Mouse A6/Twinfilin Is an Actin Monomer-Binding Protein That Localizes to the Regions of Rapid Actin Dynamics

Abstract

In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin. Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase. In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein. However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro. A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen. In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures. A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/ twinfilin localization to cytoplasm. Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.Actin is a ubiquitous protein, being found in all eukaryotic species and cell types. In muscle cells, actin filaments are stable and organized into highly ordered structures, sarcomeres. These actin filament structures, together with myosin filaments, form the framework for muscle contraction. In other cell types, actin filaments are often very dynamic and form a number of different structures called the actin cytoskeleton. In nonmuscle cells, the actin cytoskeleton is involved in multiple cell biological processes including cell morphogenesis, motility, endo-and exocytosis, and intracellular signal transduction (30).To regulate the structure and dynamics of the actin cytoskeleton, a number of actin-binding proteins have evolved in eukaryotic organisms. These proteins can specifically interact with actin monomers or actin filaments to regulate the structural and biochemical properties of actin monomers and/or filaments. From the recent increase in sequence and structural data, it has become evident that a large number of actinbinding proteins are composed of a relatively small number of protein modules that interact with actin in a specific manner (25, 32). These protein modules include motifs that specifically interact with actin monomers (thymosin-␤-4 repeat) as well as those that interact only with actin filaments (CH domain) (32).An important class of actin-binding modules is the ADF (actin-depolymerizing factor) homology (ADF-H) domain, which has been found in three functionally distinct classes of actin-binding proteins so far. The ADF-H domain is a 15-to 20-kDa protein module that is able to interact both with actin monomers and actin filaments (15). This domain was originally found in cofilin/ADF proteins, which are co...

The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

“…Twinfilin forms a 1:1 complex with G-actin, based on F-actin sedimentation assays, in which twinfilin shifts G-actin to the supernatant in a 1:1 molar ratio (Goode et al, 1998). Furthermore, the migration of twinfilin-actin complex in a sucrose gradient is consistent with a 1:1 molar ratio complex (Vartiainen et al, 2000). Yeast twinfilin inhibits the spontaneous nucleotide exchange of G-actin in a manner similar to that of ADF/cofilins (Hawkins et al, 1993;Hayden et al, 1993;Goode et al, 1998).…”

“…More recently, homologues of yeast twinfilin were identified in mammals, Drosophila melanogaster, Caenorhabditis elegans, and Schizosaccharomyces pombe, suggesting that twinfilins are ubiquitous in eukaryotes from yeast to mammals (Vartiainen et al, 2000;Wahlström et al, 2001). Twinfilins are composed of two ADF/cofilin-like (ADF-H) domains connected by a short linker region and followed by a C-terminal tail (Palmgren et al, 2002).…”

“…It is ubiquitously expressed in mouse and Drosophila tissues and is involved in cytoskeletal remodeling during development (Vartiainen et al, 2000;Wahlströ m et al, 2001). In cells, twinfilin shows diffuse cytoplasmic localization but is also concentrated to actin filament structures (Vartiainen et al, 2000;Palmgren 2001;Wahlströ m et al, 2001).…”

“…All twinfilins characterized to date are actin monomer-binding proteins that prevent actin filament assembly (Goode et al, 1998;Vartiainen et al, 2000;Wahlström et al, 2001). Twinfilin forms a 1:1 complex with G-actin, based on F-actin sedimentation assays, in which twinfilin shifts G-actin to the supernatant in a 1:1 molar ratio (Goode et al, 1998).…”

Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction

“…The second class member twinfilin is expressed in mammalian skeletal and heart muscles 24 . In vitro, twinfilin binds to actin monomers and inhibits actin polymerization 25,26 . While the players of the third class, drebrin 1 (DBN1) and drebrin-like protein (DBNL; also known as Abp1, SH3P7 and HIP-55) have been associated with the function of neurological and immunological synapses [27][28][29][30] , their role in the sarcomere was unknown.…”