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.