A lysine-rich cluster in the N-BAR domain of ARF GTPase-activating protein ASAP1 is necessary for binding and bundling actin filaments

Anjelika Gasilina,Hye-Young Yoon,Xiaoying Jian,Ruibai Luo,Paul A Randazzo

doi:10.1016/j.jbc.2022.101700

Anjelika Gasilina, Hye-Young Yoon + Show 3 more

Open Access

https://doi.org/10.1016/j.jbc.2022.101700

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Abstract

Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.

Highlights

Using a Regulated and dynamic organization of the combination of structural modeling, actin cytoskeleton plays an important role in mutagenesis and in vitro and cell-based guiding cellular processes, such as adhesion, assays, we identify a putative binding migration, proliferation, differentiation and interface between the N-BAR domain of survival [1,2,3]
We found that actin and actin filaments into higher order neutralization of charges and charge reversal structures is under control of actin-binding at positions 75, 76, and 79 of ASAP1 reduced proteins, which fall into many families, such binding of ASAP1 BAR-pleckstrin homology (PH) tandem to actin as calponin homology (CH), Wiscott-Aldrich filaments and abrogated actin bundle syndrome protein homology domain-2 (WH2), formation in vitro
Actin binding is not conserved between BAR domains of ACAP1 and ASAP1 ARFGAPs

Summary

Introduction

We and others independently showed that the BAR-PH region of ASAP1 binds to and bundles actin filaments in vitro with similar efficiency as other actin binding proteins and stress fiber alignment. K232A] ASAP1-BAR-PH-His6 triple mutant interaction with F-actin were similar to wild type protein ranging from

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