A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin- dependent kinase II holoenzyme

Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin-binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains.     

Conformational changes underlying calcium/calmodulin-dependent protein kinase II activation

Calcium/calmodulin-dependent protein kinase II (CaMKII) interprets information conveyed by the amplitude and frequency of calcium transients by a controlled transition from an autoinhibited basal intermediate to an autonomously active phosphorylated intermediate (De Koninck and Schulman, 1998). We used spin labelling and electron paramagnetic resonance spectroscopy to elucidate the structural and dynamic bases of autoinhibition and activation of the kinase domain of CaMKII. In contrast to existing models, we find that autoinhibition involves a conformeric equilibrium of the regulatory domain, modulating substrate and nucleotide access. Binding of calmodulin to the regulatory domain induces conformational changes that release the catalytic cleft, activating the kinase and exposing an otherwise inaccessible phosphorylation site, threonine 286. Autophosphorylation at Thr286 further disrupts the interactions between the catalytic and regulatory domains, enhancing the interaction with calmodulin, but maintains the regulatory domain in a dynamic unstructured conformation following dissociation of calmodulin, sustaining activation. These findings support a mechanistic model of the CaMKII holoenzyme grounded in a dynamic understanding of autoregulation that is consistent with a wealth of biochemical and functional data.     

Crystal structure of a tetradecameric assembly of the association domain of Ca2+/calmodulin-dependent kinase II

We report the crystal structure of the 143 residue association domain of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). The association domain forms a hub-like assembly, composed of two rings of seven protomers each, which are stacked head to head and held together by extensive interfaces. The tetradecameric organization of the assembly was confirmed by analytical ultracentrifugation and multiangle light scattering. Individual protomers form wedge-shaped structures from which N-terminal helical segments that connect to the kinase domain extend toward the equatorial plane of the assembly, consistent with the arrangement of the kinase domains in a second outer ring. A deep and highly conserved pocket present within the association domain may serve as a docking site for proteins that interact with CaMKII.     

An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor

The mechanism by which the epidermal growth factor receptor (EGFR) is activated upon dimerization has eluded definition. We find that the EGFR kinase domain can be activated by increasing its local concentration or by mutating a leucine (L834R) in the activation loop, the phosphorylation of which is not required for activation. This suggests that the kinase domain is intrinsically autoinhibited, and an intermolecular interaction promotes its activation. Using further mutational analysis and crystallography we demonstrate that the autoinhibited conformation of the EGFR kinase domain resembles that of Src and cyclin-dependent kinases (CDKs). EGFR activation results from the formation of an asymmetric dimer in which the C-terminal lobe of one kinase domain plays a role analogous to that of cyclin in activated CDK/cyclin complexes. The CDK/cyclin-like complex formed by two kinase domains thus explains the activation of EGFR-family receptors by homo- or heterodimerization.     

Characterization of CaMKIIα holoenzyme stability

Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long‐term memory formation and other Ca²⁺‐dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X‐ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent T_m = 36°C), which is slightly stabilized by ATP/MgCl₂ binding (apparent T_m = 40°C) and significantly stabilized by regulatory segment binding (apparent T_m = 60°C). We crystallized the kinase domain of CaMKII bound to p‐coumaric acid in the active site. This structure reveals solvent‐exposed hydrophobic residues in the substrate‐binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent T_m ~ 90°C), and the holoenzyme structure has multiple unfolding transitions ranging from ~60°C to 100°C. Using MP, we compared a CaMKIIα holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact.     

Structural basis for the inhibition of tyrosine kinase activity of ZAP-70

ZAP-70, a cytoplasmic tyrosine kinase required for T cell antigen receptor signaling, is controlled by a regulatory segment that includes a tandem SH2 unit responsible for binding to immunoreceptor tyrosine-based activation motifs (ITAMs). The crystal structure of autoinhibited ZAP-70 reveals that the inactive kinase domain adopts a conformation similar to that of cyclin-dependent kinases and Src kinases. The autoinhibitory mechanism of ZAP-70 is, however, distinct and involves interactions between the regulatory segment and the hinge region of the kinase domain that reduce its flexibility. Two tyrosine residues in the SH2-kinase linker that activate ZAP-70 when phosphorylated are involved in aromatic-aromatic interactions that connect the linker to the kinase domain. These interactions are inconsistent with ITAM binding, suggesting that destabilization of this autoinhibited ZAP-70 conformation is the first step in kinase activation.     

Regulation of protein phosphatase 2A-mediated recruitment of IQGAP1 to beta1 integrin by EGF through activation of Ca2+/calmodulin-dependent protein kinase II

Maintenance of beta1 integrin-mediated cell adhesion in quiescent human mammary epithelial (HME) cells requires protein phosphatase (PP) 2A for not only dephosphorylation of beta1 integrin but also recruitment of IQGAP1 to Rac-bound beta1 integrin. However, how PP2A-dependent regulatory machinery of cell adhesion responds to EGF remains to be elucidated. We report here that phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) at threonine 286 was involved in the beta1 integrin complex that consisted of PP2A, Rac, and IQGAP1 in quiescent HME cells. Stimulation of the cells with EGF concomitantly induced an increase in intracellular Ca2+, activation of CaMKII, and dissociation of PP2A-IQGAP1-CaMKII from beta1 integrin-Rac. Because the activation of CaMKII and dissociation of PP2A-IQGAP1-CaMKII were blocked by either Ca2+-chelator or CaMKII inhibitor, we therefore propose that EGF has the ability to abrogate the PP2A function in the maintenance of beta1 integrin-mediated cell adhesion by dissociation of PP2A-IQGAP1-CaMKII from beta1 integrin-Rac through activation of CaMKII.     

Oligomerization states of the association domain and the holoenyzme of Ca2+/CaM kinase II

Ca2+/calmodulin activated protein kinase II (CaMKII) is an oligomeric protein kinase with a unique holoenyzme architecture. The subunits of CaMKII are bound together into the holoenzyme by the association domain, a C-terminal region of approximately 140 residues in the CaMKII polypeptide. Single particle analyses of electron micrographs have suggested previously that the holoenyzme forms a dodecamer that contains two stacked 6-fold symmetric rings. In contrast, a recent crystal structure of the isolated association domain of mouse CaMKIIalpha has revealed a tetradecameric assembly with two stacked 7-fold symmetric rings. In this study, we have determined the crystal structure of the Caenorhabditis elegans CaMKII association domain and it too forms a tetradecamer. We also show by electron microscopy that in its fully assembled form the CaMKII holoenzyme is a dodecamer but without the kinase domains, either from expression of the isolated association domain in bacteria or following their removal by proteolysis, the association domains form a tetradecamer. We speculate that the holoenzyme is held in its 6-fold symmetric state by the interactions of the N-terminal approximately 1-335 residues and that the removal of this region allows the association domain to convert into a more stable 7-fold symmetric form.     

A Conserved Isoleucine Maintains the Inactive State of Bruton's Tyrosine Kinase

Despite high level of homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamics simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest that this isoleucine side chain in the Tec family kinases acts as a “wedge” that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain.     

Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression.

Phosphorylation of CREB (cyclic AMP [cAMP]- response element [CRE]-binding protein) by cAMP-dependent protein kinase (PKA) leads to the activation of many promoters containing CREs. In neurons and other cell types, CREB phosphorylation and activation of CRE-containing promoters can occur in response to elevated intracellular Ca2+. In cultured cells that normally lack this Ca2+ responsiveness, we confer Ca(2+)-mediated activation of a CRE-containing promoter by introducing an expression vector for Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV). Activation could also be mediated directly by a constitutively active form of CaMKIV which is Ca2+ independent. The CaMKIV-mediated gene induction requires the activity of CREB/ATF family members but is independent of PKA activity. In contrast, transient expression of either a constitutively active or wild-type Ca2+/calmodulin-dependent protein kinase type II (CaMKII) fails to mediate the transactivation of the same CRE-containing reporter gene. Examination of the subcellular distribution of transiently expressed CaMKIV and CaMKII reveals that only CaMKIV enters the nucleus. Our results demonstrate that CaMKIV, which is expressed in neuronal, reproductive, and lymphoid tissues, may act as a mediator of Ca(2+)-dependent gene induction.     

Substrate-selective and calcium-independent activation of CaMKII by α-actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.     

Variation in assembly stoichiometry in non‐metazoan homologs of the hub domain of Ca2+/calmodulin‐dependent protein kinase II

The multi‐subunit Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) holoenzyme plays a critical role in animal learning and memory. The kinase domain of CaMKII is connected by a flexible linker to a C‐terminal hub domain that assembles into a 12‐ or 14‐subunit scaffold that displays the kinase domains around it. Studies on CaMKII suggest that the stoichiometry and dynamic assembly/disassembly of hub oligomers may be important for CaMKII regulation. Although CaMKII is a metazoan protein, genes encoding predicted CaMKII‐like hub domains, without associated kinase domains, are found in the genomes of some green plants and bacteria. We show that the hub domains encoded by three related green algae, Chlamydomonas reinhardtii, Volvox carteri f. nagarensis, and Gonium pectoral, assemble into 16‐, 18‐, and 20‐subunit oligomers, as assayed by native protein mass spectrometry. These are the largest known CaMKII hub domain assemblies. A crystal structure of the hub domain from C. reinhardtii reveals an 18‐subunit organization. We identified four intra‐subunit hydrogen bonds in the core of the fold that are present in the Chlamydomonas hub domain, but not in metazoan hubs. When six point mutations designed to recapitulate these hydrogen bonds were introduced into the human CaMKII‐α hub domain, the mutant protein formed assemblies with 14 and 16 subunits, instead of the normal 12‐ and 14‐subunit assemblies. Our results show that the stoichiometric balance of CaMKII hub assemblies can be shifted readily by small changes in sequence.     

Calmodulin-dependent protein kinase II triggers mouse egg activation and embryo development in the absence of Ca2+ oscillations

Fertilization in mammalian eggs is accompanied by oscillatory changes in intracellular Ca(2+) concentration, which are critical for initiating and completing egg activation events and the developmental program. Ca(2+)/Camodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is postulated to be the downstream transducer of the Ca(2+) signal in many cell types. We tested the hypothesis that CaMKII is the major integrator of Ca(2+)-induced egg activation events and embryo development by microinjecting a cRNA that encodes a constitutively active (Ca(2+)-independent) mutant form of CaMKII (CA-CaMKII) into mouse eggs. Expression of this cRNA, which does not increase intracellular Ca(2+), induced a sustained rise in CaMKII activity and triggered egg activation events, including cell cycle resumption, and degradation and recruitment of maternal mRNAs; cortical granule exocytosis, however, did not occur normally. Furthermore, when mouse eggs were injected with sperm devoid of Ca(2+)-releasing activity and activated with either CA-CaMKII cRNA or by SrCl(2), similar rates and incidence of development to the blastocyst stage were observed. These results strongly suggest that CaMKII is a major integrator of the Ca(2+) changes that occur following fertilization.     

Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation

Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIδ/Ca²⁺/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIδ/Ca²⁺/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix αD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules.

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Acetylcholine increases Ca2+ influx by activation of CaMKII in mouse oocytes

IP3-induced Ca2+ release is the primary mechanism that is responsible for acetylcholine (ACh)-induced Ca2+ oscillation. However, other mechanisms remain to explain intracellular Ca2+ elevation. We here report that ACh induces Ca2+ influx via T-type Ca2+ channel by activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the ACh-induced Ca2+ influx facilitates the generation of Ca2+ oscillation in the mouse ovulated oocytes (oocytes(MII)). ACh increased Ca2+ current by 50+/-21%, and produced Ca2+ oscillation. However, the currents and Ca2+ peaks were reduced in Ca2+ -free extracellular medium. ACh failed to activate Ca2+ current and to produce Ca2+ oscillation in oocytes pretreated with KN-93, a CaMKII inhibitor. KN-92, an inactive analogue of KN93, and PKC modulators could not prevent the effect of ACh. These results show that ACh increases T-type Ca2+ current by activation of CaMKII, independent of the PKC pathway, in the mouse oocytes.     

Organization of the SH3-SH2 unit in active and inactive forms of the c-Abl tyrosine kinase

The tyrosine kinase c-Abl is inactivated by interactions made by its SH3 and SH2 domains with the distal surface of the kinase domain. We present a crystal structure of a fragment of c-Abl which reveals that a critical N-terminal cap segment, not visualized in previous structures, buttresses the SH3-SH2 substructure in the autoinhibited state and locks it onto the distal surface of the kinase domain. Surprisingly, the N-terminal cap is phosphorylated on a serine residue that interacts with the connector between the SH3 and SH2 domains. Small-angle X-ray scattering (SAXS) analysis shows that a mutated form of c-Abl, in which the N-terminal cap and two other key contacts in the autoinhibited state are deleted, exists in an extended array of the SH3, SH2, and kinase domains. This alternative conformation of Abl is likely to prolong the active state of the kinase by preventing it from returning to the autoinhibited state.     

Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations: a simple model

The rules that govern the activation and autophosphorylation of the multifunctional Ca2+-calmodulin kinase II (CaMKII) by Ca2+ and calmodulin (CaM) are thought to underlie its ability to decode Ca2+ oscillations and to control multiple cellular functions. We propose a simple biophysical model for the activation of CaMKII by Ca2+ and calmodulin. The model describes the transition of the subunits of the kinase between their different possible states (inactive, bound to Ca2+-CaM, phosphorylated at Thr(286), trapped and autonomous). All transitions are described by classical kinetic equations except for the autophosphorylation step, which is modeled in an empirical manner. The model quantitatively reproduces the experimentally demonstrated frequency sensitivity of CaMKII [Science 279 (1998) 227]. We further use the model to investigate the role of several characterized features of the kinase--as well as some that are not easily attainable by experiments--in its frequency-dependent responses. In cellular microdomains, CaMKII is expected to sense very brief Ca2+ spikes; our simulations under such conditions reveal that the enzyme response is tuned to optimal frequencies. This prediction is then confirmed by experimental data. This novel and simple model should help in understanding the rules that govern CaMKII regulation, as well as those involved in decoding intracellular Ca2+ signals.     

Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes

Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins were translocated to the area between separating chromosomes. All these results suggest that CaMKII is a multifunctional regulator of meiotic cell cycle and spindle assembly and that it may exert its effect via regulation of MPF and MAPK/p90rsk activity during the meiotic maturation and activation of pig oocytes.