The Abl-related gene (Arg) nonreceptor tyrosine kinase uses two F-actin-binding domains to bundle F-actin

Abstract

Abl family nonreceptor tyrosine kinases regulate cellular morphogenesis and motility through functional interactions with the actin cytoskeleton. Although Abl family kinases are known to contain filamentous (F)-actin-binding domains at their C termini, it is unclear how Abl family kinases regulate the structure and/or function of the actin cytoskeleton. We show here that the Abl-related kinase Arg binds with positive cooperativity to F-actin in vitro with binding saturating at a ratio of one Arg/two actin molecules. Measurements of the F-actin-binding properties of Arg deletion mutants led to the identification of a second, previously uncharacterized internal F-actin-binding domain in Arg. Purified Arg can bundle F-actin in vitro, and this bundling activity requires both F-actin-binding domains. An Arg-yellow fluorescent protein fusion protein can induce the formation of actin-rich structures at the lamellipodia of Swiss 3T3 fibroblasts. Both of Arg's F-actin-binding domains are necessary and sufficient for the formation of these actin-rich structures. Together, our data suggest that Arg can use its F-actin-bundling activity to directly regulate actin cytoskeletal structure in vivo.

Figure 1

Arg binds F-actin. (A) Diagram of purified recombinant Arg proteins. (B–F) Cosedimentation of Arg or Arg mutants with F-actin. A fixed concentration of F-actin (1 μM) was mixed with increasing concentrations of Arg from 0.125 to 1 μM (lanes 1–8). For each panel, Arg or the Arg mutants (1 μM) also were centrifuged in the absence of F-actin as a control (lanes 9–10). After ultracentrifugation at 120,000 × g for 30 min, equivalent amounts of pellet (P) and supernatant (S) fractions were subjected to SDS/PAGE followed by Coomassie blue staining. (B) Arg; (C) ArgΔGF; (D) ArgΔInt; (E) ArgΔC; (F) Arg688–1182. A plot of concentration (x axis) vs. amount bound (y axis) for each protein is shown to the right of the binding data. (G) Scatchard analysis of Arg, ArgΔGF, ArgΔInt, and Arg688–1182 binding to F-actin.

Figure 2

Arg bundles F-actin. (A–D) F-actin-bundling activity of Arg and Arg mutants. F-actin (1 μM) was mixed with 0.125–1 μM Arg or Arg mutant proteins (lanes 3–10), and the mixture was centrifuged at 10,000 × g for 10 min to pellet F-actin bundles. Equivalent amounts of the pellet (P) and supernatant (S) fractions were fractionated by SDS/PAGE followed by Coomassie blue staining. As a control in each experiment, F-actin was centrifuged without Arg or Arg mutant (lanes 1 and 2 in each panel). (A) Arg; (B) ArgΔGF; (C) ArgΔInt; (D) Arg688–1182. (E–I) Electron microscopy of F-actin in the presence of Arg (E, F), ArgΔGF (G), and Arg688–1182 (H). F-actin alone is shown also as a control (I). Mixtures of 0.5 μM phalloidin-stabilized F-actin in the absence or presence of 0.5 μM Arg or Arg mutants were incubated for 15 minutes at room temperature before negative staining with 1% uranyl acetate. Bar in E, 5 μm; bar in F, 0.1 μm. G–I were photographed at a magnification similar to F.

Figure 3

Arg contains two independent F-actin binding domains. (A) Diagram of recombinant purified GST-Arg protein fragments. (B–D) Cosedimentation of GST or GST-Arg fragments with F-actin. A fixed concentration of F-actin (1 μM) was mixed with 2.5–10 μM GST (lanes 1–6) or 1.25–10 μM GST-Arg fragment fusion protein (lanes 1–8). For C and D, GST-fusion proteins (10 μM) were centrifuged also in the absence of F-actin as a control (lanes 9–10). After ultracentrifugation at 120,000 × g for 30 min, all the pellet fractions (P) and one third of the supernatant (S) fractions were subjected to SDS/PAGE followed by Coomassie blue staining. (B) GST; (C) GST-694–930; (D) GST-1034–1182. (E) Competitive binding of F-actin-binding domains to F-actin. For each reaction, F-actin (1 μM) was mixed with GST-694–930 (10 μM) and 0–10 μM GST-1034–1182. Samples were incubated, centrifuged, and analyzed as described for C and D.

Figure 4

Arg directs the formation of actin-rich structures at the lamellipodia of Swiss 3T3 cells. Swiss 3T3 cells were transfected with Arg, Arg deletion, or Arg fragment-YFP fusions, plated on coverslips, and fixed 24 h posttransfection. F-actin was visualized with rhodamine phalloidin. (A) Colocalization of Arg-YFP and Arg deletion-YFP fusions with F-actin. (a–l) Arg-YFP; (m–o) ArgΔC-YFP; (p and r) ArgΔGF-YFP; (s–u) ArgΔInt-YFP; (v–x) YFP alone. (d–f) Enlargements of the boxed areas indicated in a–c. (j–l) Enlargements of the boxed areas indicated in g–i. (B) Colocalization of Arg fragment-YFP fusions with F-actin. (a–f) Arg688–1182-YFP; (g–i) Arg688-1034-YFP; (j–l) Arg1034–1182-YFP. (d–f) Enlargements of the boxed areas indicated in a–c.