CaMKII autophosphorylation can occur between holoenzymes without subunit exchange
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Germany
- Institute of Biology, Cellular Biophysics, Humboldt Universität zu Berlin, Germany
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Germany
- Helmholtz-Zentrum Berlin für Materialien und Energie, Macromolecular Crystallography, Germany
- Charité-Universitätsmedizin Berlin, Germany
- NeuroCure, Charité Universitätsmedizin, Germany
Figures
Figure 1 with 7 supplements
CaMKIIWT phosphorylates CaMKIIKD.
(A) Schematic representation of CaMKIIα domain arrangement (top). Yellow circles indicate phosphorylation sites. CaMKIIα holoenzyme structure (bottom, PDB:5u6y). (B) Cartoon representation of …
-
Figure 1—source data 1
Uncropped blots used in panel Figure 1E.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig1-data1-v2.zip
Figure 1—figure supplement 1
Western blot detection of CaMKIIKD phosphorylation by CaMKIIWT.
(A) Blots of pT286 detection on CaMKIIKD (4 μM) after phosphorylation with CaMKIIWT (10 nM) in the presence of ATP:Mg2+ and low Ca2+:CaM concentrations (100 nM). Each time point is done in …
-
Figure 1—figure supplement 1—source data 1
Uncropped blots used for constructing the curves in Figure 1D and Figure 1—figure supplement 1E.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
Radioactivity detection of CaMKIIKD phosphorylation by CaMKIIWT.
(A) Gels of CaMKIIKD 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 CaMKIIKD phosphorylation …
-
Figure 1—figure supplement 2—source data 1
Uncropped gels used for constructing the curves in Figure 1—figure supplement 2C.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig1-figsupp2-data1-v2.zip
Figure 1—figure supplement 3
Phosphorylation of CaMKIIKD by CaMKIIWT is concentration-dependent.
(A) Graph showing dependence of CaMKIIKD phosphorylation on CaMKIIWT concentration on a linear timescale. Each fit used the two-component exponential function (see Materials and methods for …
-
Figure 1—figure supplement 3—source data 1
Uncropped blots of examples of immunoblotting used to construct curves in Figure 1—figure supplement 3A–C.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig1-figsupp3-data1-v2.zip
Figure 1—figure supplement 4
Phosphorylation of CaMKIIKD by 0.5 nM CaMKIIWT.
(A) Replicate 1 showing western blot detection of phosphorylation development on T286 of CaMKIIKD over the course of 60 min catalyzed by 0.5 nM CaMKIIWT. Left panel is showing detection of CaMKII by …
-
Figure 1—figure supplement 4—source data 1
Uncropped blots used for constructing the curves in Figure 1—figure supplement 3A–C for 0.5 nM CaMKIIWT.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig1-figsupp4-data1-v2.zip
Figure 1—figure supplement 5
Phosphorylation of CaMKIIKD by 2 nM CaMKIIWT.
(A) Replicate 1 showing western blot detection of phosphorylation development on T286 of CaMKIIKD over the course of 60 min catalyzed by 2 nM CaMKIIWT. Left panel shows detection of CaMKII by …
-
Figure 1—figure supplement 5—source data 1
Uncropped blots used for constructing the curves in Figure 1—figure supplement 3A–C for 2 nM CaMKIIWT.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig1-figsupp5-data1-v2.zip
Figure 1—figure supplement 6
Phosphorylation of CaMKIIKD by 10 nM CaMKIIWT.
(A) Replicate 1 showing western blot detection of phosphorylation development on T286 of CaMKIIKD over the course of 60 min catalyzed by 10 nM CaMKIIWT. Left panel shows detection of CaMKII by …
-
Figure 1—figure supplement 6—source data 1
Uncropped blots used for constructing the curves in Figure 1—figure supplement 3A–C for 10 nM CaMKIIWT.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig1-figsupp6-data1-v2.zip
Figure 1—figure supplement 7
Phosphorylation of CaMKIIKD by 100 nM CaMKIIWT.
(A) Western blot detection of phosphorylation development on T286 over time when CaMKIIKD is incubated with 100 nM CaMKIIWT. Reaction at each time point is done in triplicate. The triplicates for 1, …
-
Figure 1—figure supplement 7—source data 1
Uncropped blots used for constructing the curves in Figure 1—figure supplement 3A–C for 100 nM CaMKIIWT.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig1-figsupp7-data1-v2.zip
Figure 2 with 3 supplements
Crosslinking CaMKII subunits in the hub domain does not change the rates of trans-autophosphorylation.
(A) Position of F394 residue (purple) in the hub domain of CaMKIIα (PDB: 5u6y), showing orientation towards the interface between adjacent hub domains within one hub ring. (B) Coomassie stained gel …
-
Figure 2—source data 1
Uncropped Coomassie stained gels used in panel Figure 2B.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig2-data1-v2.zip
Figure 2—figure supplement 1
Properties of CaMKIIF394BzF: kinase activity and size exclusion chromatography.
(A) Gels of CaMKIIKD phosphorylation detection by UV treated CaMKIIF394BzF. Each time point is done in triplicates. These data are used for Figure 2C. (B) Gels of CaMKIIKD phosphorylation detection …
-
Figure 2—figure supplement 1—source data 1
Uncropped images of radioactive gels used to construct the curves in Figure 2C.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig2-figsupp1-data1-v2.zip
Figure 2—figure supplement 2
Hub domain mutant CaMKIIH418BzF phosphorylates CaMKIIKD irrespective of crosslinking.
(A) Coomassie stained gel showing UV-dependent oligomerization of CaMKIIH418BzF. (B) Phosphorylation of CaMKIIKD by UV-treated or -untreated CaMKIIH418BzF. Langmuir fit determined half-maximum times …
-
Figure 2—figure supplement 2—source data 1
Uncropped images of Coomassie stained and radioactive gels used to construct the curves in Figure 2—figure supplement 2B.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig2-figsupp2-data1-v2.zip
Figure 2—figure supplement 3
Radioactivity detection of substrate phosphorylation by CaMKIIF394BzF.
(A) Gels of CaMKII substrate (GST-Syn) phosphorylation by UV-treated CaMKIIF394BzF. Each time point is done in triplicate. (B) Gels of CaMKII substrate (GST-Syn) phosphorylation by control CaMKIIF394…
-
Figure 2—figure supplement 3—source data 1
Uncropped images of radioactive gels used in Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig2-figsupp3-data1-v2.zip
Figure 3 with 1 supplement
CaMKII holoenzymes do not mix during activation.
(A) Schematic representation of the experiment performed in panel (B) and possible outcomes. (B) Western blot detection of potential CaMKIIKD-AviTag incorporation in CaMKIIF394BzF holoenzymes. Lane …
-
Figure 3—source data 1
Uncropped blots used in panel Figure 3B.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig3-data1-v2.zip
Figure 3—figure supplement 1
CaMKII holoenzymes do not mix during activation.
(A) Coomassie stained gel of CaMKIIF394BzF and CaMKIIKD crosslinking used in Figure 3B. (B) Western blot detection of potential CaMKIIWT-AviTag incorporation in CaMKIIF394BzF holoenzymes. Blue …
-
Figure 3—figure supplement 1—source data 1
Uncropped Coomassie stained gels and blots used in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig3-figsupp1-data1-v2.zip
Figure 4 with 2 supplements
Crosslinking mass spectrometry reveals inter-holoenzyme kinase domain contacts during activation.
(A) Schematic representation of crosslinking experiments and expected outcomes. In the case of subunit exchange, a flat profile of intersubunit crosslinks is expected, whereas for inter-holoenzyme …
Figure 4—figure supplement 1
XL-MS identifies interactions of CaMKII holoenzymes.
(A) Zoomed-in exemplary MS1 spectrum showing uni-isotopic (homotypic) and mixed-isotopic (heterotypic) crosslinks. The ratio of mixed-isotopic and uni-isotopic crosslinks is calculated using the …
Figure 4—figure supplement 2
Crosslinking sites involving pT286 peptides from mixed isotypes.
Kinase domain (from the 5u6y PDB structure) with regulatory domain (orange) docked. 6 Lysine residues that gave heterotypic crosslinked peptides including P-Thr286 (DSS link to Lys291) are indicated …
Figure 5 with 1 supplement
Mapping crosslinks onto holoenzyme structure.
(A) Holoenzyme structure with two neighboring subunits (green and purple) indicated (PDB: 5u6y). Hub domain is in light blue, regulatory segment (docked) in orange. (B) Basal crosslinks between …
Figure 5—figure supplement 1
Hypothetical close mode of holoenzyme interaction from MS crosslinks.
Heterotypic DSS crosslinks between kinase domains have plausible lengths (less than 20 Å) when one kinase domain from a holoenzyme is interdigitated into the structure of another holoenzyme. Hub …
Figure 6 with 2 supplements
Reversible activity-dependent colocalization of CaMKII holoenzymes.
(A) Schematic representation of CaMKII in vitro enzymatic labeling with maleimide dyes and biotin, and TIRF experimental set-up. (B) Representative TIRF images of unactivated (basal) CaMKIIWT sample …
Figure 6—figure supplement 1
Activation stimuli are necessary for CaMKIIWT holoenzyme colocalization.
(A) Absence of colocalization between differently labeled CaMKIIWT holoenzymes in the absence of Mg2+:ATP. (B) Absence of colocalization between differently labeled CaMKIIWT holoenzymes upon …
Figure 6—figure supplement 2
CaMKIIKD holoenzymes cluster only in the presence of activated CaMKIIWT holoenzymes.
(A) Absence of colocalization between CaMKIIKD and CaMKIIWT in basal conditions. (B) Colocalization of CaMKIIKD and CaMKIIWT upon activation. (C) Absence of colocalization between differently …
Figure 7 with 2 supplements
Mass photometry detects clusters of holoenzymes forming upon activation.
(A) Mass distribution of 400 nM CaMKIIWT under basal conditions (red curve). Blue curve is multi- Gaussian fit (shown separately on lower graph). Red curve in upper graph is the fit residual. (B) …
Figure 7—figure supplement 1
CaMKIIWT forms higher order clusters during activation.
(A) Two replicates of the particle mass distribution of 400 nM CaMKIIWT under basal conditions (red curve). Blue curve is multi-Gaussian fit (shown separately on lower graph). Red curve in upper …
Figure 7—figure supplement 2
CaMKIIKD fails to form higher order clusters during activation.
(A) Particle mass distribution of 400 nM CaMKIIKD under basal conditions. (B) Particle mass distribution of 400 nM CaMKIIKD under activating conditions. (C) Western blot detection of pT286 on CaMKIIK…
-
Figure 7—figure supplement 2—source data 1
Uncropped blots used in Figure 7—figure supplement 2C.
- https://cdn.elifesciences.org/articles/86090/elife-86090-fig7-figsupp2-data1-v2.zip
Additional files
-
Supplementary file 1
Crosslinked peptides identified by MS X-linking.
(A) Homotypic crosslinks (basal, 30 min). (B) Homotypic crosslinks (basal, 150 min). (C) Heterotypic crosslinks (basal, 30 min). (D) Heterotypic crosslinks (basal, 150 min). (E) Homotypic crosslinks (activated, 30 min). (F) Homotypic crosslinks (activated, 150 min). (G) Heterotypic crosslinks (activated, 30 min). (H) Heterotypic crosslinks (activated, 150 min) (I) pT286 Heterotypic peptides (30 min). (J) pT286 Heterotypic peptides (150 min).
- https://cdn.elifesciences.org/articles/86090/elife-86090-supp1-v2.pdf
-
MDAR checklist
- https://cdn.elifesciences.org/articles/86090/elife-86090-mdarchecklist1-v2.pdf
