Ca2+ and Calmodulin Regulate the Binding of Filamin A to Actin Filaments

Fumihiko Nakamura,John H Hartwig,Thomas P Stossel,Pawel T Szymanski

doi:10.1074/jbc.m502203200

Fumihiko Nakamura, John H Hartwig + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m502203200

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Abstract

Filamin A (FLNa) cross-links actin filaments (F-actin) into three-dimensional gels in cells, attaches F-actin to membrane proteins, and is a scaffold that collects numerous and diverse proteins. We report that Ca(2+)-calmodulin binds the actin-binding domain (ABD) of FLNa and dissociates FLNa from F-actin, thereby dissolving FLNa.F-actin gels. The FLNa ABD has two calponin homology domains (CH1 and CH2) separated by a linker. Recombinant CH1 but neither FLNa nor its ABD binds Ca(2+)-calmodulin in the absence of F-actin. Extending recombinant CH1 to include the negatively charged region linker domain makes it, like full-length FLNa, unable to bind Ca(2+)-calmodulin. Ca(2+)-calmodulin does, however, dissociate the FLNa ABD from F-actin provided that the CH2 domain is present. These findings identify the first evidence for direct regulation of FLNa, implicating a mechanism whereby Ca(2+)-calmodulin selectively targets the FLNa.F-actin complex.

Highlights

Regulation of Filamin A (FLNa) dimerization, the actin-binding domain (ABD), or the structure of the intervening ␤-sheet repeats could affect cross-linking of the F-actin of FLNa, purified FLNa constitutively cross-links actin filaments in vitro, and no direct regulation of this activity has emerged in 30 years of research on this protein
The potency of the FLNa F-actin cross-linking activity resides in the large size (280 kDa) of the subunits composed of a string of 24 ␤-pleated sheet repeats, because much higher concentrations of other dimeric F-actin cross-linking proteins with nearly identical ABDs and binding affinities for F-actin are necessary to create gels of actin filaments of identical length
The amount of FLNa required to gel actin in the presence of 2 mM calcium was slightly higher, presumably

Summary

Introduction

Regulation of FLNa dimerization, the ABD, or the structure of the intervening ␤-sheet repeats could affect cross-linking of the F-actin of FLNa, purified FLNa constitutively cross-links actin filaments in vitro, and no direct regulation of this activity has emerged in 30 years of research on this protein. Actin filament-severing proteins such as members of the gelsolin and actin-depolymerizing factor/cofilin family could dismantle such actin networks [3, 4]. Plausible, this mechanism does not explain why a large fraction of cellular FLNa is free of F-actin in vivo. Tel.: 617-355-9000; Fax: 617-355-9016; E-mail: fnakamura@ rics.bwh.harvard.edu. 2 The abbreviations used are: FLNa, filamin A; ABD, actin-binding domain; ABS, actinbinding site; CaM, calmodulin; CBB, Coomassie Brilliant Blue; CH, calponin homology; GST, glutathione S-transferase; HA, hemagglutinin; mAb, monoclonal antibody; N-ABD, N-terminal actin-binding domain; C-ABD, C-terminal actin-binding domain

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