McCoy F , Darbandi R , Chen SI , Eckard L , Dodd K , Jones K , Baucum AJ , Gibbons JA , Lin SH , Colbran RJ , Nutt LK .

P30CA021765 NCI NIH HHS , R01 MH063232 NIMH NIH HHS, P30 CA021765 NCI NIH HHS , T32 MH065215 NIMH NIH HHS

	FIGURE 1. Activity and dephosphorylation of CaMKII is regulated metabolically. A, G6P inhibits apoptosis in egg extracts. Egg extracts were incubated in the absence or presence of G6P, and samples were taken at the indicated times and analyzed for caspase 3/7 activation using the caspase 3/7 GLO assay (Promega). n = > 3 independent experiments. B, egg extract contains multiple isoforms of CaMKII. Egg extract was treated with or without G6P, and samples were taken and immunoblotted for pan-CaMKII and pCaMKII Thr-286/287. GAPDH was used as a loading control. n = > 3 independent experiments. C, G6P maintains phosphorylation of endogenous CaMKII Thr-286/287. Egg extracts were incubated at room temperature in the absence (upper panel) or presence (lower panel) of G6P. Samples were taken at the indicated times and immunoblotted for CaMKIIα and pCaMKII Thr-286/287. n = > 3 independent experiments. D, G6P inhibits recombinant CaMKIIα Thr-286 dephosphorylation. GST-CaMKIIα (TT305/6AA) bound to glutathione-Sepharose was incubated in egg extract in the presence of G6P and [γ-32P]ATP. Beads were washed and then incubated in a fresh aliquot of egg extract in the absence or presence of G6P. At the indicated times, samples were taken and analyzed for GST-CaMKIIα (TT305/6AA) phosphorylation status by SDS-PAGE, Coomassie Blue staining, and autoradiography. n = > 3 independent experiments.
	FIGURE 2. Inhibition or depletion of PP1 inhibits CaMKII dephosphorylation and apoptosis. A, depletion of PP1 and PP2A inhibits recombinant CaMKIIα Thr-286 dephosphorylation. Upper panel, GST-CaMKIIα (TT305/6AA) was phosphorylated in egg extract in the presence of G6P as in Fig. 1A. Beads were collected, washed, and then incubated in egg extract depleted with Sepharose or microcystin-Sepharose (MC-LR). At the indicated times, samples were taken and analyzed by SDS-PAGE, Coomassie Blue staining, and autoradiography. Lower panel, depletion of PP1 and PP2A was confirmed by immunoblotting for PP1β, PP2A, and GAPDH as a loading control. n = > 3 independent experiments. Seph, Sepharose. B, depletion of PP1 and PP2A inhibits endogenous CaMKII Thr-286/287 dephosphorylation. Upper panel, egg extracts were depleted as in A. Samples were taken at the indicated times and immunoblotted for CaMKIIα and pCaMKII Thr-286/287. Lower panel, depletion of PP1 and PP2A was confirmed by immunoblotting for PP1β, PP2A, and GAPDH as a loading control. n = 3 independent experiments. C, depletion of PP1 and PP2A inhibits apoptosis. At the indicated times, samples were taken from egg extract depleted with Sepharose or MC-LR and analyzed for caspase 3/7 (C3/7) activation using a caspase 3/7 GLO assay (Promega). n = > 3 independent experiments. D, inhibition of PP1 and PP2A with okadaic acid inhibits recombinant CaMKIIα Thr-286 dephosphorylation. Left panel, GST-CaMKIIα (TT305/6AA) was phosphorylated in egg extract in the presence of G6P as in Fig. 1A. Beads were collected, washed, and then incubated in a fresh aliquot of egg extracts containing 1 nM or 10 μM okadaic acid. Samples were taken at the indicated times and analyzed as A. Right panel, the same experiment as displayed in the left panel, conducted independently. n = > 3 independent experiments. E, inhibition of PP1 and PP2A with okadaic acid inhibits endogenous CaMKII Thr-286/287 dephosphorylation. Egg extracts were incubated at room temperature in the presence of 10 μM okadaic acid. Samples were taken at the indicated times and immunoblotted for CaMKIIα and pCaMKII Thr-286/287. n = 2 independent experiments. F, inhibition of PP1 and PP2A with okadaic acid inhibits apoptosis. Egg extracts were incubated in the absence or presence of okadaic acid (1 nM or 10 μM), and samples were taken at the indicated times and analyzed for C3/7 activation using a caspase 3/7 GLO assay (Promega). n = 3 independent experiments.
	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.
	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.
	FIGURE 5. A metabolically regulated factor inhibits PP1 dephosphorylation of CaMKII. A, CaMKII bound to caspase 2 is capable of phosphorylating caspase 2. GST-caspase 2 pro-domain (Pro C2) (WT or S135A) bound to glutathione-Sepharose was incubated in egg extract to bind CaMKII. GST-Pro C2 bound to endogenous CaMKII was retrieved, washed, and incubated in kinase buffer containing [γ-32P]ATP in the absence or presence of 500 μM CaCl2. Beads were washed, eluted, and analyzed for GST-Pro C2 phosphorylation by SDS-PAGE, Coomassie Blue staining, and autoradiography. n = > 3 independent experiments. B, CaMKII and calmodulin are required for Ca2+-induced phosphorylation of caspase 2. CaMKII or calmodulin (CAM) were depleted from egg extracts using CAM-Sepharose or calmodulin binding peptide-Sepharose, respectively. Control depletions were carried out with Sepharose alone. Left panel, selective depletion of CaMKII, and CAM was confirmed by immunoblotting for CaMKIIα, CAM, and GAPDH as a loading control. Right panel, GST-Pro C2 bound to glutathione-Sepharose was incubated in control, CaMKII-depleted, or CAM-depleted egg extracts in the absence or presence of 500 μM CaCl2 and [γ-32P]ATP. Beads were washed and analyzed as in A. n = > 3 independent experiments. C, caspase 2 binds active CaMKII in the presence of G6P. GST-Pro C2 bound to glutathione-Sepharose was incubated in egg extract in the presence of G6P. At the indicated times, beads were collected, washed, eluted, and immunoblotted for pCaMKII Thr-286/287 and GST as a loading control. n = > 3 independent experiments. D, CaMKII bound to caspase 2 is dephosphorylated rapidly when removed from egg extract. GST-Pro C2 bound to glutathione-Sepharose was incubated in egg extract containing G6P. Beads were then collected, washed, and incubated in phosphatase buffer. At the indicated times, beads were collected and analyzed for pCaMKII Thr-286/287 as in C. n = > 3 independent experiments. E, inhibition of PP1 and PP2A with okadaic acid inhibits dephosphorylation of endogenous CaMKII bound to caspase-2. Left panel, GST-Pro C2 bound to glutathione-Sepharose was incubated in egg extracts and G6P in the presence of 10 μM okadaic acid. Beads were collected, washed, and incubated in phosphatase buffer in the absence or presence of 10 μM okadaic acid. At the indicated times, beads were collected and analyzed for pCaMKII Thr-286/287 as in C. n = 3 independent experiments. F, depletion of PP1 and PP2A inhibits dephosphorylation of endogenous CaMKII bound to caspase 2. Left panel, GST-Pro C2 bound to glutathione-Sepharose was incubated in cytosolic fractions of egg extract depleted with Sepharose or microcystin-Sepharose (MC-LR) in the presence of G6P. Beads were collected, washed, and then incubated in phosphatase buffer. At the indicated times, beads were collected and analyzed for pCaMKII Thr-286/287 as in C. Right panel, selective depletion of PP1 and PP2A was confirmed by immunoblotting for PP1β, PP2A, and GAPDH as a loading control. n = 3 independent experiments. G, depletion of PP1 inhibits dephosphorylation of endogenous CaMKII bound to caspase 2. Left panel, GST-Pro C2 bound to glutathione-Sepharose was incubated in egg extract depleted with GST-neurabin in the presence of G6P. Beads were collected, washed, and incubated in phosphatase buffer. At the indicated times, beads were collected and analyzed for pCaMKII Thr-286/287 as in C. Right 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.

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