Figure 5. CaMKII activation induced by CoA requires basal Ca²⁺/CAM.

A) CoA-Sepharose was incubated with indicated concentrations of rat brain purified CaMKII ± CAM in kinase buffer. CAM-Sepharose was incubated with 100 nM of CaMKII as a positive control (last lane). Beads were immunoblotted for CaMKII. n≥3.

B) The experiment from panel A was repeated using recombinant mouse CaMKIIα, and in the presence of 50 µM EGTA. n≥3.

C) CoA-Sepharose was incubated with 65.6 nM of mouse CaMKIIα (left panel), or 65.6 nM human GST-CaMKIIγ (right panel) in kinase buffer in the presence of 2 mM EGTA. Beads were analyzed as in panel A. n=2.

D) GST-pro C2 was incubated in kinase buffer with [γ-³²P]ATP, mouse CaMKIIα, CAM and either CoA, acetyl CoA (Ac. CoA) or CoA-propan-2-1 (all 10 mM), in the presence of 50 µM EGTA. Beads were analyzed by autoradiography. n=2 (also see Fig. S3).

E) GST-pro C2 was incubated in kinase buffer with [γ -³²P]ATP and rat brain purified CaMKII, ± CAM, 10 mM CoA or 500 µM CaCl₂. Beads analyzed as in panel D. n≥3.

F) GST-pro C2 was incubated in kinase buffer with [γ-³²P]ATP, mouse CaMKIIα (left), human GST-CaMKIIγ (right), CAM and 10 mM CoA, in the presence of 100 µM EGTA. Beads were analyzed as in panel D. n=2.

G) GST-pro C2 was incubated in kinase buffer with [γ-³²P]ATP, mouse CaMKIIα, CAM, 10 mM CoA and indicated concentrations of EGTA. Beads were analyzed as in panel D. n=3.

H) GST-pro C2 was incubated in kinase buffer with [γ-³²P]ATP, CaMKIIα, CAM, CoA, ± indicated concentrations of CaCl₂, and either 100 µM EGTA or 1 mM EGTA. Beads were analyzed as in panel D. n=3.

I) GST-pro C2 was incubated with [γ-³²P]ATP, CaMKIIα, CAM and indicated concentrations of CoA, in the presence of 50 µM EGTA. Beads were analyzed as in panel D. Each data point represents mean ± S.E. of 2.

J) The same experiment as in panel H, assessing the phosphorylation of CaMKIIα. Each data point represents mean ± S.E. of 2.