FIGURE 3.

Depletion of PP1 inhibits apoptosis and dephosphorylation of CaMKII. A, recombinant neurabin binds all isoforms of PP1 but not PP2A. GST or GST-neurabin (Neu) bound to glutathione-Sepharose or microcystin conjugated to Sepharose (MC-LR) was incubated in egg extract. The beads were washed, eluted, and immunoblotted for PP1α, PP1β, PP1γ, PP2A, and GST as a loading control. n = > 3 independent experiments. B, depletion of PP1 inhibits caspase 3/7 activation. Egg extracts were depleted of PP1 with indicated concentrations of GST-neurabin. Immunoblotting confirmed the selective depletion of PP1 isoforms, but not PP2A, by GST-neurabin. Actin was used as a loading control. C, samples were taken from egg extracts depleted with GST or GST-neurabin at the indicated times and analyzed for caspase 3/7 activation using a caspase 3/7 Glo assay (Promega). n = 2 independent experiments. D, depletion of PP1 inhibits caspase 2 processing. Eggs extracts were depleted using GST or GST-neurabin bound to glutathione-Sepharose. Upper panel, egg extract depleted with GST or GST-neurabin was incubated with in vitro-translated (IVT) ³⁵S-labeled full-length caspase 2 (C2). Samples were taken at the indicated times and analyzed for C2 processing by SDS-PAGE and autoradiography. Processing of caspase 2 is indicated by cleavage of full-length caspase 2 (∼45 kDa) to lower molecular weight cleavage products (∼30 kDa and ∼15 kDa). Note the 6-h time point in GST 6 versus Neu 6 (lanes 5 and 6). Lower panel, selective depletion of PP1, but not PP2A, was confirmed by immunoblotting for PP1β, PP2A, and GAPDH as a loading control. n = 3 independent experiments. E, depletion of PP1 inhibits recombinant CaMKIIα Thr-286 dephosphorylation. Upper panel, GST-CaMKIIα (TT305/6AA) bound to glutathione-Sepharose was incubated in egg extract in the presence of [γ-³²P]ATP as in Fig. 1A. Beads were collected, washed, and then incubated in egg extract depleted with GST or GST-neurabin. At the indicated times, samples were taken and analyzed by SDS-PAGE, Coomassie Blue staining, and autoradiography. Lower panel, the same experiment as displayed in the upper panel, conducted independently. n = > 3 independent experiments. F, depletion of PP1 inhibits endogenous CaMKII Thr-286/287 dephosphorylation. Upper panel, egg extracts were depleted of PP1 using GST-neurabin as in Fig. 2B. Samples were taken at the indicated times and immunoblotted for CaMKIIα and pCaMKII Thr-286/287. Long and short exposures of pCaMKII (Thr-286/287) are shown. Lower panel, selective depletion of PP1, but not PP2A, was confirmed by immunoblotting for PP1β, PP2A, and GAPDH as a loading control. n = 2 independent experiments.