We recently identified a novel actin monomer-binding protein from the budding yeast S. cerevisiae
. This protein is composed of two cofilin-like repeats (ADF-H domains) and was therefore named twinfilin (10
). In our database searches, we have also identified homologues of yeast twinfilin in mammals, C. elegans
, and S. pombe
and Fig. ). However, previous biochemical studies with a human homologue of twinfilin suggested that it was a tyrosine kinase (4
In this study, we have reexamined the putative tyrosine kinase activity of purified mouse A6/twinfilin protein, which is 95% homologous to the human A6 tyrosine kinase at the amino acid level. We did not find any evidence for the tyrosine kinase activity with this protein (Fig. ). In this study, we used the same experimental setup (buffers and substrate) as used earlier by Beeler et al. (4
). Because Beeler et al. (4
) also used a recombinant protein produced in E. coli
in their kinase assays, it is unlikely that the difference in source of the A6/twinfilin protein would explain these discrepancies in results. It is also notable that our recombinant protein appeared to be homogenous, monomeric, and active in other biochemical assays (see Materials and Methods and Fig. and ). Therefore, we feel that it is unlikely that these proteins would be tyrosine kinases. This conclusion is also supported by the fact that A6/twinfilins do not show any sequence homology to known protein kinases or have any sequence motifs typical for kinases.
Various biochemical results in our work with mouse A6/twinfilin show that it has actin-related activities similar to those previously reported for yeast twinfilin (10
). Both yeast and mouse proteins appear to form a complex with actin monomers and prevent the actin filament assembly. However, mouse A6/twinfilin appears to be somewhat less efficient in increasing the critical concentration for actin polymerization compared to yeast twinfilin (Fig. C and D) (10
). Migration of the actin-A6/twinfilin complex in sucrose gradients indicate that this protein forms a 1:1 ratio complex with actin monomer (Fig. ). This is in agreement with our previous studies with yeast twinfilin, which showed that twinfilin can prevent the assembly of (sequester) an equal molar amount of actin monomers in solution (10
). In future studies, it will be important to examine why the twinfilin molecule needs two ADF-H domains in order to interact with one molecule of actin and to determine the molecular mechanism whereby it prevents actin filament assembly.
In our previous studies with yeast twinfilin, it was difficult to judge whether this protein interacts with actin filaments, because a fraction of yeast twinfilin pelleted on its own in actin filament cosedimentation assays (10
). However, because the mouse recombinant A6/twinfilin is fully soluble in buffers used in actin cosedimentation assays, we were able to demonstrate that A6/twinfilin does not have a detectable affinity to actin filaments (Fig. A and B). This is also in agreement with the fact that only those residues that have previously been shown to be important for both actin monomer and actin filament binding in yeast cofilin (13
) are conserved in both ADF-H domains in A6/twinfilin proteins (Fig. ). In contrast, the yeast cofilin residues that are important specifically for actin filament interactions are not conserved in A6/twinfilin proteins (Fig. ).
To understand the role of A6/twinfilin in the mouse, we first examined its expression patterns in adult mouse tissues (Fig. A). A6/twinfilin mRNA appears to be expressed in most adult mouse tissues examined in this study. However, A6/twinfilin has only very low expression levels in skeletal muscle and spleen. The majority of actin filaments in skeletal muscle are organized into sarcomeres and are capped at both ends of the filaments (33
). Therefore, these cells are expected to have less actin filament turnover than other cell types and consequently probably lower requirements for proteins involved in actin filament turnover. We have recently also examined the expression patterns of the three different cofilin/ADF mRNAs in adult mouse tissues and noticed that, similarly to A6/twinfilin, skeletal muscle and spleen are the tissues where expression levels of these cofilin mRNAs are lowest (P. Lappalainen et al. unpublished data). Because the role of cofilin in vivo is to increase actin filament turnover (8
), the actin filament dynamics in these tissues probably are slow. Therefore, the expression pattern of A6/twinfilin mRNA in adult mouse tissues is in agreement with its possible role in actin filament turnover in vivo.
We used cultured cells to study the localization of A6/twinfilin at the cellular level. In NIH 3T3 and N18 cells, A6/twinfilin shows a strong cytoplasmic staining but is also concentrated to certain F-actin-rich structures at the cell cortex (Fig. and ). In previous studies, we localized twinfilin in yeast by using a twinfilin-GFP fusion protein. In these experiments the GFP-twinfilin fusion protein showed a diffuse cytoplasmic staining in yeast (10
). However, in the course of our work with mouse A6/twinfilin we also used a GFP-mouse A6/twinfilin construct to localize this protein in various mouse cell lines. As observed with the GFP-twinfilin fusion protein in yeast, the mouse GFP-A6/twinfilin fusion protein showed a diffuse cytoplasmic staining, with no obvious colocalization with the cortical actin cytoskeleton, as can be observed when an antibody is used (data not shown). These results indicate that as a GFP fusion protein, twinfilin does not localize correctly in yeast and mouse cells. We speculate that the GFP at the C terminus of these constructs would interfere twinfilin's interaction with other protein(s) required for its correct localization. Alternatively, the possible elevated expression levels of these GFP constructs could also be responsible for these artifactual localization results. In support of these results, we have recently also examined twinfilin localization in yeast cells by using a polyclonal antibody. These experiments showed that also in yeast the native twinfilin localizes mainly to the cortical actin cytoskeleton (S. Palmgren and P. Lappalainen, unpublished data).
It is important to note that although A6/twinfilin in mouse cells colocalizes with F-actin structures, it does not localize to all actin filament structures. As shown in Fig. and , A6/twinfilin does not colocalize with stress fibers or with certain other cortical actin filament structures in RhoA-induced cells. Interestingly, A6/twinfilin staining in NIH 3T3 cells overlaps with G-actin staining as visualized with fluorescent DNase I (Fig. and ). Because high concentrations of G-actin are believed to localize to the areas of rapid actin filament turnover in cells (9
), the A6/twinfilin localization in these cells is in agreement with its possible role in actin filament turnover. The role of A6/twinfilin in actin filament dynamics is also supported by the overexpression data (Fig. ), which shows that high concentration of A6/twinfilin in NIH 3T3 cells leads to a decrease in the amount of stress fibers and simultaneous appearance of abnormal actin filament structures in these cells.
As shown in Fig. , A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Therefore, it is possible that the A6/twinfilin localization to these cellular compartments is regulated by these small GTPases. In support of this hypothesis, we also showed that in NIH 3T3 cells expressing an activated form of Rac1(V12), A6/twinfilin localizes strongly to the cortical areas in cells, whereas in cells expressing a dominant negative form of Rac1(V12,N17) A6/twinfilin localizes predominantly to the perinuclear area of the cytoplasm (Fig. ). Therefore, we speculate that the GTP-bound form of Rac1 could localize A6/twinfilin to membrane ruffles by a direct activation (posttranslational modification) of A6/twinfilin. Alternatively, the Rac1-induced localization of A6/twinfilin could be mediated by an interaction with some other downstream protein(s) of the Rac1 signaling cascade. It has recently been demonstrated that dephosphorylation of another ADF-H domain protein, cofilin, leads to its localization from cytoplasm to nucleus and to the cortical actin cytoskeleton (19
). It is therefore interesting that in addition to the cortical localization, the expression of Rac1(V12) in NIH 3T3 cells also appears to lead to an increased nuclear localization of A6/twinfilin (Fig. ).
Previous reports have shown that Rac1 induces membrane ruffles and lamellipodium formation in fibroblasts (27
). Two downstream effectors of Rac1 on the actin cytoskeleton have been identified so far. Rac1 may lead to an activation of an actin filament-nucleating Arp2/3 protein complex through interactions with WASP (Wiscott-Aldrich syndrome protein) family proteins (17
). Active Arp2/3 complex promotes actin filament nucleation and assembly in cells (20
). Rac1 also regulates cofilin phosphorylation through LIM kinases (1
). Because phosphorylation of cofilin leads to its inactivation (19
), Rac1 also seems to promote the formation of actin filament structures through a decrease in the rate of actin filament depolymerization. Therefore, the overall effect of Rac1 activation appears to be an increase in actin assembly at the cell cortex. Because Rac1 seems to play a role in A6/twinfilin localization in these cells, we speculate that twinfilin would also be involved in actin assembly. Its role in actin assembly may be to localize actin monomers at the sites of rapid actin filament polymerization. Alternatively, twinfilin might form a complex with some other protein(s) to promote actin filament assembly by a more complex mechanism.
In conclusion, we have shown that mouse A6 protein is a homologue of yeast twinfilin and that it has biochemical activities similar to those of the yeast protein, including a 1:1 actin monomer sequestering activity. In contrast to earlier reports, we did not find any tyrosine kinase activity with this protein. Furthermore, we showed that in mouse cell lines A6/twinfilin localizes to the G-actin-rich cortical actin structures and that its localization to these areas may be regulated by the small GTPase Rac1. These results suggest that A6/twinfilin is involved in the regulation of actin filament dynamics at the cortical actin cytoskeleton.