Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis

AbstractDuring clathrin mediated endocytosis (CME), membrane scission is achieved by the concerted action of dynamin and its interacting partners. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamin’s efficient recruitment and function. First, we show in dynamin triple knock-out cells that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, are partially capable of rescuing CME. However, mutating two motifs largely prevents that ability. To support this observation, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent onesin vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME 2 to 3 times more effectively than with monovalent ones. Finally, the frequency of endocytic events decreases with competing peptides or hypomorphic rescue mutants but the kinetics of dynamin recruitment is unaffected. This suggests that PRD-SH3 interactions act upstream of dynamin accumulation at the neck of nascent vesicles. We conclude from these data that dynamin drives vesicle scissionviamultivalent interactionsin vivo.

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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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The human GRB2 and Drosophila Drk genes can functionally replace the Caenorhabditis elegans cell signaling gene sem-5.

Molecular Biology of the Cell ◽

10.1091/mbc.4.11.1175 ◽

1993 ◽

Vol 4 (11) ◽

pp. 1175-1188 ◽

Cited By ~ 41

Author(s):

M J Stern ◽

L E Marengere ◽

R J Daly ◽

E J Lowenstein ◽

M Kokel ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Cell Signaling ◽

Tyrosine Kinases ◽

Growth Factor Receptor ◽

Sh3 Domains ◽

Signaling Systems ◽

Src Homology ◽

And Function

Mutations in the Caenorhabditis elegans gene sem-5 affect cell signaling processes involved in guiding a class of cell migrations and inducing vulval cell fates. The sem-5 sequence encodes a protein comprised almost exclusively of SH2 and SH3 domains (SH, src homology region) that are found together in many signaling proteins and nonreceptor tyrosine kinases. A human protein, GRB2, was identified by its ability to associate with the activated human epidermal growth factor receptor (hEGFR). The GRB2 and Sem-5 proteins share an identical architecture of their SH2 and SH3 domains and 58% amino acid sequence identity. Here we demonstrate that GRB2 and a Drosophila sem-5-like gene Drk can specifically rescue sem-5 mutants. We also show that Sem-5, like GRB2, can bind to the activated hEGFR in vitro. We further correlate the abilities of several mutant variants of GRB2 and Sem-5 to bind to the hEGFR in vitro with their abilities to functionally replace sem-5 in vivo. These data indicate that GRB2 and Drk are functional homologues of Sem-5 and demonstrate the high degree of conservation of both structure and function between signaling systems throughout evolution.

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Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2720-2729.1992 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2720-2729

Author(s):

L Caron ◽

N Abraham ◽

T Pawson ◽

A Veillette

Keyword(s):

T Cell ◽

Protein Phosphorylation ◽

Phospholipase C ◽

Interleukin 2 ◽

Cell Receptor ◽

Amino Terminal ◽

Src Homology ◽

Phospholipase C Gamma ◽

Domain 3

To understand the mechanism(s) by which p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56lck with the plasma membrane, completely abolished the ability of F505 p56lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.

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FKBP51 Modulates Hippocampal Size and Function in Post-translational Regulation of Parkin

10.21203/rs.3.rs-493867/v1 ◽

2021 ◽

Author(s):

Bin Qiu ◽

Zhaohui Zhong ◽

Shawn Righter ◽

Yuxue Xu ◽

Jun Wang ◽

...

Keyword(s):

Hippocampal Neurons ◽

Translational Regulation ◽

Disease Onset ◽

Fold Increase ◽

Knock Out ◽

Fk506 Binding Protein ◽

Protein Levels ◽

And Function

Abstract FK506-binding protein 51 (encoded by Fkpb51) has been associated with stress-related mental illness. To identify its function, we studied the morphological consequences of Fkbp51 deletion. Artificial Intelligence-assist morphological analysis identified that Fkbp51 knock-out (KO) mice possess more elongated CA and DG but shorter in height in coronal section when compared to WT. Primary cultured Fkbp51 KO hippocampal neurons were shown to exhibit larger dendritic outgrowth than wild-type (WT) controls, pharmacological manipulation experiments suggest that this may occur through regulation of microtubule-associated protein. Both in vitro primary culture and in vivo labeling support that FKBP51 regulates microtubule-associated protein expression. Furthermore, in the absence of differences in mRNA expression, Fkbp51 KO hippocampus exhibited decreases in βIII-tubulin, MAP2, and Tau protein levels, but a greater than 2.5-fold increase in Parkin protein. Overexpression and knock-down FKBP51 demonstrated that FKBP51 negatively regulates Parkin in a dose-dependent and ubiquitin-mediated manner. These results indicate a potential novel post-translational regulatory of Parkin by FKBP51 and significance of their interaction on disease onset.

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Coordinated autoinhibition of F-BAR domain membrane binding and WASp activation by Nervous Wreck

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1524412113 ◽

2016 ◽

Vol 113 (38) ◽

pp. E5552-E5561 ◽

Cited By ~ 17

Author(s):

Tatiana B. Stanishneva-Konovalova ◽

Charlotte F. Kelley ◽

Tania L. Eskin ◽

Emily M. Messelaar ◽

Steven A. Wasserman ◽

...

Keyword(s):

Bound States ◽

Membrane Binding ◽

Structural Basis ◽

Synaptic Growth ◽

Sh3 Domains ◽

Actin Organization ◽

Bar Domain ◽

Src Homology

Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. Here we used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott–Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. Our results define structural rearrangements in Nwk that control F-BAR–membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo.

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The development of functional B lymphocytes in conditional PU.1 knock-out mice

Blood ◽

10.1182/blood-2005-01-0283 ◽

2005 ◽

Vol 106 (6) ◽

pp. 2083-2090 ◽

Cited By ~ 55

Author(s):

Matthew Polli ◽

Aleksandar Dakic ◽

Amanda Light ◽

Li Wu ◽

David M. Tarlinton ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Fetal Liver ◽

Cell Receptor ◽

Interleukin 4 ◽

Knock Out ◽

B Lineage ◽

And Function

Abstract An abundance of research has entrenched the view that the Ets domain containing transcription factor PU.1 is fundamental to the development and function of B lymphocytes. In this study, we have made use of a conditional PU.1 allele to test this notion. Complete deletion of PU.1 resulted in the loss of B cells and all other lineage-positive cells in the fetal liver and death between E18.5 and birth; however, specific deletion of PU.1 in the B lineage had no effect on B-cell development. Furthermore, deletion of PU.1 in B cells did not compromise their ability to establish and maintain an immune response. An increased level of apoptosis was observed in vitro upon B-cell receptor (BCR) cross-linking; however, this was partially rescued by interleukin-4 (IL-4). These findings suggest that PU.1 is not essential for the development of functional B lymphocytes beyond the pre-B stage. (Blood. 2005;106:2083-2090)

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Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck.

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2720 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2720-2729 ◽

Cited By ~ 65

Author(s):

L Caron ◽

N Abraham ◽

T Pawson ◽

A Veillette

Keyword(s):

T Cell ◽

Protein Phosphorylation ◽

Phospholipase C ◽

Interleukin 2 ◽

Cell Receptor ◽

Amino Terminal ◽

Src Homology ◽

Phospholipase C Gamma ◽

Domain 3

To understand the mechanism(s) by which p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56lck with the plasma membrane, completely abolished the ability of F505 p56lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.

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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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A Single-Amino-Acid Substitution in the TvbS1 Receptor Results in Decreased Susceptibility to Infection by Avian Sarcoma and Leukosis Virus Subgroups B and D and Resistance to Infection by Subgroup E In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.02206-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2097-2105 ◽

Cited By ~ 23

Author(s):

Markéta Reinišová ◽

Filip Šenigl ◽

Xueqian Yin ◽

Jiří Plachý ◽

Josef Geryk ◽

...

Keyword(s):

Cultured Cells ◽

Genetic Defect ◽

Single Amino Acid ◽

Receptor Protein ◽

Susceptibility To Infection ◽

Rich Domain ◽

Domain 3 ◽

Avian Sarcoma

ABSTRACT The avian sarcoma and leukosis virus (ASLV) family of retroviruses contains five highly related envelope subgroups (A to E) thought to have evolved from a common viral ancestor in the chicken population. Three genetic loci in chickens determine the susceptibility or resistance of cells to infection by the subgroup A to E ASLVs. Some inbred lines of chickens display phenotypes that are somewhere in between either efficiently susceptible or resistant to infection by specific subgroups of ASLV. The tvb gene encodes the receptor for subgroups B, D, and E ASLVs. The wild-type TvbS1 receptor confers susceptibility to subgroups B, D, and E ASLVs. In this study, the genetic defect that accounts for the altered susceptibility of an inbred chicken line, line M, to infection by ASLV(B), ASLV(D), and ASLV(E) was identified. The tvb gene in line M, tvb r2 , encodes a mutant TvbS1 receptor protein with a substitution of a serine for a cysteine at position 125 (C125S). Here, we show that the C125S substitution in TvbS1 significantly reduces the susceptibility of line M cells to infection by ASLV(B) and ASLV(D) and virtually eliminates susceptibility to ASLV(E) infection both in cultured cells and in the incidence and growth of avian sarcoma virus-induced sarcomas in chickens. The C125S substitution significantly reduces the binding affinity of the TvbS1 receptor for the subgroup B, D, and E ASLV envelope glycoproteins. These are the first results that demonstrate a possible role of the cysteine-rich domain 3 in the function of the Tvb receptors.

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