Activation State-Dependent Substrate Gating in Ca²⁺/Calmodulin-Dependent Protein Kinase II

Figure 2

Maximally active CaMKII holoenzymes enhance the phosphorylation of PSD proteins over monomeric CaMKII. (a–d) Isolated rat PSDs were phosphorylated in low (EGTA) or high calcium (Ca²⁺/CaM) to assess endogenous, copurified kinases. PSDs were also phosphorylated with the addition of equal molar catalytic subunits of T²⁸⁷ autophosphorylated CaMKII multimer (CaMKII_holo^+P = diamond) or monomer (CaMKII_m^+P = circle). (a) Coomassie-stained gel illustrating the PSD profile and the purified proteins used in the reaction. Square indicates CaM. (b) Phosphorimaging of the SDS-PAGE showing ³²P-phosphoprotein bands. (c) Phosphorylation profile scaled to total Coomassie-stained protein. Molecular weight markers indicated along the scaled axis (pixels) representing the scaled lane of the SDS-PAGE. (d) Total phosphorylation for each condition assessed using the area under the curve (AUC) as arbitrary intensity units. (e) Quantitation of Ca²⁺-dependent (Ca²⁺/CaM) and Ca²⁺-independent (EGTA) enzymatic activity (katal/mol) of CaMKII measured via soluble peptide assays with radioactive (γ³²P) ATP. CaMKII, monomer or multimer, was autophosphorylated at T²⁸⁷ prior to the PSD phosphorylation (and activity measurement) (see Materials and Methods) (). Error bars denote ± s.d.

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