Kuriyan Lab Logo        
Luke Chao
       Stamp


Intersubunit capture of regulatory segments is a component of cooperative CaMKII activation


Luke H Chao, Patricia Pellicena, Sebastian Deindl, Lauren A Barclay, Howard Schulman & John Kuriyan


Nature Structural & Molecular Biology Published online: 7 February 2010 | doi:10.1038/nsmb.1751 (local copy)

Abstract / Figures from the paper / Supplemental Material / Coordinates



Abstract:

The dodecameric holoenzyme of calcium-calmodulin-dependent protein kinase II (CaMKII) responds to high-frequency Ca2+ pulses to become Ca2+ independent. A simple coincidence-detector model for Ca2+-frequency dependency assumes noncooperative activation of kinase domains. We show that activation of CaMKII by Ca2+-calmodulin is cooperative, with a Hill coefficient of ~3.0, implying sequential kinase-domain activation beyond dimeric units. We present data for a model in which cooperative activation includes the intersubunit 'capture' of regulatory segments. Such a capture interaction is seen in a crystal structure that shows extensive contacts between the regulatory segment of one kinase and the catalytic domain of another. These interactions are mimicked by a natural inhibitor of CaMKII. Our results show that a simple coincidence-detection model cannot be operative and point to the importance of kinetic dissection of the frequency-response mechanism in future experiments.


Figures from the paper.

Click on the small image to get a bigger one.

First figure from paper

Figure 1: Cartoon schematic of models for CaMKII activation. (a) In a coincidence-detection model, Ca2+-calmodulin binding and activation occurs stochastically. Activation without cooperativity allows for CaMKII transphosphorylation to occur only in response to coincident and adjacent Ca2+-calmodulin binding events. It is not known if autophosphorylation is bidirectional for nearest-neighbor subunits. (b) Binding of Ca2+-calmodulin is cooperative, and autoinhibited kinase domains form a dimer in the crystal. In a holoenzyme comprising autoinhibited dimers, Ca2+-calmodulin binding to one kinase in the dimer would release the Ca2+-calmodulin binding site of a second kinase domain. (c) This work demonstrates that activation of CaMKII is cooperative; thus, CaMKII is not a simple coincidence detector. The level of cooperativity observed indicates sequential activation of kinase domains beyond dimers, where activation of a kinase subunit potentiates activation beyond a second subunit (shown as gray-blue subunits). (d) The capture of regulatory segments as substrates for adjacent kinase domains increases the cooperativity of activation. Only the activation and potentiation through subunit capture are shown, with any subsequent autophosphorylation omitted for simplicity.
Illustrator file, local only


First figure from paper

Figure 2: Cooperativity of CaMKII activation by Ca2+-calmodulin. (a) Schematic diagrams of the CaMKII domain structure and regulatory segment arrangement in the autoinhibited state. Left, domain structure of CaMKII. The regulatory segment is enlarged, highlighting three elements: the R1 element, which contains the regulatory phosphorylation site Thr286; the R2 element, which clamps the regulatory segment to the kinase domain in the autoinhibited state; and the R3 element, which includes the calmodulin-recognition motif. Below, schematic diagram of the structure of the autoinhibited kinase domain: schematic of arrangement of autoinhibited dimers of the kinase domain in the CaMKII holoenzyme, with individual kinase domain enlarged. In the autoinhibited state, the R1 element is sequestered in a cleft below helix αD, and the R2 element positions helix αD to prevent substrate access to the active site. (b) Cooperative activation of C. elegans CaMKII holoenzyme occurs with a Hill coefficient of 3.0 ± 0.3. C. elegans holoenzyme phosphorylation of the peptide syntide was measured as a function of calmodulin concentration. (c) The Hill coefficient for C. elegans CaMKII activation for constructs with various linker lengths. The velocity of substrate phosphorylation at varying calmodulin concentrations is shown. The linker modifications within residues 314-340 are indicated in the schematic at left. In the +6 linker, six flexible residues were introduced to the middle of the linker region. In the Δ17 linker, 17 residues were deleted from the middle of the linker region. In the Δ26 linker, all 26 residues were removed from the linker region. (d) Cooperativity of calmodulin activation is reduced by autocamtide for the C. elegans CaMKII Δ17 deletion construct. Activity of CaMKII Δ17 was measured toward syntide and autocamtide and plotted on a velocity (v) versus log10[CaM] plot (right) and a log10 [v/(Vmax - v)] versus log10 [CaM] plot (left). All error bars and ± terms expressed are s.e.m.
Illustrator file, local only


First figure from paper

Figure 3: Crystal structure of the CaMKII enzyme-substrate complex. (a) Schematic diagram of the crystallized CaMKII enzyme-substrate complex. The construct contains the kinase domain of CaMKII and the R1 portion of the regulatory segment. The structure shown (at right) is that of crystal form A. (b) The CaMKII enzyme-substrate-complex active site is in the active conformation. Comparison of the major active-site components of the CaMKII enzyme-substrate complex with that of protein kinase A in the active state (PDB 1ATP). (c) Docking sites used by the R1 element are indicated as docking sites A, B and C and are colored red, green and gold, respectively, on a surface representation of the kinase domain. The R1 element is shown in a sticks representation. At right, an electrostatic surface potential representation (produced with Adaptive Poisson-Boltzmann Solver tools) of the CaMKII kinase domain in the enzyme-substrate complex illustrates a negatively charged region (red) encompassing docking sites B and C that is used by residues in the R1 element (basic residues shown in blue, hydrophobic residues shown in black).
Illustrator file, local only


First figure from paper

Figure 4: Capture of the regulatory segment results in cooperative activation of a monomeric kinase domain. (a) The activity of monomeric C. elegans CaMKII kinase domain toward syntide at varying calmodulin concentrations. The monomeric kinase domain does not show cooperative activation (data from a representative Ca2+-calmodulin activation response shown). (b) Presence of a decoy kinase domain that is competent for capture of the R1 element results in cooperativity in the calmodulin activation of monomeric kinase domain. The decoy was added in 10 times molar excess (100 nM) and has no enzyme activity because of mutations (D135N and K42M) introduced in the active site. (c) Mutations in docking site B of the decoy eliminate cooperative activation of the monomeric kinase domain. Data for three mutations (I205K, I101D and F98E, each introduced separately) are shown. (d) Mutation of residues (A280D and I281D, introduced together) in the R1 element in the C. elegans CaMKII holoenzyme results in a reduction in the Hill coefficient for Ca2+-calmodulin activation. All error bars and ± terms expressed are s.e.m.
Illustrator file, local only


First figure from paper

Figure 5: Structure of the CaMKII inhibitor CaMKIINtide bound to the kinase domain. Left, schematic diagram of the CaMKII kinase domain and the docking regions occupied by CaMKIINtide. Right, surface representation of the CaMKII kinase domain with the CaMKIINtide peptide bound; critical residues are highlighted. Basic residues (Arg43 and Lys46 (rat numbering for CaMKIINtide40)) occupy docking sites C and B, Leu47 and Ile50 occupy docking site B and a pseudosubstrate recognition mode of interaction is observed in docking site A (Arg52 at the P-3 site, Val56 at the P+1 site).
Illustrator file, local only


First figure from paper

Figure 6: A hypothetical mechanism for CaMKII activation. If the time interval between Ca2+ spikes is long compared to the dissociation time of Ca2+-calmodulin (low frequency), initial binding is presumed to lead to a separation of kinase domains; however, transphosphorylation of Thr286 does not occur before Ca2+-calmodulin dissociates. If the interval between Ca2+ spikes is short compared to the Ca2+-calmodulin dissociation time (high-frequency regime) Ca2+-calmodulin remains bound to the first pair of kinase domains long enough for a second, slow step to occur, in which the activated kinase domains capture the regulatory segments of adjacent kinase domains. This potentiates the binding of Ca2+-calmodulin to those domains with increased affinity, resulting in the phosphorylation of Thr286 and acquisition of autonomy (Ca2+-calmodulin-independent activity).
Illustrator file, local only




Supplemental Material


Supplemental Methods


Coordinates


Coordinates in the Protein Data Bank:
3KK8
3KK9
3KL8