Dynamin-2 Phosphorylation as A Critical Regulatory Target of Bin1 and GSK3α for Endocytosis in Muscle

Tight regulation of endocytosis ensures accurate control of cellular signaling and membrane dynamics, which are crucial for tissue morphogenesis and functions. Mutations of Bin1 and dynamin-2 (Dyn2), proteins that generate membrane curvature and sever endocytic invaginations, respectively, cause progressive hereditary myopathy. Here, we show that Bin1 inhibits Dyn2 via direct interaction of its SRC Homology 3 (SH3) domain with the proline-rich domain (PRD) of Dyn2. Phosphorylation of S848 of Dyn2 by GSK3α, a kinase downstream of insulin signaling, relieves Dyn2 from the inhibition of Bin1 and promotes endocytosis in muscle. Mutations of Bin1 associated with centronuclear myopathy disrupt its inhibition of Dyn2, thereby exaggerating Dyn2 fission activity and causing excessive fragmentation of T-tubules in the muscle cells. Our work reveals how Bin1-Dyn2 interaction fine-tunes membrane remodeling at the molecular level, and lay the foundation for future exploration of endocytic regulation and hereditary muscle diseases.

Download Full-text

Role of the Proline-rich Domain of Dynamin-2 and Its Interactions with Src Homology 3 Domains during Endocytosis of the AT1Angiotensin Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m200778200 ◽

2002 ◽

Vol 277 (24) ◽

pp. 21650-21656 ◽

Cited By ~ 22

Author(s):

Márta Szaszák ◽

Zsuzsanna Gáborik ◽

Gábor Turu ◽

Peter S. McPherson ◽

Adrian J. L. Clark ◽

...

Keyword(s):

Src Homology 3 ◽

Rich Domain ◽

Src Homology ◽

Dynamin 2

Download Full-text

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.

Download Full-text

Insights into the structure and protein–protein interactions of the pro-apoptotic protein ASPP2

Biochemical Society Transactions ◽

10.1042/bst0350966 ◽

2007 ◽

Vol 35 (5) ◽

pp. 966-969 ◽

Cited By ~ 10

Author(s):

S. Rotem ◽

C. Katz ◽

A. Friedler

Keyword(s):

Intermolecular Interactions ◽

Protein Interactions ◽

Intramolecular Interaction ◽

Protein Protein Interactions ◽

Sh3 Domains ◽

Src Homology 3 ◽

Rich Domain ◽

C Terminus ◽

Apoptotic Protein ◽

Src Homology

ASPP (apoptosis-stimulating protein of p53) 2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. Here, we provide an overview of the structure and protein–protein interactions of ASPP2. The C-terminus of ASPP2 contains Ank (ankyrin) repeats and an SH3 domain (Src homology 3 domain). The Ank–SH3 domains mediate interactions between ASPP2 and numerous proteins involved in apoptosis such as p53 and Bcl-2. The proline-rich domain of ASPP2 is unfolded in its native state, but was not shown to mediate intermolecular interactions. Instead, it makes an intramolecular domain–domain interaction with the Ank–SH3 C-terminal domains of ASPP2. This intramolecular interaction between the unstructured proline-rich domain and the structured Ank–SH3 domains in ASPP2, which is possible due to the unfolded nature of the proline-rich domain, is proposed to have an important role in regulating the intermolecular interactions of ASPP2 with its partner proteins.

Download Full-text

Direct Interaction between the Cytoplasmic Tail of ADAM 12 and the Src Homology 3 Domain of p85α Activates Phosphatidylinositol 3-Kinase in C2C12 Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m101162200 ◽

2001 ◽

Vol 276 (27) ◽

pp. 24466-24472 ◽

Cited By ~ 36

Author(s):

Qing Kang ◽

Yi Cao ◽

Anna Zolkiewska

Keyword(s):

Direct Interaction ◽

Cytoplasmic Tail ◽

C2c12 Cells ◽

Phosphatidylinositol 3 Kinase ◽

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology ◽

Adam 12 ◽

Phosphatidylinositol 3

Download Full-text

Induced Expression and Association of the Mona/Gads Adapter and Gab3 Scaffolding Protein during Monocyte/Macrophage Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.22.11.3744-3756.2002 ◽

2002 ◽

Vol 22 (11) ◽

pp. 3744-3756 ◽

Cited By ~ 30

Author(s):

Caroline Bourgin ◽

Roland P. Bourette ◽

Sylvie Arnaud ◽

Yan Liu ◽

Larry R. Rohrschneider ◽

...

Keyword(s):

Bone Marrow Cells ◽

Sh2 Domain ◽

Scaffolding Protein ◽

Mouse Bone Marrow ◽

Macrophage Differentiation ◽

Src Homology 3 ◽

Rich Domain ◽

Src Homology ◽

Macrophage Colony ◽

Carboxy Terminal

ABSTRACT Mona/Gads is a Grb2-related, Src homology 3 (SH3) and SH2 domain-containing adapter protein whose expression is restricted to cells of hematopoietic lineage (i.e., monocytes and T lymphocytes). During monocyte/macrophage differentiation, Mona is induced and interacts with the macrophage colony-stimulating factor receptor, M-CSFR (also called Fms), suggesting that Mona could be involved in developmental signaling downstream of the M-CSFR by recruiting additional signaling proteins to the activated receptor. Our present results identify Mona as a specific partner protein for the DOS/Gab family member Gab3 in monocytic/macrophage development. Mona does not interact with Gab2; however, Gab3 also forms a complex with the Mona-related adapter Grb2. Glutathione S-transferase pull-down experiments demonstrate that the Mona and Gab3 interaction utilizes the carboxy-terminal SH3 domain of Mona and the atypical proline-rich domain of Gab3. Mona is known to interact with the phosphorylated Y697 site of the M-CSFR. The M-CSFR mutation Y697F exhibited qualitative and quantitative abnormalities in receptor and Gab3 tyrosine phosphorylation, and Mona induction was greatly reduced. The Y807F M-CSFR mutation is defective in differentiation signaling, but not growth signaling, and also fails to induce Mona protein expression. During M-CSF-stimulated macrophage differentiation of mouse bone marrow cells, Mona and Gab3 expression is coinduced, these proteins interact, and Mona engages in multimolecular complexes. These data suggest that association of Mona and Gab3 plays a specific role in mediating the M-CSFR differentiation signal.

Download Full-text

Kinetics of Src Homology 3 Domain Association with the Proline-rich Domain of Dynamins

Journal of Biological Chemistry ◽

10.1074/jbc.m501745200 ◽

2005 ◽

Vol 280 (24) ◽

pp. 23147-23156 ◽

Cited By ~ 44

Author(s):

Elena Solomaha ◽

Frances L. Szeto ◽

Mohammed A. Yousef ◽

H. Clive Palfrey

Keyword(s):

Src Homology 3 ◽

Rich Domain ◽

Src Homology 3 Domain ◽

Src Homology ◽

Kinetics Of

Download Full-text

Endophilin Recruitment via GPCR Interactions Enables Membrane Curvature Generation in the Absence of Anionic Lipids

10.1101/2020.07.11.198937 ◽

2020 ◽

Author(s):

Samsuzzoha Mondal ◽

Imania Powers ◽

Karthik Narayan ◽

Samuel Botterbusch ◽

Tobias Baumgart

Keyword(s):

Electrostatic Interactions ◽

Membrane Lipids ◽

Direct Interaction ◽

Sh3 Domain ◽

Membrane Curvature ◽

Receptor Internalization ◽

Intracellular Loop ◽

Bar Domain ◽

Src Homology ◽

Anionic Lipids

AbstractThe Bin/Amphiphysin/Rvs (BAR) family protein endophilin plays key roles in membrane curvature generation during endocytosis of cellular receptors. The Src homology 3 (SH3) domain of endophilin interacts with the proline rich third intracellular loop (TIL) of various G-protein coupled receptors (GPCRs). While electrostatic interactions between BAR domain and anionic membrane lipids have been considered to be the major driving force in curvature generation, it is unclear how the direct interaction between TIL and SH3 affects this function and its coupling with receptor internalization. Here we show that TIL mediated interactions alone not only recruit endophilin to the membrane but also facilitate curvature sorting and curvature generating behavior of endophilin. To demonstrate this, we designed model membranes with covalently lipid-conjugated TIL and lipids without net negative charge so that endophilin was recruited exclusively via SH3/TIL interactions. We find curvature generation and curvature sorting under those conditions. Furthermore, we show that TIL interacts electrostatically with membranes in the presence of anionic lipids and that this interaction can interfere with binding of SH3. Overall, our study suggests that an interplay between TIL, charged membranes, BAR domain, and SH3 domain mediate membrane curvature generation to regulate receptor endocytosis following receptor stimulation.

Download Full-text