FIGURE 6.

ArgBP2γ region B interacts with α-actinin. A, the schematic illustrates the constructs used and amino acid sequence alignment of regions that are conserved (B in red and C in orange) comparing human, rat, and zebrafish ArgBP2. B, proteins encoding FLAG-tagged ArgBP2γ, ponsin, Vinexin, ArgBP2γ-ΔB, ArgBP2γ-ΔC, or ArgBP2γ-(S259A) as indicated were transfected into COS-7 cells. The 2% SDS-soluble cell lysate and 2% Triton X-100-soluble cell lysate were probed for either FLAG or α-actinin. The solubility of α-actinin was reduced only in the presence of ArgBP2γ and ArgBP2γ-ΔC. C, FLAG-tagged ArgBP2γ, ArgBP2γ-ΔB, ArgBP2γ-C, ArgBP2γ-(S259A), or ponsin were mixed with an equal volume of cell lysate containing GFP-α-actinin and subjected to anti-FLAG immunoprecipitation. GFP-α-actinin bound FLAG-ArgBP2γ only in the presence of domain B. Ponsin displayed poor binding to GFP-α-Actinin despite its ∼4-fold higher expression compared with ArgBP2γ. D, the schematic shows the domain structure of α-actinin. Various domains of α-actinin were tested for their ability to interact with endogenous ArgBP2; only CH1 appeared necessary to bind ArgBP2. E, FLAG-ArgBP2γ binding to α-actinin is enhanced by F-actin. The Triton X-100-soluble lysates were treated latrunculin A, phalloidin, or Ca²⁺ (10 μm) for 30 min prior to anti-FLAG immunoprecipitation (IP). F, the lysates from COS-7 cells expressing FLAG-ArgBP2, U2-OS cells transfected with either control siRNA or actinin siRNA were probed for actin and actinin levels. The actinin siRNA reduced the level of actinin by 70%. FLAG-ArgBP2γ containing lysates were mixed with those from control or α-actinin siRNA knockdown cells. The level of actin co-immunoprecipitated with ArgBP2γ was reduced when lysates contained less α-actinin. G, ArgBP2 was tested for F-actin co-sedimentation (see “Experimental Procedures”) with or without recombinant α-actinin (CH1 + 2). The top panel shows the blot for the GST-HA-ArgBP2 protein (as indicated) that includes the B domain, and the bottom panel shows recombinant GST-HA-ArgBP2 lacking domain B. The recombinant protein was clarified before incubation with buffer alone, with F-actin alone, or with F-actin + α-actinin(CH1 + 2) as indicated by the horizontal bar. Equal volumes of supernatant (S) or re-suspended pellet (P) fractions (100,000 × g, 1 h) were analyzed by SDS-PAGE and Western blotting.