Tài liệu Báo cáo khoa học: Inorganic phosphate regulates the binding of cofilin to actin filaments pdf

Inorganic phosphate regulates the binding of cofilin to

actin filaments

Andras Muhlrad1

, Dmitry Pavlov2

, Y. Michael Peyser1 and Emil Reisler2

1 Institute of Dental Sciences, School of Dental Medicine, Hebrew University of Jerusalem, Israel

2 Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, USA

Actin dynamics, the polymerization and depolymeriza￾tion of actin filaments and formation of ordered actin

assemblies, is critical to many events of cell motility,

including the movement of whole cells, cell division,

vesicular transport and exo- and endocytosis. The

essential processes of actin dynamics are closely regula￾ted in the cell by a large number of actin binding pro￾teins and small molecules. The large family of actin

depolymerizing factor (ADF)⁄ cofilin (AC) proteins [1]

has a central role in regulating actin dynamics. These

small proteins, which are ubiquitous in all eukaryotic

cells, increase the depolymerization and nucleation of

actin filaments and accelerate their treadmilling [2].

AC proteins accelerate the turnover of actin filaments

by severing them [3–6], thereby increasing the number

of the free pointed and barbed ends, or by increasing

monomer dissociation from the pointed end of fila￾ments [7], and ⁄ or by both processes [8–10]. The action

of these proteins on actin is promoted in most cases by

increasing pH [4], and is regulated by phosphatidyl

inositides [11,12] and their phosphorylation (except for

yeast cofilin) by kinases [13,14].

The binding of ADF⁄ cofilin to F-actin induces large

conformational changes in the structure of actin fila￾ments, including changes in their mean twist [15], and

the weakening of longitudinal contacts between proto￾mers along the long-pitch helix [16,17] and lateral contacts

between the two strands [18,19]. According to electron

microscopy evidence, the weakening of longitudinal

contacts in F-actin is due to conformational changes in

subdomain 2 of actin, including the disordering of the

DNase I binding loop (D-loop) [20]. Solution studies

also showed cofilin induced conformational changes in

actin, particularly in subdomain 2 and its D-loop. The

fluorescence intensity of probes [tetramethyl rhodamine

cadaverine (TRC) and dansyl diethylamine] attached to

Gln41 on the D-loop decreased dramatically upon bind￾ing of cofilin to F-actin [17,21]. Moreover, subtilisin

Keywords

actin; cofilin; collisional quenching;

fluorescence; limited proteolysis

Correspondence

A. Muhlrad, Institute of Dental Sciences,

School of Dental Medicine, Hebrew

University of Jerusalem, PO Box 12272,

Jerusalem 91120, Israel

Fax: +972 2 675 8561

Tel: +972 2 675 7587

E-mail: [email protected]

(Received 3 January 2006, accepted 8

February 2006)

doi:10.1111/j.1742-4658.2006.05169.x

Inorganic phosphate (Pi) and cofilin ⁄ actin depolymerizing factor proteins

have opposite effects on actin filament structure and dynamics. Pi stabilizes

the subdomain 2 in F-actin and decreases the critical concentration for

actin polymerization. Conversely, cofilin enhances disorder in subdomain 2,

increases the critical concentration, and accelerates actin treadmilling. Here,

we report that Pi inhibits the rate, but not the extent of cofilin binding to

actin filaments. This inhibition is also significant at physiological concen￾trations of Pi, and more pronounced at low pH. Cofilin prevents conforma￾tional changes in F-actin induced by Pi, even at high Pi concentrations,

probably because allosteric changes in the nucleotide cleft decrease the

affinity of Pi to F-actin. Cofilin induced allosteric changes in the nucleotide

cleft of F-actin are also indicated by an increase in fluorescence emission

and a decrease in the accessibility of etheno-ADP to collisional quenchers.

These changes transform the nucleotide cleft of F-actin to G-actin-like.

Pi regulation of cofilin binding and the cofilin regulation of Pi binding to

F-actin can be important aspects of actin based cell motility.

Abbreviations

AC, ADF ⁄ cofilin; ADF, actin depolymerizing factor; D-loop, DNase I binding loop; Ksv, Ksv ¼ [(F0 ⁄ F))1]Æquencher M)1

, where F0 and F are

fluorescence values in the presence and absence of quencher respectively; Pi, inorganic phosphate; TRC, tetramethyl rhodamine cadaverine.

1488 FEBS Journal 273 (2006) 1488–1496 ª 2006 The Authors Journal compilation ª 2006 FEBS