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

Autor:	Gasilina A; Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, District of Columbia, USA., Yoon HY; Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA., Jian X; Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA., Luo R; Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA., Randazzo PA; Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. Electronic address: randazzp@mail.nih.gov.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2022 Mar; Vol. 298 (3), pp. 101700. Date of Electronic Publication: 2022 Feb 08.
DOI:	10.1016/j.jbc.2022.101700
Abstrakt:	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. Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2022. Published by Elsevier Inc.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu