FIGURE 4.

Caspase 2 acts as scaffold for, and binds directly to, PP1 and CaMKII. A, FLAG-tagged caspase 2 and CaMKII coprecipitate with recombinant calmodulin. GST or GST-calmodulin (CAM) bound to glutathione-Sepharose was incubated in egg extract containing in vitro-translated FLAG-caspase 2 (C2) full-length in the absence or presence of G6P. Beads were washed, eluted, and immunoblotted for FLAG, CaMKIIα, and pCaMKII Thr-286/287. n = 3 independent experiments. B, CaMKII coprecipitates with full-length caspase 2. GST or GST-C2 (full-length) conjugated to glutathione-Sepharose was incubated in egg extract. Beads were washed, eluted, and immunoblotted for CaMKIIα and GST as a loading control. *, GST-full-length C2; **, GST-full-length C2 (C-terminal truncation, see “Experimental Procedures”). n = > 3 independent experiments. C, CaMKIIα binds directly to recombinant full-length caspase-2. GST, GST-C2 (full-length) or GST-CAM bound to glutathione-Sepharose was incubated in kinase buffer with the indicated concentrations of recombinant CaMKIIα. Beads were washed, eluted, and immunoblotted for CaMKIIα and GST as a loading control. *, GST-full-length C2 (fifth through eighth lanes); **, GST-CAM (tenth lane); ***, GST. n = 3 independent experiments. D, CaMKIIα binds directly to recombinant caspase 2 pro-domain. GST, GST-active C2 (containing the catalytic domain of caspase 2), GST-Pro C2, or GST-CAM was incubated in kinase buffer with indicated concentrations of recombinant CaMKIIα and analyzed as in F. n = 3 independent experiments. E, CaMKII coprecipitates with recombinant PP1. GST or GST-PP1 (α, β, or γ) bound to glutathione-Sepharose was incubated in egg extract. Beads were washed, eluted, and immunoblotted for CaMKIIα. GST-PP1 (α, β, or γ) was detected by Coomassie staining and used as a loading control. *, GST-PP1; **, GST. n = > 3 independent experiments. F, in vitro-translated (IVT) caspase 2 coprecipitates with recombinant PP1. GST or GST-PP1 (α, β, or γ) bound to glutathione-Sepharose was incubated in cytosolic fractions of egg extract containing in vitro-translated ³⁵S-labeled, full-length C2. Beads were washed, eluted, and analyzed by SDS-PAGE and autoradiography. GST and GST-PP1 (α, β, or γ) were detected by Coomassie staining and used as a loading control. *, GST-PP1; **, GST. n = 3 independent experiments. G, in vitro-translated caspase 2 binds directly to recombinant PP1. GST or GST- PP1 (α, β, or γ) conjugated to glutathione-Sepharose was incubated in kinase buffer containing in vitro-translated ³⁵S-labeled, full-length C2. Beads were washed, eluted, and analyzed by SDS-PAGE and autoradiography. GST and GST-PP1 (α, β, or γ) were detected by Coomassie staining and used as loading control. *, GST-PP1; **, GST. n = 2 independent experiments. H, PP1γ binds directly to full-length caspase 2. GST or GST-C2 (full-length) conjugated to glutathione-Sepharose was incubated in kinase buffer containing the indicated concentrations of purified PP1γ. Beads were washed, eluted, and immunoblotted for PP1γ. GST and GST-C2 (full-length) were detected by Coomassie staining and used as a loading control. *, GST-C2 (full-length); **, GST. n = 2 independent experiments.