Figure 1. CaMKII^WT phosphorylates CaMKII^KD.

(A) Schematic representation of CaMKIIα domain arrangement (top). Yellow circles indicate phosphorylation sites. CaMKIIα holoenzyme structure (bottom, PDB:5u6y). (B) Cartoon representation of activation of CaMKII by calcium:calmodulin (top) and proposed mechanism for spread of kinase activity (bottom). Yellow circles indicate phosphorylation sites. (C) Schematic representation of experiment performed in panel (D). (D) Kinase activity of CaMKII^WT (10 nM) against CaMKII^KD (4 μM). Half-time of maximum phosphorylation (t_1⁄2=1.1 ± 0.2 min) determined by western blot, using an antibody against pT286. These data were fit with a single component, see Figure 1—figure supplement 3 for a fit to a repeated experiment with two components. The shading represents standard deviation (SD) from the mean value of three technical replicates. (E) Kinase activity of EGFP-CaMKII^WT and CaMKII^KD-AviTag in HEK cells and HEK cell lysates. ‘Basal’: activity in cells; ‘activated in vitro’: activity in cell lysates supplemented with 1 μM purified Ca²⁺:CaM and 0.5 mM ATP:Mg²⁺. Lane 1 – autophosphorylation of EGFP-CaMKII^WT in singly transfected cells, lane 2 – autophosphorylation of CaMKII^KD-AviTag in singly transfected cells, lane 3 – autophosphorylation of EGFP-CaMKII^WT and CaMKII^KD-AviTag in co-transfected cells, lane 4 – stimulated kinase activity of EGFP-CaMKII^WT from lysate in lane 1 and CaMKII^KD-AviTag from lysate in lane 2, incubated together at 4 °C for 1 min, lane 5 - stimulated kinase activity of EGFP-CaMKII^WT from lysate in lane 1 and CaMKII^KD-AviTag from lysate in lane 2, incubated together at 37 °C for 1 min, lane 6 - stimulated kinase activity of EGFP-CaMKII^WT from lysate in lane 1 and CaMKII^KD-AviTag from lysate in lane 2, incubated together at 37 °C for 10 min, lane 7 - stimulated kinase activity of EGFP-CaMKII^WT from lysate in lane 1 and 8 μM purified CaMKII^KD-AviTag, incubated together at 37 °C for 10 min, lane 8 - stimulated kinase activity of EGFP-CaMKII^WT from lysate in lane 1 37 °C for 10 min.

Figure 1—figure supplement 1. Western blot detection of CaMKII^KD phosphorylation by CaMKII^WT.

(A) Blots of pT286 detection on CaMKII^KD (4 μM) after phosphorylation with CaMKII^WT (10 nM) in the presence of ATP:Mg²⁺ and low Ca²⁺:CaM concentrations (100 nM). Each time point is done in triplicate. (B) Blots of pT286 detection on CaMKII^KD (4 μM), after phosphorylation with CaMKII^WT (10 nM) in the presence of ATP:Mg²⁺ and high Ca²⁺:CaM concentrations (2 μM). Each time point is done in triplicate. (C) Western blot showing there is no phosphorylation signal from pT286 antibody on CaMKII^KD (4 μM) when CaMKII^WT is omitted from the kinase reaction. (D) Western blot showing that 10 nM CaMKII^WT (as included on the gels in panels A and B) gives no phosphorylation signal from the pT286 antibody. (E) Raw densitometry data from blots in panels A and B fitted with Langmuir function. Half-maximum times for CaMKII^KD phosphorylation by CaMKII^WT under low Ca²⁺:CaM conditions is t_1/2 = 0.9 ± 0.4 min, and under high Ca²⁺:CaM conditions is t_1/2 = 1.1 ± 0.2 min. The shading represents standard deviation (SD) from the mean value of three technical replicates.

Figure 1—figure supplement 2. Radioactivity detection of CaMKII^KD phosphorylation by CaMKII^WT.

(A) Gels of CaMKII^KD phosphorylation detection in the presence of ATP:Mg²⁺ and low Ca²⁺:CaM concentrations (100 nM). Each time point is done in triplicates. (B) Gels of CaMKII^KD phosphorylation detection in the presence of ATP: Mg²⁺ and high Ca²⁺:CaM concentrations (2 μM). Each time point is done in triplicates. (C) Raw densitometry data from gels in A and B fitted with Langmuir function. Half-maximum times for CaMKII^KD phosphorylation by CaMKII^WT under low Ca²⁺:CaM conditions is t_1/2 = 1.16 ± 0.6 min, and under high Ca²⁺:CaM conditions is t_1/2 = 4.3 ± 1.13 min. The shading represents standard deviation (SD) from the mean value of three technical replicates.

Figure 1—figure supplement 3. Phosphorylation of CaMKII^KD by CaMKII^WT is concentration-dependent.

(A) Graph showing dependence of CaMKII^KD phosphorylation on CaMKII^WT concentration on a linear timescale. Each fit used the two-component exponential function (see Materials and methods for details). In these ‘free fits’. the rates r₁ and k₂ were allowed to vary between the four fits (10 free parameters in all, including the amplitudes). Note that we repeated the experiments at 10 nM CaMKII^WT (essentially the same as Figure 1D). Because the phosphorylation reaction was so fast at 100 nM CamKII^WT, we fitted only a single component (phosphorylation rate was 2.5 min^–1). Fit parameters (±1 SD) were as follows: 10 nM fit A₁=0.27 ± 0.06; k₂=0.043 ± 0.006 nM^–1 min^–1; r₁ = 0.035 ± 0.009 min^–12 nM fit A₁=0.6 ± 0.1; k₂=0.007 ± 0.003 nM^–1 min^–1; r₁ = 0.44 ± 0.13 min^–10.5 nM fit A₁=0.5 ± 0.3; k₂=0.02 ± 0.03 nM^–1 min^–1; r₁ = 0.17 ± 0.11 min^–1 Same free fit as panel (B) on a logarithmic timescale to reveal the two components. (C) Global fit (5 free parameters) using common values of rates r₁ and k₂ for each curve. r₁=0.41 min^–1 and k₂=0.017 nM^–1 min^–1. For the 100 nM fit, the amplitude A₁ was set to zero. Note that at 100 nM, the first order rate k₂ * 100 nM is about 3 x faster (1.7 min^–1) than the rate estimated for concentration-independent phosphorylation. (D) Representative western blots of the time course of phosphorylation on T286 of CaMKII^KD by CaMKII^WT at different concentrations (concentrations of proteins used is indicated on the left side of each panel). Lanes which are marked with ‘kinase-dead (1 hr)' show that no pT286 signal can be detected on CaMKII^KD (4 µM) after 1 hr of incubation without CaMKII^WT. Lanes labeled with concentration of CaMKII^WT demonstrate that the wild-type protein is invisible for anti-pT286 antibody at the reaction amounts loaded on these gels for all except for 100 nM WT lane, where a phosphorylation signal is detected after 1 h of incubation of CaMKII^WT with activation stimuli, but without CaMKII^KD. However, the signal is about 20% of the signal detected on CaMKII^KD after 1 hr. The bottom blot shows pT286 detection of triplicate reactions at 1, 2, 5 and a single reaction at 10 min. The other blots used to construct the curves in (A–C) are in Figure 1—figure supplements 4–7.

Figure 1—figure supplement 4. Phosphorylation of CaMKII^KD by 0.5 nM CaMKII^WT.

(A) Replicate 1 showing western blot detection of phosphorylation development on T286 of CaMKII^KD over the course of 60 min catalyzed by 0.5 nM CaMKII^WT. Left panel is showing detection of CaMKII by anti-pan CaMKII antibody. Middle panel is showing detection of pT286 signal on CaMKII by anti-pT286 antibody. Right panel is showing merged signal of pan-CaMKII and pT286 signal. Lane labeled with ‘kinase-dead’ is showing that although CaMKII^KD can be detected by pan antibody, the signal on T286 (middle panel) is absent after 60 min of the kinase reaction, when CaMKII^WT is excluded from the reaction. Lane labeled with ‘0.5 nM WT’ demonstrates that the wild-type protein is invisible for anti-pan and anti-pT286 antibodies after 60 min of kinase reaction without CaMKII^KD at the protein amount loaded on this gel. (B) Replicate 2 showing western blot detection of phosphorylation development on T286 of CaMKII^KD over the course of 60 min catalyzed by 0.5 nM CaMKII^WT. The labeling of the blots is the same like in (A). (C) Replicate 3 showing western blot detection of phosphorylation development on T286 of CaMKII^KD over the course of 60 min catalyzed by 0.5 nM CaMKII^WT. The labeling of the blots is the same like in (A) and (B).

Figure 1—figure supplement 5. Phosphorylation of CaMKII^KD by 2 nM CaMKII^WT.

(A) Replicate 1 showing western blot detection of phosphorylation development on T286 of CaMKII^KD over the course of 60 min catalyzed by 2 nM CaMKII^WT. Left panel shows detection of CaMKII by anti-pan CaMKII antibody. Middle panel shows detection of pT286 signal on CaMKII by anti-pT286 antibody. Right panel shows merged signal of pan-CaMKII and pT286 signal. Lane labeled with ‘kinase-dead’ shows that although CaMKII^KD can be detected by pan antibody, the signal on T286 (middle panel) is absent after 60 min of the kinase reaction, when CaMKII^WT is excluded from the reaction. Lane labeled with ‘2 nM WT’ demonstrates that the wild-type protein is invisible for anti-pan and anti-pT286 antibodies after 60 min of kinase reaction without CaMKII^KD at the protein amount loaded on this gel. Lane labeled with ‘100 nM WT’ demonstrates that the wild-type protein is somewhat visible for anti-pan and anti-pT286 antibodies after 60 min of kinase reaction without CaMKII^KD at the protein amount loaded on this gel, but this signal represents about 20% of the pT286 signal coming from reactions incubated with CaMKII^KD. (B) Replicate 2 showing western blot detection of phosphorylation development on T286 of CaMKII^KD over the course of 60 min catalyzed by 2 nM CaMKII^WT. The labeling of the blots is the same as in (A). (C) Replicate 3 showing western blot detection of phosphorylation development on T286 of CaMKII^KD over the course of 60 min catalyzed by 2 nM CaMKII^WT. The labeling of the blots is the same as in (A) and (B).

Figure 1—figure supplement 6. Phosphorylation of CaMKII^KD by 10 nM CaMKII^WT.

(A) Replicate 1 showing western blot detection of phosphorylation development on T286 of CaMKII^KD over the course of 60 min catalyzed by 10 nM CaMKII^WT. Left panel shows detection of CaMKII by anti-pan CaMKII antibody. Middle panel shows detection of pT286 signal on CaMKII by anti-pT286 antibody. Right panel shows merged signal of pan-CaMKII and pT286 signal. Lane labeled with ‘kinase-dead’ is showing that although CaMKII^KD can be detected by pan antibody, the signal on T286 (middle panel) is absent after 60 min of the kinase reaction, when CaMKII^WT is excluded from the reaction. Lane labeled with ‘10 nM WT’ demonstrates that the wild-type protein is invisible for anti-pan and anti-pT286 antibodies after 60 min of kinase reaction without CaMKII^KD at the protein amount loaded on this gel. (B) Replicate 2 showing western blot detection of phosphorylation development on T286 of CaMKII^KD over the course of 60 min catalyzed by 10 nM CaMKII^WT. The labeling of the blots is the same as in (A). (C) Replicate 3 showing western blot detection of phosphorylation development on T286 of CaMKII^KD over the course of 60 min catalyzed by 10 nM CaMKII^WT. The labeling of the blots is the same as in (A) and (B).

Figure 1—figure supplement 7. Phosphorylation of CaMKII^KD by 100 nM CaMKII^WT.

(A) Western blot detection of phosphorylation development on T286 over time when CaMKII^KD is incubated with 100 nM CaMKII^WT. Reaction at each time point is done in triplicate. The triplicates for 1, 2, and 5 min were loaded one after another on the same gel. Lane labeled with ‘10’ is a single reaction at 10 min time point. Lane labeled with ‘kinase-dead’ is showing that although CaMKII^KD can be detected by pan antibody, the signal on T286 (middle panel) is absent after 60 min of the kinase reaction, when CaMKII^WT is excluded from the reaction. (B) Western blot detection of phosphorylation development on T286 over time when CaMKII^KD is incubated with 100 nM CaMKII^WT. Reaction at each time point is done in triplicate. The duplicate for 10 min time point and then triplicates for 15, 30, and 60 min were loaded one after another on the same gel.