Glia maturation factor (GMF) interacts with Arp2/3 complex in a nucleotide state-dependent manner

Abstract

Glia maturation factor (GMF) is a member of the actin-depolymerizing factor (ADF)/cofilin family. ADF/cofilin promotes disassembly of aged actin filaments, whereas GMF interacts specifically with Arp2/3 complex at branch junctions and promotes debranching. A distinguishing feature of ADF/cofilin is that it binds tighter to ADP-bound than to ATP-bound monomeric or filamentous actin. The interaction is also regulated by phosphorylation at Ser-3 of mammalian cofilin, which inhibits binding to actin. However, it is unknown whether these two factors play a role in the interaction of GMF with Arp2/3 complex. Here we show using isothermal titration calorimetry that mammalian GMF has very low affinity for ATP-bound Arp2/3 complex but binds ADP-bound Arp2/3 complex with 0.7 μM affinity. The phosphomimetic mutation S2E in GMF inhibits this interaction. GMF does not bind monomeric ATP- or ADP-actin, confirming its specificity for Arp2/3 complex. We further show that mammalian Arp2/3 complex nucleation activated by the WCA region of the nucleation-promoting factor N-WASP is not affected by GMF, whereas nucleation activated by the WCA region of WAVE2 is slightly inhibited at high GMF concentrations. Together, the results suggest that GMF functions by a mechanism similar to that of other ADF/cofilin family members, displaying a preference for ADP-Arp2/3 complex and undergoing inhibition by phosphorylation of a serine residue near the N terminus. Arp2/3 complex nucleation occurs in the ATP state, and nucleotide hydrolysis promotes debranching, suggesting that the higher affinity of GMF for ADP-Arp2/3 complex plays a physiological role by promoting debranching of aged branch junctions without interfering with Arp2/3 complex nucleation.

Keywords: ADP; ATP; Actin; Arp2/3 Complex; GMF; Isothermal Titration Calorimetry; Protein Phosphorylation.

FIGURE 1.

Analysis by ITC of the binding of GMFγ to Arp2/3 complex. Experiments were conducted at 20 °C. Arp2/3 complex (or actin-latrunculin B) in the cell at 8–10 μm (or 13–15 μm) was titrated with a 14-fold molar excess of GMFγ in 7-μl injections (7-s injections, with an interval of 200 s between injections). A, titration of GMFγ into ADP-Arp2/3 complex. The data were fit to a binding isotherm derived from the integrated heats of binding plotted against the molar ratio of ligand (GMFγ) added to ADP-Arp2/3 complex in the cell after subtracting the heat of dilution. The best fit parameters (solid black line) correspond to a one-site binding model with a dissociation constant of 0.7 μm. B, titration of GMFγ into ATP-Arp2/3 complex. The data could not be fit to a binding isotherm. C, titration of GMFγ into buffer (control experiment). D, titration of GMFγ into ADP-actin (black squares) and ATP-actin (green squares). Note that these two titrations look similar to that of GMFγ into buffer, indicating a complete lack of interaction. E, titration of GMFγ_S2E into ADP-Arp2/3 complex. The data could not be fit to a binding isotherm. F, titration of GMFγ_S2A into ADP-Arp2/3 complex. Each titration was repeated at least two times, and four times for that shown in A. In A, errors are reported as S.E., whereas for the other titrations, errors were derived from curve fitting.

FIGURE 2.

GMFγ does not inhibit actin polymerization by Arp2/3 complex. Shown are the time courses of the fluorescence increase upon polymerization of 2 μm actin (6% pyrene-labeled) alone (black line) and with addition of the indicated proteins (colored lines). A, effect of different concentrations of GMFγ on actin polymerization induced by 20 nm Arp2/3 complex activated by 200 nm WCA_N-WASP. Polymerization rates at 50% polymerization are shown. The lag time (measured as the time to 10% polymerization) was 190 s. B, effect of different concentrations of GMFγ on actin polymerization induced by 20 nm Arp2/3 complex activated by 200 nm WCA_WAVE. Lag times were 229, 245, 277, and 292 s for 0, 1, 2, and 4 μm GMF, respectively. Each measurement was performed three times; one representative curve is shown. Errors are reported as S.E. a.u., arbitrary units.