Examining the Specificity of Src Homology 3 Domain–Ligand Interactions with Alkaline Phosphatase Fusion Proteins

Yamabhai, Montarop; Kay, Brian K.

doi:10.1006/abio.1997.2040

Cited by 34 publications

(25 citation statements)

References 42 publications

(10 reference statements)

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“…This motif is supplemented with a partially overlapping second PXXP motif (PPKPKLK). Phage display screens with peptide libraries suggested that the SH3 domain of Abp1 (SH3P7) similar to that of cortactin prefers ϩPPPpKP-motifs (Yamabhai and Kay, 1997). These preferences match exactly with the conserved Abp1 SH3 domain recognition sequence identified in both ProSAPs.…”

Section: Discussionsupporting

confidence: 55%

Linkage of the Actin Cytoskeleton to the Postsynaptic Density via Direct Interactions of Abp1 with the ProSAP/Shank Family

Qualmann

Boeckers²,

Jeromin³

et al. 2004

J. Neurosci.

122

127

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Section: Discussionsupporting

confidence: 55%

Linkage of the Actin Cytoskeleton to the Postsynaptic Density via Direct Interactions of Abp1 with the ProSAP/Shank Family

Qualmann

Boeckers²,

Jeromin³

et al. 2004

J. Neurosci.

122

127

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“…In silico analyses of the sequences of ProSAP1 and ProSAP2 in conjunction with hints on Abp1 SH3 domain-binding preferences from peptide screening (Yamabhai and Kay, 1997) led to the prediction of at least five sites in ProSAP1. Among those, the deduced binding site from amino acids 946 -957 (Qualmann et al, 2004), which we experimentally confirmed by both demonstrating that a peptide containing this site (ProSAP1 930 -972) binds and that a C-terminal deletion mutant lacking the amino acids 946 -957 (ProSAP1 528 -1252 ⌬946 -957) does not bind to the Abp1 SH3 domain (Fig.…”

Section: A Prosap1 Mutant Lacking All Abp1 Binding Sites Is Unable Tomentioning

confidence: 99%

The Actin-Binding Protein Abp1 Controls Dendritic Spine Morphology and Is Important for Spine Head and Synapse Formation

Haeckel¹,

Ahuja²,

Gundelfinger³

et al. 2008

J. Neurosci.

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Polymerization and organization of actin into complex superstructures, including those found in dendritic spines, is indispensable for structure and function of neuronal networks. Here we show that the filamentous actin (F-actin)-binding protein 1 (Abp1), which controls Arp2/3 complex-mediated actin nucleation and binds to postsynaptic scaffold proteins of the ProSAP (proline-rich synapse-associated protein 1)/Shank family, has a profound impact on synaptic organization. Overexpression of the two Abp1 F-actin-binding domains increases the length of thin, filopodia-like and mushroom-type spines but dramatically reduces mushroom spine density, attributable to lack of the Abp1 Src homology 3 (SH3) domain. In contrast, overexpression of full-length Abp1 increases mushroom spine and synapse density. The SH3 domain alone has a dominant-negative effect on mushroom spines, whereas the density of filopodia and thin, immature spines remains unchanged. This suggests that both actin-binding and SH3 domain interactions are crucial for the role of Abp1 in spine maturation. Indeed, Abp1 knockdown significantly reduces mushroom spine and synapse density. Abp1 hereby works in close conjunction with ProSAP1/Shank2 and ProSAP2/Shank3, because Abp1 effects were suppressed by ProSAP2 RNA interference and the ProSAP/ Shank-induced increase of spine head width is further promoted by Abp1 cooverexpression and reduced on Abp1 knockdown. Also, interfering with the formation of functional Abp1-ProSAP protein complexes prevents ProSAP-mediated spine head extension. Spine head extension furthermore depends on local Arp2/3 complex-mediated actin polymerization, which is controlled by Abp1 via the Arp2/3 complex activator N-WASP (neural Wiskott-Aldrich syndrome protein). Abp1 thus plays an important role in the formation and morphology control of synapses by making a required functional connection between postsynaptic density components and postsynaptic actin dynamics.

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“…Microtiter Plate Assays for Peptide Binding to EH Domains-Various peptide sequences were fused genetically to the N terminus of secreted alkaline phosphatase (AP) using published methods (34). Microtiter plate wells, coated with approximately 1 g of GST fusions to EH domains of Intersectin, End3p, or Pan1p, were incubated with different peptide-AP fusions for 1 h. After washing, the amount of enzyme retained in the wells is measured with a colorimetric assay using pnitrophenyl phosphate and a microtiter plate spectrophotometer (Molecular Devices, Sunnyvale, CA) at a wavelength of 405 nm.…”

Section: Isolation Ofmentioning

confidence: 99%

“…Evidence is presented in support of the interaction of Intersectin with two protein components of the endocytic machinery, dynamin and synaptojanin, as well as several novel putative clathrin-binding proteins. (34). Plasmids bearing the cDNA inserts were rescued from the isolated phage by Cre-mediated excision, and the inserts were sequenced by fluorescent dideoxynucleotide sequencing.…”

mentioning

confidence: 99%

Intersectin, a Novel Adaptor Protein with Two Eps15 Homology and Five Src Homology 3 Domains

Yamabhai¹,

Hoffman²,

Hardison³

et al. 1998

Journal of Biological Chemistry

Self Cite

278

308

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We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX 1-2 NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.The EH 1 domain has recently been described as a protein interaction module involved in endocytosis (1). The domain was first discovered in Eps15, a tyrosine kinase phosphorylation substrate of the epidermal growth factor receptor (2). Eps15 is a ϳ145,000 Da protein with three EH repeats and has been shown to be a component of endocytic vesicle intermediates (3-5). Biochemical analysis of the Eps15 EH domains have shown that they are likely involved in protein-protein interactions: far-Western blotting and affinity chromatography experiments demonstrate that a number of cellular proteins can bind to the Eps15 EH fusion protein (2), and recently, several potential cellular ligands have been identified (6). Within the Saccharomyces cerevisiae genome there are five EH domaincontaining proteins, two of which, Pan1 and End3, have been shown to have roles in endocytosis (7-9). Another protein interaction module is the Src homology 3 (SH3) domain. This domain is 50 -70 amino acids long and is present in numerous signal transduction and cytoskeletal proteins (10, 11). Examination of the ligand specificity of SH3 domains has revealed that they recognize proline-rich sequences containing the core peptide sequence PXXP (12, 13). SH3 domains have proposed roles in directing the assembly of NADPH oxidase subunits (14), modulating the activity of phosphatidylinositol 3Ј-kinase (15) and the GTPase activity of dynamin (16), as well as lo...

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Examining the Specificity of Src Homology 3 Domain–Ligand Interactions with Alkaline Phosphatase Fusion Proteins

Cited by 34 publications

References 42 publications

Linkage of the Actin Cytoskeleton to the Postsynaptic Density via Direct Interactions of Abp1 with the ProSAP/Shank Family

Linkage of the Actin Cytoskeleton to the Postsynaptic Density via Direct Interactions of Abp1 with the ProSAP/Shank Family

The Actin-Binding Protein Abp1 Controls Dendritic Spine Morphology and Is Important for Spine Head and Synapse Formation

Intersectin, a Novel Adaptor Protein with Two Eps15 Homology and Five Src Homology 3 Domains

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