Allosteric N-WASP activation by an inter-SH3 domain linker in Nck

Actin filament networks assemble on cellular membranes in response to signals that locally activate neural Wiskott–Aldrich-syndrome protein (N-WASP) and the Arp2/3 complex. An inactive conformation of N-WASP is stabilized by intramolecular contacts between the GTPase binding domain (GBD) and the C helix of the verprolin-homology, connector-helix, acidic motif (VCA) segment. Multiple SH3 domain-containing adapter proteins can bind and possibly activate N-WASP, but it remains unclear how such binding events relieve autoinhibition to unmask the VCA segment and activate the Arp2/3 complex. Here, we have used purified components to reconstitute a signaling cascade driven by membrane-localized Src homology 3 (SH3) adapters and N-WASP, resulting in the assembly of dynamic actin networks. Among six SH3 adapters tested, Nck was the most potent activator of N-WASP–driven actin assembly. We identify within Nck a previously unrecognized activation motif in a linker between the first two SH3 domains. This linker sequence, reminiscent of bacterial virulence factors, directly engages the N-WASP GBD and competes with VCA binding. Our results suggest that animals, like pathogenic bacteria, have evolved peptide motifs that allosterically activate N-WASP, leading to localized actin nucleation on cellular membranes.

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ANKRD54 preferentially selects Bruton’s Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library

PLoS ONE ◽

10.1371/journal.pone.0174909 ◽

2017 ◽

Vol 12 (4) ◽

pp. e0174909 ◽

Cited By ~ 1

Author(s):

Manuela O. Gustafsson ◽

Dara K. Mohammad ◽

Erkko Ylösmäki ◽

Hyunseok Choi ◽

Subhash Shrestha ◽

...

Keyword(s):

Tyrosine Kinase ◽

Sh3 Domain ◽

Bruton’S Tyrosine Kinase ◽

Bruton's Tyrosine Kinase ◽

Src Homology 3 ◽

Src Homology

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Src Homology 3 (SH3) Domain

Encyclopedic Reference of Immunotoxicology ◽

10.1007/3-540-27806-0_1377 ◽

2006 ◽

pp. 600-600

Keyword(s):

Sh3 Domain ◽

Src Homology 3 ◽

Src Homology

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Src-Dependent Phosphorylation of ASAP1 Regulates Podosomes

Molecular and Cellular Biology ◽

10.1128/mcb.01781-06 ◽

2007 ◽

Vol 27 (23) ◽

pp. 8271-8283 ◽

Cited By ~ 71

Author(s):

Sanita Bharti ◽

Hiroki Inoue ◽

Kapil Bharti ◽

Dianne S. Hirsch ◽

Zhongzhen Nie ◽

...

Keyword(s):

Splice Variants ◽

Phosphorylation Site ◽

Sh3 Domain ◽

Tyrosine Kinase Signaling ◽

Bar Domain ◽

Src Homology 3 ◽

3T3 Fibroblasts ◽

Src Homology ◽

Nih 3T3 ◽

Interfering Rna

ABSTRACT Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.

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Interaction of Grb2 SH3 domain with UVRAG in an Alzheimer’s disease–like scenario

Biochemistry and Cell Biology ◽

10.1139/bcb-2014-0001 ◽

2014 ◽

Vol 92 (3) ◽

pp. 219-225 ◽

Cited By ~ 1

Author(s):

Kasturi Roy ◽

Oishee Chakrabarti ◽

Debashis Mukhopadhyay

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Growth Factor Receptor ◽

Adaptor Protein ◽

Sh3 Domain ◽

Binding Motifs ◽

Src Homology 3 ◽

Src Homology ◽

Cellular Compartmentalization

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein which participates in trafficking pathways alongside its role in signaling. Proteins important for actin remodeling and cellular compartmentalization contain SRC Homology 3 (SH3) binding motifs that interact with Grb2. While studying the Grb2–amyloid precursor protein (APP) intracellular domain (AICD) interaction in Alzheimer’s disease cell line models, it was seen that Grb2 colocalized to compartments that mature into autophagosomes. The entrapping of AICD in the Grb2 vesicles and its clearance via autophagosomes was a survival contrivance on the part of the cell. Here, we report that Grb2, when in excess, interacts with ultraviolet radiation resistance-associated gene protein (UVRAG) under excess conditions of AICD–Grb2 or Grb2. The N-terminal SH3 domain of Grb2 specifically interacts with UVRAG, unlike the C-terminal SH3 domain. This interaction helps to understand the role of Grb2 in the autophagic maturation of vesicles.

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Asef2 Functions as a Cdc42 Exchange Factor and Is Stimulated by the Release of an Autoinhibitory Module from a Concealed C-Terminal Activation Element

Molecular and Cellular Biology ◽

10.1128/mcb.01608-06 ◽

2006 ◽

Vol 27 (4) ◽

pp. 1380-1393 ◽

Cited By ~ 39

Author(s):

Michael J. Hamann ◽

Casey M. Lubking ◽

Doris N. Luchini ◽

Daniel D. Billadeau

Keyword(s):

Regulatory Mechanism ◽

Sh3 Domain ◽

Terminal Segment ◽

Binding Region ◽

Exchange Factor ◽

Src Homology 3 ◽

Cell Metastasis ◽

Src Homology ◽

And Migration ◽

Exchange Activity

ABSTRACT Asef (herein called Asef1) was identified as a Rac1-specific exchange factor stimulated by adenomatous polyposis coli (APC), contributing to colorectal cancer cell metastasis. We investigated Asef2, an Asef1 homologue having a similar N-terminal APC binding region (ABR) and Src-homology 3 (SH3) domain. Contrary to previous reports, we found that Asef1 and Asef2 exchange activity is Cdc42 specific. Moreover, the ABR of Asef2 did not function independently but acted in tandem with the SH3 domain to bind APC. The ABRSH3 also bound the C-terminal tail of Asef2, allowing it to function as an autoinhibitory module within the protein. Deletion of the C-terminal tail did not constitutively activate Asef2 as predicted; rather, a conserved C-terminal segment was required for augmented Cdc42 GDP/GTP exchange. Thus, Asef2 activation involves APC releasing the ABRSH3 from the C-terminal tail, resulting in Cdc42 exchange. These results highlight a novel exchange factor regulatory mechanism and establish Asef1 and Asef2 as Cdc42 exchange factors, providing a more appropriate context for understanding the contribution of APC in establishing cell polarity and migration.

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Crystal structure of a Src-homology 3 (SH3) domain

Nature ◽

10.1038/359851a0 ◽

1992 ◽

Vol 359 (6398) ◽

pp. 851-855 ◽

Cited By ~ 311

Author(s):

Andrea Musacchio ◽

Martin Noble ◽

Richard Pauptit ◽

Rik Wierenga ◽

Matti Saraste

Keyword(s):

Crystal Structure ◽

Sh3 Domain ◽

Src Homology 3 ◽

Src Homology

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Properties of phagocyte NADPH oxidase p47-phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid

Biochemical Journal ◽

10.1042/bj20021629 ◽

2003 ◽

Vol 373 (1) ◽

pp. 221-229 ◽

Cited By ~ 16

Author(s):

Guihong PENG ◽

Jin HUANG ◽

Mellonie BOYD ◽

Michael E. KLEINBERG

Keyword(s):

Arachidonic Acid ◽

Nadph Oxidase ◽

Sh3 Domain ◽

Free System ◽

Cell Free System ◽

Src Homology 3 ◽

Rich Domain ◽

Phagocyte Nadph Oxidase ◽

Px Domain ◽

Src Homology

In an early step in the assembly of the phagocyte NADPH oxidase, p47-phox translocates from the cytosol to the membrane, mediated by engagement of the N-termini of two p47-phox Src homology 3 (SH3) domains with a proline-rich region (PRR) in the p22-phox subunit of cytochrome b558. In response to phagocyte activation, several serine residues in a C-terminal arginine/lysine-rich domain of p47-phox are phosphorylated, leading to changes in the conformation of p47-phox and exposure of its N-terminal SH3 domain that is normally masked by internal association with the arginine/lysine-rich domain. We report that triple alanine substitutions at Asp-217, Glu-218 and Glu-223 in a short sequence that links the tandem p47-phox SH3 domains unmasked the N-terminal SH3 domain, similar to the effects of aspartic acid substitutions at Ser-310 and Ser-328 in the arginine/lysine-rich region. Recombinant p47-phox proteins with mutations in either the linker region or the arginine/lysine-rich domain were active in the absence of arachidonic acid stimulation in a cell-free NADPH oxidase system consisting of recombinant p67-phox, Rac1–guanosine 5′-[γ-thio]triphosphate and neutrophil membranes. Supplementing neutrophil membranes with phosphoinositides or other negatively charged phospholipids markedly enhanced cell-free superoxide generation by these p47-phox mutants in the absence of arachidonic acid, to levels equivalent to those generated by wild-type p47-phox following arachidonic acid activation. This enhancement may be related to recruitment to the membrane of p47-phox mediated by a novel secondary phox homology (PX) domain binding site that broadly recognizes phospholipids. No specific enhancement by specific phosphorylated phosphatidylinositols was found to suggest a dominant role for the p47-phox primary PX domain binding site. Truncated p47-phox S310D S328D lacking the C-terminal PRR was inactive in the cell-free system without arachidonic acid, but was fully active with arachidonic acid. This suggests that activation of NADPH oxidase in an arachidonate-free cell-free system requires association of the p47-phox C-terminal PRR with the p67-phox C-terminal SH3 domain.

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Coupling of the murine protein tyrosine phosphatase PEST to the epidermal growth factor (EGF) receptor through a Src homology 3 (SH3) domain-mediated association with Grb2

Oncogene ◽

10.1038/sj.onc.1201008 ◽

1997 ◽

Vol 14 (14) ◽

pp. 1643-1651 ◽

Cited By ~ 38

Author(s):

Alain Charest ◽

John Wagner ◽

Mei Kwan ◽

Michel L Tremblay

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Protein Tyrosine Phosphatase ◽

Egf Receptor ◽

Tyrosine Phosphatase ◽

Sh3 Domain ◽

Protein Tyrosine ◽

Src Homology 3 ◽

Src Homology ◽

Epidermal Growth

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Src Homology 3 (SH3) Domain

10.1007/springerreference_33015 ◽

2011 ◽

Keyword(s):

Sh3 Domain ◽

Src Homology 3 ◽

Src Homology

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Intracellular mechanisms of molecular recognition and sorting for transport of large extracellular matrix molecules

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609571113 ◽

2016 ◽

Vol 113 (41) ◽

pp. E6036-E6044 ◽

Cited By ~ 58

Author(s):

Yoshihiro Ishikawa ◽

Shinya Ito ◽

Kazuhiro Nagata ◽

Lynn Y. Sakai ◽

Hans Peter Bächinger

Keyword(s):

Extracellular Matrix ◽

Protein Transport ◽

Transmembrane Protein ◽

Sh3 Domain ◽

Src Homology 3 ◽

Transport Vesicles ◽

Collagen Molecules ◽

Src Homology ◽

Copii Vesicles ◽

Golgi Organization

Extracellular matrix (ECM) proteins are biosynthesized in the rough endoplasmic reticulum (rER) and transported via the Golgi apparatus to the extracellular space. The coat protein complex II (COPII) transport vesicles are approximately 60–90 nm in diameter. However, several ECM molecules are much larger, up to several hundreds of nanometers. Therefore, special COPII vesicles are required to coat and transport these molecules. Transmembrane Protein Transport and Golgi Organization 1 (TANGO1) facilitates loading of collagens into special vesicles. The Src homology 3 (SH3) domain of TANGO1 was proposed to recognize collagen molecules, but how the SH3 domain recognizes various types of collagen is not understood. Moreover, how are large noncollagenous ECM molecules transported from the rER to the Golgi? Here we identify heat shock protein (Hsp) 47 as a guide molecule directing collagens to special vesicles by interacting with the SH3 domain of TANGO1. We also consider whether the collagen secretory model applies to other large ECM molecules.

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