A direct interaction with calponin inhibits the actin-nucleating activity of gelsolin

Abstract

Gelsolin and calponin are well-characterized cytoskeletal proteins that are abundant and widely expressed in vertebrate tissues. It is also becoming apparent, however, that they are involved in cell signalling. In the present study, we show that gelsolin and calponin interact directly to form a high-affinity (K(d)=16 nM) 1:1 complex, by the use of fluorescent probes attached to both proteins, by affinity chromatography and by immunoprecipitation. These methods show that gelsolin can form high-affinity complexes with two calponin isoforms (basic h1 and acidic h3). They also show that gelsolin binds calponin through regions that have been identified previously as being calponin's actin-binding sites. Moreover, gelsolin does not interact with calponin while calponin is bound to F-actin. Reciprocal experiments to find calponin-binding sites on gelsolin show that these are in both the N- and C-terminal halves of gelsolin. Calponin has minimal effects on actin severing by gelsolin. In contrast, calponin markedly affects the nucleation activity of gelsolin. The maximum inhibition of nucleation by gelsolin was 50%, which was achieved with a ratio of two calponins for every gelsolin. Thus the interaction of calponin with gelsolin may play a regulatory role in the formation of actin filaments through modulation of gelsolin's actin-binding function and through the prevention of calponin's actin-binding activities.

Calponin binds G‐actin and F‐actin with similar affinity

“…The binding of monomeric actin within the calponin regulatory region is likely to prevent it binding F‐actin and a number of other proteins that calponin binds to. One of these binding partners is gelsolin, this interaction is mediated through the same small region that encompasses the actin binding sites, ABS1 and ABS2 and we have shown that calponin cannot bind gelsolin and F‐actin simultaneously [6]. We anticipate that the binding of G‐actin will also prevent calponin binding to gelsolin, thereby preventing the formation of the gelsolin calponin complex.…”

“…The shift in the emission fluorescence spectra is plotted versus G‐actin concentrations. Inset, quantitative analysis of the above data for the interaction between acrylodan labeled‐calponin and G‐actin was performed by plotting 1/(1− X ) versus C / X ∗ E where C corresponds to concentration of G‐actin expressed in μM and E to concentration of calponin fixed at 0.37 μM [6]. The data fit an extrapolated value of 22 nm for the maximum emission shift.…”

Calponin in Non-Muscle Cells

“…It also binds Ca 2? -calmodulin [1,2], tropomyosin [26], myosin [27,28], the 20-kDa regulatory light chain of myosin [29], desmin [30,31], tubulin [32], caldesmon [33], caltropin [34], gelsolin [35], and phospholipids [36].…”

Regulation of Actin Dynamics by Protein Kinase R Control of Gelsolin Enforces Basal Innate Immune Defense

“…Toward this, an in vitro actin polymerization assay was performed as described elsewhere (Ferjani et al, 2006). In brief, rhodamine-labeled nonnucleated actin monomers were incubated with recombinant functional GSN protein (GSN1-6) or, as a control, a truncated nonfunctional GSN peptide (GSN3-4).…”