Regulation of Actin Binding and Actin Bundling Activities of Fascin by Caldesmon Coupled with Tropomyosin

Abstract

Human fascin is an actin-bundling protein and is thought to play a role in the formation of microfilament bundles of microspikes and stress fibers in cultured cells. To explore the regulation of fascin-actin interaction, we have examined the effects of culture cell caldesmon and tropomyosin (TM) on actin binding activity of human fascin. Caldesmon alone or TM alone has little or no effect on the actin binding of fascin. However, caldesmon together with TM completely inhibits actin binding of human fascin. When calmodulin is added, the inhibition of fascin-actin interaction by caldesmon and TM becomes Ca 2؉ dependent because Ca 2؉ /calmodulin blocks actin binding of caldesmon. Furthermore, as phosphorylation of caldesmon by cdc2 kinase inhibits actin binding of caldesmon, phosphorylation can also control actin binding of fascin in the presence of TM. As expected by the inhibition of fascin-actin binding, caldesmon coupled with TM also inhibits actin bundling activity of fascin. Whereas smooth muscle caldesmon alone or TM alone shows no effect, caldesmon together with TM completely inhibits actin bundling activity of fascin. This inhibition is again Ca 2؉ dependent when calmodulin is added to the system. These results suggest important roles for caldesmon and TM in the regulation of the function of human fascin.Fascins belong to a unique family of actin-bundling proteins (1, 2), which include sea urchin fascin (3-8), HeLa 55-kDa actin-bundling protein (9, 10), and the gene products of Drosophila singed (11-13). All of these proteins make F-actin aggregate side-by-side into bundles (4, 8 -10) and are localized in the structures containing actin bundles including filopodia and stress fibers of cultured cells (1, 9, 10, 14), bristles of Drosophila, actin bundles of Drosophila nurse cells (11, 13), and microspikes and microvilli of sea urchin eggs and coelomocytes (6, 7). Some of these structures such as filopodia and microspikes are known to be dynamic structures responding to various biological signals. For example, the fertilization of sea urchin eggs induces the formation of fascin-containing microvilli on their surfaces (7). Filopodia contain fascin-actin bundles and are actively extending and retracting during cell movement of fibroblasts. Fascin should thus be involved in the assembly and disassembly of actin bundles in such structures. However, the mechanisms for the regulation of actin binding of fascin are not well understood.One way to regulate fascin-actin interaction is phosphorylation. We have shown that human fascin is phosphorylated at Ser-39 in vivo in human neuroblastoma cells upon treatment with 12-O-tetradecanoylphorbal-13-acetate, a tumor promoter (16). The same site is phosphorylated by protein kinase C, which results in the inhibition of actin binding of fascin (16,17). However, the stoichiometry of fascin phosphorylation by protein kinase C is low, suggesting that kinases other than protein kinase C may be involved. Furthermore, the stoichiometry of in vivo phosphorylation is also low in the a...

Dual Actin-bundling and Protein Kinase C-binding Activities of Fascin Regulate Carcinoma Cell Migration Downstream of Rac and Contribute to Metastasis

“…Indeed, several molecular processes including the balance between Rac and Rho activities and microtubule targeting coordinately regulate protrusions and focal adhesions (Sander et al, 1999;Wittmann et al, 2003;Goldberg and Kloog, 2006). Although we did not observe alterations of the cell body actin cytoskeleton upon fascin depletion, effects on focal adhesions and thereby cell migration could also be a consequence of a lack of competition between fascin and tropomyosin for binding to contractile actomysin filaments (Ishikawa et al,1998). It is highly likely that disruption of a key intracellular mechanical link would alter the forces exerted on focal adhesions by the actin cytoskeleton.…”

The actin filament-associated protein AFAP-110 is an adaptor protein that modulates changes in actin filament integrity

“…A paradox of these results is that most of the known actin ®lament cross-linking proteins are negatively regulated by PKC phosphorylation which dramatically decreases the abilities of Fascin, MARCKS and VASP to cross-link actin ®laments (Bubb et al, 1999;Harbeck et al, 2000;Hartwig et al, 1992;Huang et al, 1997;Ishikawa et al, 1998;Yamakita et al, 1996). Src phosphorylation similarly inhibits the ability of cortactin to cross-link actin ®laments (Huang et al, 1997).…”

Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function

“…Tropomyosin binding to gelsolin-capped actin filaments has been reported to remove gelsolin from the barbed end and then anneal filaments (49,50). Care was taken to minimize these processes that are proportional to the actin and tropomyosin concentrations and inversely proportional to filament length (50,51).…”

“…Best known for its function in cooperative regulation of actinmyosin interaction (7,8), tropomyosin is now recognized for its role in regulating actin filament stability (9). Tropomyosin stiffens actin filaments (10) and offers protection against severing and depolymerization by gelsolin (11,12), cofilin (13)(14)(15)(16), and DNase I (17). Tropomyosin also inhibits the rate of depolymerization from the pointed end (18,19) and inhibits Arp2/3 complex-nucleated branching (20).…”