FIGURE 3:

The phosphorylation state of Asl-A controls Plk4 activity. (A) Asl-A-13A enhances Slimb binding to Plk4, whereas Asl-A-13PM diminishes this interaction. Samples of anti-GFP immunoprecipitates were prepared from lysates of S2 cells treated as described in Figure 2B. Immunoblots were probed for GFP, V5, Slimb, and α-tubulin. (B, C) Graphs show relative amounts of Asl-A-V5 (B) or Slimb (C) bound to Plk4-GFP. Values were measured by densitometry of anti-V5 or anti-Slimb immunoblots of IP samples, normalized to the respective Plk4-GFP band intensity, and plotted relative to WT control. n = 3 experiments. ns, not significantly different. (D) Asl-A mutants modulate Plk4 autophosphorylation state. Samples of anti-GFP immunoprecipitates were prepared from lysates of S2 cells treated as described in Figure 2B except, in this experiment, cells were cotransfected with inducible Plk4-myc. Immunoblots were probed for GFP, myc, V5, and α-tubulin. Dashed lines mark Plk4-myc with different electrophoretic mobilities, indicating changes in phosphorylation state. (E) Asl-A-13PM suppresses Plk4-dependent Ana2 phosphorylation. Anti-GFP immunoprecipitates were prepared from lysates of S2 cells expressing GFP-Ana2, nondegradable Plk4-ND-myc, and the indicated Asl-V5 construct. Samples of anti-GFP immunoprecipitates were prepared from lysates of S2 cells treated as described in Figure 2B. Immunoblots were probed with anti-GFP to detect total GFP-Ana2 levels and with anti-pS318 to detect phospho-Ana2.