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

Figure 3

Enhanced phosphorylation of intermediate and weak SPOT substrates with increasing number of active subunits per holoenzyme. (a) Time-dependent inactivation of CaMKII by basal autophosphorylation. Ca²⁺/CaM-stimulated activity of CaMKII_holo expressed in molar terms (katal/mol) and measured via standard soluble peptide (AC-2) assays with radioactive (γ³²P) ATP. Enzymatic activity was measured following preinactivation reactions of various times (see Materials and Methods); mean ± s.d. (). By calculating the average enzymatic activity per subunit in a fully activatable tetradecameric holoenzyme (i.e., 0 min of inactivation), the average number of activated subunits per holoenzyme was determined for each condition. (b, c) Phosphorylation ([³²P] phosphate incorporation) of immobilized CaMKII substrates (GluN2B, Syntide, GluA1, and Vimentin synthesized via SPOTs) by CaMKII_holo exhibiting 1, 5, or 14 activated subunits per holoenzyme. Substrates ranked from the lowest to the highest K_m. (b) Raw SPOT phosphorylation (PSL/mm²) represented as mean ± s.d. (). (c) SPOT phosphorylation normalized by the number of subunits activated per holoenzyme and represented as mean ± s.d. (). One-way ANOVA of log-normalized data with Holm-Šídák posttest compared to GluN2B within each group (, ).

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