Dataset Information

Heterogeneity in human hippocampal CaMKII transcripts reveals allosteric hub-dependent regulation.

ABSTRACT: Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in Ca²⁺ signaling throughout the body. In the hippocampus, CaMKII is required for learning and memory. Vertebrate genomes encode four CaMKII homologs: CaMKII?, CaMKII?, CaMKII?, and CaMKII?. All CaMKIIs consist of a kinase domain, a regulatory segment, a variable linker region, and a hub domain, which is responsible for oligomerization. The four proteins differ primarily in linker length and composition because of extensive alternative splicing. Here, we report the heterogeneity of CaMKII transcripts in three complex samples of human hippocampus using deep sequencing. We showed that hippocampal cells contain a diverse collection of over 70 CaMKII transcripts from all four CaMKII-encoding genes. We characterized the Ca²⁺/CaM sensitivity of hippocampal CaMKII variants spanning a broad range of linker lengths and compositions. The effect of the variable linker on Ca²⁺/CaM sensitivity depended on the kinase and hub domains. Moreover, we revealed a previously uncharacterized role for the hub domain as an allosteric regulator of kinase activity, which may provide a pharmacological target for modulating CaMKII activity. Using small-angle x-ray scattering and single-particle cryo-electron microscopy (cryo-EM), we present evidence for extensive interactions between the kinase and the hub domains, even in the presence of a 30-residue linker. Together, these data suggest that Ca²⁺/CaM sensitivity in CaMKII is homolog dependent and includes substantial contributions from the hub domain. Our sequencing approach, combined with biochemistry, provides insights into understanding the complex pool of endogenous CaMKII splice variants.

SUBMITTER: Sloutsky R

PROVIDER: S-EPMC7654443 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Heterogeneity in human hippocampal CaMKII transcripts reveals allosteric hub-dependent regulation.

Sloutsky Roman R Dziedzic Noelle N Dunn Matthew J MJ Bates Rachel M RM Torres-Ocampo Ana P AP Boopathy Sivakumar S Page Brendan B Weeks John G JG Chao Luke H LH Stratton Margaret M MM

Science signaling 20200721 641

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in Ca<sup>2+</sup> signaling throughout the body. In the hippocampus, CaMKII is required for learning and memory. Vertebrate genomes encode four CaMKII homologs: CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ. All CaMKIIs consist of a kinase domain, a regulatory segment, a variable linker region, and a hub domain, which is responsible for oligomerization. The four proteins differ primarily in linker length and composition becau ...[more]

PMID: 32694170

Similar Datasets

Project description:Ca2+ signaling plays a key role in physiological processes such as memory formation and cardiac function. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is the primary kinase that responds to Ca2+ inputs in these cells. There are four CaMKII paralogs in mammals which are alternatively spliced in the variable linker region to create upwards of 70 different variants. In this study, we systematically studied different linker regions and determined that the position of charged residues within the linker region modulates the Ca2+/CaM sensitivity of the holoenzyme. We present an X-ray crystal structure of full-length CaMKIIδ that shows a domain-swapped conformation of the subunits within the dodecameric holoenzyme. In this structure, the kinase domain of one subunit is docked onto the hub domain of a different subunit, providing an additional interface within the holoenzyme. Mutations at the equatorial and lateral interfaces revealed that the kinase-hub interaction dissociates as the hub-hub interfaces are disturbed, which led alterations in the stoichiometry of CaMKII holoenzyme and Ca2+/CaM sensitivity. Molecular dynamics simulations of linker-containing domain-swapped and non-domain-swapped CaMKIIs reveal that the domain-swapped configuration facilitates an interaction between the calmodulin binding domain and the variable linker region, such that dynamic electrostatic forces between charges on these segments can modulate the equilibrium between the compact and extended conformational states of the holoenzyme. Small angle X-ray scattering data confirms that a negatively charged linker CaMKII holoenzyme adopts a more compact conformation compared to a positively charged linker. These data support a model where patches of charged linker residues interact with the calmodulin binding domain to allosterically regulate sensitivity to Ca2+/CaM. Our findings provide a new framework for understanding CaMKII structure and allosteric regulation by the variable linker region in Ca2+-sensitive cells.

Project description:Calcium/calmodulin-dependent protein kinase II (CaMKII), an abundant protein in neurons, is involved in synaptic plasticity and learning. CaMKII associates with multiple proteins located at or near the postsynaptic density (PSD), and CaMKII is known to translocate from cytoplasm to PSD under excitatory conditions. The present study examined the laminar distribution of CaMKII at the PSD by immunogold labeling in dissociated hippocampal cultures under low calcium (EGTA or APV), control, and stimulated (depolarization with high K+ or NMDA) conditions. The patterns of CaMKII distribution are classified with particular reference to the two layers of the PSD: (1) the PSD core, a layer within ~ 30-40 nm to the postsynaptic membrane, and (2) the PSD pallium, a deeper layer beyond the PSD core, ~ 100-120 nm from the postsynaptic membrane. Under low calcium conditions, a subpopulation (40%) of synapses stood out with no CaMKII labeling at the PSD, indicating that localization of CaMKII at the PSD is sensitive to calcium levels. Under control conditions, the majority (~ 60-70%) of synapses had label for CaMKII dispersed evenly in the spine, including the PSD and the nearby cytoplasm. Upon stimulation, the majority (60-75%) of synapses had label for CaMKII concentrated at the PSD, delineating the PSD pallium from the cytoplasm. Median distance of label for CaMKII to postsynaptic membrane was higher in low calcium samples (68-77 nm), than in control (59-63 nm) and stimulated samples (49-53 nm). Thus, upon stimulation, not only more CaMKII translocated to the PSD, but they also were closer to the postsynaptic membrane. Additionally, there were two relatively infrequent labeling patterns that may represent intermediate stages of CaMKII distribution between basal and stimulated conditions: (1) one type showed label preferentially localized near the PSD core where CaMKII may be binding to NR2B, an NMDA receptor concentrated at the PSD core, and (2) the second type showed label preferentially in the PSD pallium, where CaMKII may be binding to Shank, a PSD scaffold protein located in the PSD pallium. Both of these distribution patterns may portray the initial stages of CaMKII translocation upon synaptic activation. In addition to binding to PSD proteins, the concentrated CaMKII labeling at the PSD under heightened excitatory conditions could also be formed by self-clustering of CaMKII molecules recruited to the PSD. Most importantly, these accumulated CaMKII molecules do not extend beyond the border of the PSD pallium, and are likely held in the pallium by binding to Shank under these conditions.

Dataset Information

Heterogeneity in human hippocampal CaMKII transcripts reveals allosteric hub-dependent regulation.

Publications

Heterogeneity in human hippocampal CaMKII transcripts reveals allosteric hub-dependent regulation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets