1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Flexible linkers in CaMKII control the balance between activating and inhibitory autophosphorylation

  1. Moitrayee Bhattacharyya
  2. Young Kwang Lee
  3. Serena Muratcioglu
  4. Baiyu Qiu
  5. Priya Nyayapati
  6. Howard Schulman
  7. Jay T Groves  Is a corresponding author
  8. John Kuriyan  Is a corresponding author
  1. Department of Molecular and Cell Biology, University of California, Berkeley, United States
  2. California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, United States
  3. Howard Hughes Medical Institute, University of California, Berkeley, United States
  4. Department of Chemistry, University of California, Berkeley, United States
  5. Panorama Institute of Molecular Medicine, United States
  6. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States
Research Article
Cite this article as: eLife 2020;9:e53670 doi: 10.7554/eLife.53670
10 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Structural organization and Ca2+/CaM-dependent activation of CaMKII.

(a) CaMKII is organized as a holoenzyme with kinase domains connected to a central dodecameric/tetradecameric hub by a regulatory segment and a flexible linker, referred to as the kinase-hub linker. All the domains are labeled and this color scheme used will be maintained throughout. The kinase-hub linker is the principle difference between the four CaMKII isoforms: α/β/γ/δ. (b) Crystal structure of the autoinhibited kinase domain from human CaMKII-δ (PDB ID: 2VN9) (Rellos et al., 2010). The regulatory segment places Thr 306 optimally for cis-phosphorylation, while Thr 286, at the base of the kinase can only be phosphorylated in trans. (c) Schematic diagram showing the design principle for all the constructs used in this study. (d) Depiction of the four possible phosphorylation states of CaMKII.

Figure 1—figure supplement 1
Amino acid sequences for the kinase-hub linker in the four isoforms of human CaMKII.
Figure 2 with 1 supplement
Mammalian expression-based single-molecule Total Internal Reflection Fluorescence (TIRF) assay.

(a) Schematic diagram showing the experimental setup. Biotinylated mEGFP-CaMKII overexpressed in HEK 293T cells was pulled down directly from diluted cell lysate, allowing visualization at a single-molecule resolution. The immobilization onto glass substrates functionalized with streptavidin relies on the interaction between biotinylated CaMKII and streptavidin. Autophosphorylation status of CaMKII holoenzymes can be measured using phosphospecific primary antibodies and Alexa-labeled secondary antibodies. (b) Representative single-molecule TIRF images showing mEGFP-CaMKII holoenzymes (green dots), phosphorylation at Thr 286 (red dots) and phosphorylation at Thr 305/306 (purple dots) from left to right. A 3-color merge of these images reports on the fraction of CaMKII holoenzymes that are phosphorylated at Thr 286 and/or Thr 305/306. (c) Fraction of CaMKII-α that shows detectable phosphorylation at Thr 286 is plotted for different Ca2+/CaM concentrations ranging from 0.02 μM to 5 μM. The cartoon at the bottom depicts two extreme cases, where only a few holoenzymes are phosphorylated or where most holoenzymes are phosphorylated. (d) Distribution of intensity for pThr 286 (561 nm), at different Ca2+/CaM concentrations, for CaMKII-α holoenzymes with detectable phosphorylation (see Materials and methods for details of normalization). The cartoon at the bottom shows that a right-shift in the peak value of the intensity histogram represents a higher extent of phosphorylation within a CaMKII holoenzyme.

Figure 2—figure supplement 1
Validation of phosphospecific antibodies.

Mutation/deletion of epitopes for pThr 286 (left) and pThr 305/306 (right)-specific antibodies leads to no detection of phosphosignal at the respective sites.

Figure 3 with 3 supplements
Autophosphorylation status of activated CaMKII-α and CaMKII-β/β*.

(a) Comparison of the extent of autophosphorylation (intensity histogram) within CaMKII-α and CaMKII-β holoenzymes at the activating site (Thr 286) (left panel) and the inhibitory site (Thr 305/306) (right panel). The insets show the fraction of holoenzymes that exhibit any detectable phosphorylation for the corresponding phosphosite in CaMKII-α and CaMKII-β. (b) Comparison of the extent of autophosphorylation (intensity histograms) between CaMKII-α and CaMKII-β* holoenzymes at the activating site (Thr 286) (left panel) and the inhibitory site (Thr 305/306) (right panel). The insets show the fraction of holoenzymes that exhibit any detectable phosphorylation for the corresponding phosphosite in CaMKII-α and CaMKII-β* (see Materials and methods for details of normalization). (c) Autophosphorylation status of CaMKII after activation in HEK 293T cells using ionomycin. Fractions of CaMKII-α and CaMKII-β* that show detectable phosphorylation at Thr 286 are plotted for different conditions. (d) Fractions of CaMKII-α and CaMKII-β* that show detectable phosphorylation at Thr 305/306 are plotted for different conditions. + / - depicts the presence or absence of ionomycin and/or phosphatase inhibitors.

Figure 3—figure supplement 1
Inhibitory autophosphorylation status of activated CaMKII-α.

(a) Schematic diagram showing the experimental set up. CaMKII was activated, followed by a wash to remove the components of the activation buffer. The pre-activated CaMKII (species A) is then treated with Mg2+-ATP for 30 min to generate species B. (b) Fraction of CaMKII-α holoenzymes with detectable phosphorylation at Thr 305/306 is plotted for each species. (c) Intensity distribution of pThr 305/306 (640 nm) for species A and species B with detectable phosphorylation . The area under each intensity histogram is scaled by the fraction of holoenzymes that show no detectable phosphorylation under that condition (see Materials and methods for details of normalization).

Figure 3—figure supplement 2
Autophosphorylation status of activated CaMKII-γ* and CaMKII-δ*.

Comparison of the extent of autophosphorylation (intensity histogram) among CaMKII-γ*, CaMKII-δ*, CaMKII-α, and CaMKII-β* holoenzymes at the activating site (Thr 286) (left panel) and the inhibitory site (Thr 305/306) (right panel). The insets show the fraction of holoenzymes that exhibit any detectable phosphorylation for the corresponding phosphosite in CaMKII-γ* and CaMKII-δ* (see Materials and methods for details of normalization).

Figure 3—figure supplement 3
Autophosphorylation status of activated CaMKII-β/β* variants.

(a) Comparison of the extent of phosphorylation at the activating site (left panel) and the inhibitory site (right panel) for CaMKII-β and CaMKII-β’E holoenzymes with detectable phosphorylation. (b) Comparison of the extent of phosphorylation at the activating site (left panel) and the inhibitory site (right panel) for CaMKII-β and CaMKII-β’E* holoenzymes with detectable phosphorylation. (c) Comparison of the extent of phosphorylation at the activating site (left panel) and the inhibitory site (right panel) for CaMKII-α, CaMKII-β and CaMKII-α* holoenzymes with detectable phosphorylation (see Materials and methods for details of normalization).

Inhibitory autophosphorylation in CaMKII-α-CaMKII-β* heterooligomers.

(a) Schematic diagram showing that co-expression of GFP-CaMKII-α and mCherry-CaMKII-β* leads to the formation of heterooligomers. (b) Bar graph showing the fraction of holoenzymes that show detectable phosphorylation at the inhibitory site (Thr 305/306), which increases as the ratio of CaMKII-β* increases. (c) Intensity histogram for the homooligomers and heterooligomers. As the ratio of CaMKII-β* increases, there is a right-shift in the peak value of the intensity histogram (see Materials and methods for details of normalization).

Figure 5 with 1 supplement
A simplified schematic diagram showing the key pathways for autophosphorylation at the activating and inhibitory sites, in the absence or presence of Ca2+/CaM.

While Thr 305/306 can get phosphorylated both in cis in the absence of Ca2+/CaM or in trans in the presence of Ca2+/CaM, autophosphorylation of Thr 286 can only happen in trans in the presence of Ca2+/CaM. Ca2+/CaM shows a rapid association and dissociation until CaMKII gets phosphorylated at Thr 286, when its affinity for Ca2+/CaM increases by about 1000-fold. A detailed description of all the different reactions and conditions that form the basis of our kinetic model is provided in the Appendix.

Figure 5—figure supplement 1
Results from simulations of a simple kinetic model for autophosphorylation in CaMKII using Berkeley Madonna.

Plot showing the production of all species bearing pThr 286 or pThr 305/306 over simulation time, when (a) the linker-length is short with faster rates of trans-autophosphorylation and when (b) the linker-length is long and the rates of trans-autophosphorylation are 10-fold slower.

Effect of λ-phosphatase on the phosphorylation status of CaMKII when the kinase is active.

(a) Schematic diagram showing the experimental set up. CaMKII was activated in the presence of 0, 200, 400, or 800 units of λ-phosphatase for 45 min. (b–c) Bar graph showing the fraction of CaMKII-α and CaMKII-β* holoenzymes that shows detectable phosphorylation at the activating site (Thr 286) and the inhibitory site (Thr 305/306), respectively, in the presence of λ-phosphatase. (d) Intensity distribution of pThr 286 (561 nm) signal for CaMKII-α (left panel) and CaMKII-β* (right panel) holoenzymes with detectable phosphorylation in the presence of λ-phosphatase. (e) Intensity distribution of pThr 305/306 (640 nm) signal for CaMKII-α (left panel) and CaMKII-β* (right panel) holoenzymes with detectable phosphorylation in the presence of λ-phosphatase (see Materials and methods for details of normalization).

Figure 7 with 3 supplements
Effect of λ-phosphatase on the dephosphorylation kinetics when kinase activity is switched off.

(a) Schematic diagram showing the experimental set up. CaMKII was activated, followed by a wash to remove the components of the activation buffer, and then saturating amounts of λ-phosphatase/PP1α (400–800 units) were added for 0, 3, 15, or 30 min. (b) Plot showing the fraction of α and β* holoenzymes that exhibits detectable phosphorylation at the inhibitory site (Thr 305/306) upon treatment with λ-phosphatase for defined time-points. The fractions at 3, 15, or 30 min for α and β* are normalized by the corresponding activated version that has not been exposed to any λ-phosphatase (0 min time-point, whose value is set to 1.0). (c) Same as (b) but for the activating site (Thr 286). (d) Intensity distribution for pThr 286 (561 nm) for CaMKII-α (left panel) and CaMKII-β* (right panel) holoenzymes with detectable phosphorylation after 0, 3, 15, or 30 min of λ-phosphatase treatment (see Materials and methods for details of normalization).

Figure 7—figure supplement 1
Effect of phosphatases on dephosphorylation kinetics when kinase activity is switched off.

(a) Fraction of CaMKII-α/β* holoenzymes with detectable phosphorylation at Thr 305/306 (left) and Thr 286 (right) after 0, 3, 15, or 30 min of treatment with λ-phosphatase, when the kinase activity is switched off. (b) Same as (a), but PP1α phosphatase was used. (c) Intensity distribution for pThr 286 (561 nm) for CaMKII-α (left panel) and CaMKII-β* (right panel) holoenzymes with detectable phosphorylation at Thr 286 after 0, 3, 15, or 30 min of PP1α treatment (see Materials and methods for details of normalization).

Figure 7—figure supplement 2
Measurement of dephosphorylation kinetics in solution.

(a) Schematic diagram depicting the experimental design to measure dephosphorylation kinetics in-solution using λ-phosphatase. (b) Fraction of CaMKII-α/β* holoenzymes with detectable phosphorylation at Thr 305/306 (left) and Thr 286 (right) after 0, 3, 15, or 30 min of treatment with λ-phosphatase in-solution, when the kinase activity is switched off by staurosporine. (c) Intensity distribution for pThr 286 (561 nm) for CaMKII-α (left panel) and CaMKII-β* (right panel) holoenzymes with detectable phosphorylation at Thr 286 after 0, 3, 15, or 30 min of λ-phosphatase treatment (see Materials and methods for details of normalization).

Figure 7—figure supplement 3
Inhibition of CaMKII kinase activity by staurosporine.

Fraction of CaMKII-α/β* holoenzymes with detectable phosphorylation at Thr 286 (left) and Thr 305/306 (right) after 60 min of treatment with the activation buffer (see Materials and methods) in the absence and presence of 100 μM staurosporine.

Figure 8 with 1 supplement
Effect of addition of Ca2+/CaM on the rates of dephosphorylation at the activating site.

(a) Schematic diagram showing the experimental set up. CaMKII was activated, followed by wash to remove the components of the activation buffer and then saturating amounts of λ-phosphatase were added for 3, 15, or 30 min, in the presence and absence of Ca2+/CaM. (b) Plot showing the fraction of CaMKII-α holoenzymes with detectable phosphorylation at the activating site (Thr 286, right panel) after 0 min (activated control) and 3, 15, or 30 min of treatment with saturating amounts of λ-phosphatase in the absence (green trace) and presence (pink trace) of Ca2+/CaM. The fractions at 3, 15, or 30 min are normalized with respect to activated CaMKII that has not been exposed to any λ-phosphatase (0 min, whose value is set to 1.0). (c) Intensity distribution for pThr 286 (561 nm) for CaMKII-α holoenzymes with detectable phosphorylation, upon 0, 3, 15, or 30 min of λ-phosphatase treatment in the presence of 5 μM Ca2+/CaM (see Materials and methods for details of normalization).

Figure 8—figure supplement 1
Effect of addition of Ca2+/CaM on the rates of dephosphorylation at the autonomy site.

(a) Fraction of CaMKII-β* holoenzymes with detectable phosphorylation at Thr 286, upon 3 min of λ-phosphatase treatment in the absence and presence of 5 μM Ca2+/CaM. (b) Intensity distribution of pThr 286 (561 nm) for CaMKII-β* holoenzymes with detectable phosphorylation, under the same conditions as in (a) (see Materials and methods for details of normalization).

Recovery of phosphorylation at the activating site by subthreshold concentrations of Ca2+/CaM.

(a) Schematic diagram showing the experimental set up. CaMKII was activated, followed by a wash to remove the components of the activation buffer and then saturating amounts of λ-phosphatase were added for 3–5 min. The sample was then washed to remove the λ-phosphatase, followed by further treatment with subthreshold concentrations of Ca2+/CaM (25 nM) for 30 min and the autophosphorylation status was measured. (b) Cartoon representation of the different species generated after each treatment. Each species is color-coded and the color schemes are maintained throughout the plots. (c) Fraction of CaMKII-α holoenzymes with detectable phosphorylation at Thr 286 is plotted for each species. (d) Intensity distribution of pThr 286 (561 nm) for only those CaMKII-α holoenzymes that show any detectable Thr 286 phosphorylation, for the different species of interest as described in (b).

Appendix 1—figure 1
A summary of the reactions considered in the kinetic model.

K denotes the kinase (CaMKII) and C denotes CaM. Phosphorylation on Thr 286 or Thr 305/306 is denoted by 286 and 306, respectively.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene (human)CaMKII-αUniprot_ID: Q9UQM7
Gene (human)CaMKII-βUniprot_ID: Q13554
Gene (human)CaMKII-β’EUniprot_ID: Q13554-3
Gene (human)CaMKII-γUniprot_ID: Q13555
Gene (human)CaMKII-δUniprot_ID: Q13557
Recombinant DNA reagentpET21a-BirAAddgeneplasmid # 20857for biotinylation of the AVI tag in HEK 293T cells
Recombinant DNA reagentEYFP-CaMAddgeneplasmid # 47603coexpressed for in cell activation of CaMKII
Recombinant DNA reagentpEGFP-C1 (plasmid)Clontech, Mountain View, CAvector backbone for inserting the CaMKII genes
Recombinant DNA reagentpSNAPf (plasmid)New England Biolabs, MAN9183Svector backbone
Cell line (human)HEK 293TUC Berkeley cell culture facilityauthenticated using STR profiling and tested negative for mycoplasma
Antibodyanti-CaMKII (phospho T286); mouse monoclonalAbcamab1710951:500
Antibodyanti-CaMKII (phospho T306); rabbit polyclonalPhosphoSolutionsp1005-3061:500
Peptide, recombinant proteinPoly-L-lysine PEG (PLL:PEG)SuSoS, Dübendorf, SwitzerlandPLL(20)-g[3.5]- PEG(2)preparation of flow chambers
Peptide, recombinant
protein
streptavidinSigma-AldrichS0677functionalize the glass substrates for capturing biotinylated CaMKII
Peptide, recombinant proteincalmodulinSigma-AldrichC4874activation of CaMKII
Peptide, recombinant
protein
λ-phosphataseNew England Biolabs, MAP0753Lphosphatase
Peptide, recombinant proteinPP1αEMD Millipore,
Burlington, MA
14–595phosphatase
Chemical compound, drugPEG-BiotinSuSoS, Dübendorf, SwitzerlandPLL(20)-g[3.5]- PEG(2)/PEG(3.4)- biotin(50%)preparation of flow chambers
Chemical compound, drug1% protease inhibitor cocktailSigmaP8340protease inhibitor cocktail for lysis buffer
Chemical compound, drug0.5% phosphatase inhibitor cocktail 2 and 3SigmaP0044 and P5726phosphatase inhibitor cocktails for lysis buffer
Chemical compound, drugstaurosporineAbcamab120056kinase inhibitor
Chemical compound, drugcyclosporin ASigma-Aldrich30024phosphatase inhibitor
Chemical compound, drugokadaic acidAbcamab141831phosphatase inhibitor
Software, algorithmFIJI (ImageJ)open access software, see https://imagej.net/Fiji/Downloadsimage processing
Software, algorithmin-house Matlab codesopen access, see Source code 1 image processing
Othersticky-Slide VI 0.4Ibidi80608flow chambers
Otherglass coverslipsIbidi10812functionalized substrates

Data availability

All data generated or analyzed during this study are summarized in the manuscript, figures, appendix, and supplementary files. The in-house Matlab programs that are used for data analysis are provided as Source code file 1 and is open-access.

Additional files

Source code 1

In-house Matlab programs that are used for data analyses are provided as an open source package.

A readme file and a test dataset is included for clarity.

https://cdn.elifesciences.org/articles/53670/elife-53670-code1-v2.zip
Transparent reporting form
https://cdn.elifesciences.org/articles/53670/elife-53670-transrepform-v2.docx

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