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

Okrut, Julia; Prakash, Sumit; Wu, Qiong; Taunton, Jack

doi:10.1073/pnas.1510876112

Cited by 30 publications

(26 citation statements)

References 59 publications

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“…Arp2/3 was purchased from Cytoskeleton, Inc. Profilin, cofilin, and utrophin actin binding domain (1-261) were purified as previously described (Bieling et al, 2016). 35 Motility assay Similar to a previously described motility assay (Okrut, Prakash, Wu, Kelly, & Taunton, 2015), 2 µl of 0.5% 3 µm streptavidin polystyrene beads (Bangs Laboratories) are incubated with 1 µM biotin-p14 cytoplasmic tail peptide in 10 mM HEPES (pH 7.5), 1 mg/ml BSA and 50 mM KCl 40 for 10 min at room temperature. Peptide-coated beads are diluted eight-fold into motility buffer (10 mM HEPES, 2 mM MgCl2, 50 mM KCl, 50 mM NaCl, 1 mg/ml BSA, 2.5 mM ATP, 5 mM TCEP), containing 0.1 µM Grb2 (20% labeled), 0.2 µM N-WASP, 9 µM actin, 0.075 µM arp2/3, 0.05 µM capping protein, 2.6 µM profilin, 3.5 µM cofilin and incubated for 15 min at room temperature while rotating.…”

Section: Drug Treatmentmentioning

confidence: 99%

A viral fusogen hijacks the actin cytoskeleton to drive cell-cell fusion

Chan

Son

Schmid

et al. 2019

Preprint

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Cell-cell fusion, which is essential for tissue development and used by some viruses to form pathological syncytia, is typically driven by fusogenic membrane proteins with tall (>10 nm) ectodomains that undergo conformational changes to bring apposing membranes in close contact 15 prior to fusion. Here we report that a viral fusogen with a short (<2 nm) ectodomain, the reptilian orthoreovirus p14, accomplishes the same task by hijacking the actin cytoskeleton. We show that the cytoplasmic domain of p14 triggers N-WASP-mediated assembly of a branched actin network, directly coupling local force generation with a short membrane-disruptive ectodomain.This work reveals that overcoming energetic barriers to cell-cell fusion does not require 20 conformational changes of tall fusogens but can instead be driven by harnessing the host cytoskeleton. Impact Statement:A viral fusogen drives cell-cell fusion by hijacking the actin machinery to directly couple actin assembly with a short fusogenic ectodomain. 25

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Section: Drug Treatmentmentioning

confidence: 99%

A viral fusogen hijacks the actin cytoskeleton to drive cell-cell fusion

Chan

Son

Schmid

et al. 2019

Preprint

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“…8). First, N-WASp relies on an Nck SH3 interdomain linker region for its ability to recruit and activate Arp2/3 (Okrut et al, 2015), and this linker also contributes to phase separation (Banjade et al, 2015). Next, the high-affinity binding of N-WASp to Nck disrupts the SH3-mediated autoinhibition of Nck (Takeuchi et al, 2010), which may in turn allow subsequent binding of low-affinity interactors, such as dynamin.…”

Section: Discussionmentioning

confidence: 99%

Multivalent nephrin–Nck interactions define a threshold for clustering and tyrosine-dependent nephrin endocytosis

Martín

New

Phippen

et al. 2020

Journal of Cell Science

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Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the nephrin-Nck (Nck1 or Nck2) complex. Phosphorylation on multiple tyrosine residues within the tail of the nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of nephrin clustering are not well understood. Here, we demonstrate that nephrin phosphorylation regulates the formation of membrane clusters in podocytes. We also reveal a connection between clustering and endocytosis, which appears to be driven by threshold levels of nephrin tyrosine phosphorylation and Nck SH3 domain signaling. Finally, we expose an in vivo correlation between transient changes in nephrin tyrosine phosphorylation, nephrin localization and integrity of the glomerular filtration barrier during podocyte injury. Altogether, our results suggest that nephrin phosphorylation determines the composition of effector proteins within clusters to dynamically regulate nephrin turnover and podocyte health.

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“…the Wave Regulatory Complex). Upstream signaling molecules stimulate the activity of these NPFs by disruption auto-inhibition, either allosterically (Rohatgi et al, 2000;Padrick et al, 2008) or competitively (Okrut et al, 2015). The ability of LC3 to promote actin nucleation by JMY, however, does not involve disrupting an autoinhibitory interaction, but rather the enhancement of a previously unsuspected nucleation activity associated with JMY's N-terminal LIR sequence.…”

Section: Discussionmentioning

confidence: 99%

LC3 and STRAP regulate actin filament assembly by JMY during autophagosome formation

Hu¹,

Mullins²

2018

Preprint

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The actin regulator JMY creates filament networks that move membranes during autophagy. We find that, in unstarved cells, JMY is inhibited by interaction with the STRAP protein, but upon starvation JMY is recruited away from STRAP and activated by LC3. AbstractDuring autophagy actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in non-starved cells, cytoplasmic JMY co-localizes with STRAP, a regulator of JMY's nuclear functions, on non-motile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMY's de novo actin nucleation activity via a cryptic actin-binding sequence near JMY's Nterminus, and STRAP inhibits JMY's ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3-and JMY-dependent actin network formation on membranes, and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMY's actin assembly activities in trans during autophagy.

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

Cited by 30 publications

References 59 publications

A viral fusogen hijacks the actin cytoskeleton to drive cell-cell fusion

A viral fusogen hijacks the actin cytoskeleton to drive cell-cell fusion

Multivalent nephrin–Nck interactions define a threshold for clustering and tyrosine-dependent nephrin endocytosis

LC3 and STRAP regulate actin filament assembly by JMY during autophagosome formation

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