ◽

Anna Potempska ◽

Kwang Soo Kim ◽

...

Keyword(s):

Tyrosine Kinases ◽

Binding Protein ◽

Membrane Skeleton ◽

General Mechanism ◽

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology

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Intracellular targeting, interaction with Src homology 3 (SH3) domains and rolipram-detected conformational switches in cAMP-specific PDE4A phosphodiesterase

Biochemical Society Transactions ◽

10.1042/bst0250374 ◽

1997 ◽

Vol 25 (2) ◽

pp. 374-381 ◽

Cited By ~ 12

Author(s):

M. D. Houslay ◽

G. Scotland ◽

S. Erdogan ◽

E. Huston ◽

S. Mackenzie ◽

...

Keyword(s):

Sh3 Domains ◽

Src Homology 3 ◽

Intracellular Targeting ◽

Src Homology ◽

Conformational Switches

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Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox

Biochemical Journal ◽

10.1042/bj3170919 ◽

1996 ◽

Vol 317 (3) ◽

pp. 919-924 ◽

Cited By ~ 59

Author(s):

Frans B. WIENTJES ◽

George PANAYOTOU ◽

Emer REEVES ◽

Anthony W. SEGAL

Keyword(s):

Nadph Oxidase ◽

Binding Assays ◽

Phagocytic Cells ◽

Sh3 Domains ◽

Cytosolic Proteins ◽

Src Homology 3 ◽

Vitro Binding ◽

Src Homology ◽

Flavocytochrome B

The NADPH oxidase of neutrophils and other bone-marrow-derived phagocytic cells is a multi-component system consisting of a flavocytochrome b in the plasma membrane and at least four cytosolic proteins. Three of the cytosolic proteins contain src homology 3 (SH3) domains, two each in p47phox and p67phox, and one in p40phox. All three translocate from the cytosol to the flavocytochrome in the membrane upon stimulation of the cells. A small G-protein, p21rac, is also involved in activation of the oxidase. The three cytosolic phox proteins occur as a complex in the cytosol and the strongest interaction appeared to be between p67phox and p40phox. We have investigated the interaction between p40phox and the other two cytosolic phox proteins by in vitro binding assays. An affinity-bead approach was used as well as a biosensor technique (surface plasmon resonance). We observed the strongest attachment between p40phox and p67phox where the binding was between the N-terminal half of p67phox and the C-terminal half of p40phox, and did not appear to involve SH3 domains and proline-rich sequences. p40phox also bound p47phox but more weakly than it did p67phox.

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Author response: A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones

10.7554/elife.06935.026 ◽

2015 ◽

Author(s):

Matthias Siebert ◽

Mathias A Böhme ◽

Jan H Driller ◽

Husam Babikir ◽

Malou M Mampell ◽

...

Keyword(s):

Binding Protein ◽

Author Response ◽

High Affinity ◽

Active Zones

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αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts

Biochemical Journal ◽

10.1042/bj20041502 ◽

2005 ◽

Vol 388 (2) ◽

pp. 631-638 ◽

Cited By ~ 35

Author(s):

Björn ROTTER ◽

Odile BOURNIER ◽

Gael NICOLAS ◽

Didier DHERMY ◽

Marie-Christine LECOMTE

Keyword(s):

Binding Protein ◽

Focal Adhesions ◽

Membrane Skeleton ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Binding Protein ◽

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of α- and β-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent α-spectrin subunit in non-erythroid cells, αII-spectrin, contains two particular spectrin repeats in its central region, α9 and α10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the α9-α10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the α10 repeat of αII-spectrin whereas EVL interacts with the Src homology 3 domain located within the α9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between αII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

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A Deubiquitinating Enzyme UBPY Interacts with the Src Homology 3 Domain of Hrs-binding Protein via a Novel Binding Motif PX(V/I)(D/N)RXXKP

Journal of Biological Chemistry ◽

10.1074/jbc.m007251200 ◽

2000 ◽

Vol 275 (48) ◽

pp. 37481-37487 ◽

Cited By ~ 168

Author(s):

Masaki Kato ◽

Keiji Miyazawa ◽

Naomi Kitamura

Keyword(s):

Binding Protein ◽

Binding Motif ◽

Deubiquitinating Enzyme ◽

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology

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A Novel Phosphorylation-Dependent RNase Activity of GAP-SH3 Binding Protein: a Potential Link between Signal Transduction and RNA Stability

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.3956 ◽

1998 ◽

Vol 18 (7) ◽

pp. 3956-3965 ◽

Cited By ~ 126

Author(s):

Imed-eddine Gallouzi ◽

Fabienne Parker ◽

Karim Chebli ◽

Florence Maurier ◽

Emmanuel Labourier ◽

...

Keyword(s):

Binding Protein ◽

Protein A ◽

Rna Stability ◽

Chinese Hamster ◽

Rnase Activity ◽

Lung Fibroblasts ◽

Src Homology 3 ◽

Quiescent Cells ◽

Potential Link ◽

Src Homology

ABSTRACT A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serum-stimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity. Indeed, G3BP harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3′-untranslated region of human c-myc mRNA. The endoribonuclease activity of G3BP can initiate mRNA degradation and therefore represents a link between a RasGAP-mediated signaling pathway and RNA turnover.

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Intersectin associates with synapsin and regulates its nanoscale localization and function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715341114 ◽

2017 ◽

Vol 114 (45) ◽

pp. 12057-12062 ◽

Cited By ~ 19

Author(s):

Fabian Gerth ◽

Maria Jäpel ◽

Arndt Pechstein ◽

Gaga Kochlamazashvili ◽

Martin Lehmann ◽

...

Keyword(s):

Synaptic Vesicles ◽

Synapsin I ◽

Wild Type ◽

Src Homology 3 ◽

Reserve Pool ◽

Hippocampal Synapses ◽

Important Regulator ◽

Src Homology ◽

Elevated Activity ◽

And Function

Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery.

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