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

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: 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 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.

Download Full-text

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

10.1101/721589 ◽

2019 ◽

Cited By ~ 4

Author(s):

Roman Sloutsky ◽

Noelle Dziedzic ◽

Matthew J. Dunn ◽

Rachel M. Bates ◽

Ana P. Torres-Ocampo ◽

...

Keyword(s):

Learning And Memory ◽

Kinase Domain ◽

The Body ◽

Complex Samples ◽

X Ray Scattering ◽

Human Hippocampus ◽

Conserved Genes ◽

New Finding ◽

Dependent Protein ◽

The Brain

AbstractCa2+-calmodulin dependent protein kinase II (CaMKII) plays a central role in Ca2+ signaling throughout the body. Specifically in the hippocampus, CaMKII is required for learning and memory. CaMKII is encoded by four highly conserved genes in vertebrates: α, β, γ, and δ. AllCaMKIIs are comprised of a kinase domain, regulatory segment, variable linker region, and hub domain responsible for oligomerization. The four genes differ primarily in linker length and composition due to extensive alternative splicing. Here, we unambiguously report the heterogeneity of CaMKII transcripts in 3 complex samples of human hippocampus using Illumina sequencing. Our results show that hippocampal cells contain a diverse collection of 70 CaMKII transcripts from all four CaMKII genes. We characterized the Ca2+/CaM sensitivity of hippocampal CaMKII variants spanning a broad range of linker lengths and compositions. We demonstrate that the effect of the variable linker on Ca2+/CaM sensitivity is conditional on kinase and hub domains. Moreover, we reveal a novel role for the hub domain as an allosteric regulator of kinase activity, which may provide a new pharmacological target for modulating CaMKII activity. Using small angle X-ray scattering and single-particle electron cryo-microscopy, we present evidence for extensive interaction between the kinase and the hub domain, even in the presence of a 30-residue linker. Taken together, we propose that Ca2+/CaM sensitivity in CaMKII is gene-dependent and includes significant contributions from the hub. Our sequencing approach combined with biochemistry provides new insights into understanding the complex pool of endogenous CaMKII.One Sentence SummaryCaMKII is a well-conserved protein that is essential for learning and memory. When CaMKII is mutated in a mouse, this mouse has difficulty learning and remembering how to get through a maze. The hippocampus is the part of the brain required for memory. Here, we used a specific experiment to determine every type of CaMKII that is in a human hippocampus. We found 70 different types and then asked how these differences affect CaMKII function. These data provide evidence that an assembly domain of CaMKII plays an unexpected role regulating its activity. This new finding helps us better understand endogenous CaMKII in the brain and provides a new mechanism for modulating CaMKII activity.

Download Full-text

Cryo-EM structure of the DNA-PK holoenzyme

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1707386114 ◽

2017 ◽

Vol 114 (28) ◽

pp. 7367-7372 ◽

Cited By ~ 37

Author(s):

Humayun Sharif ◽

Yang Li ◽

Yuanchen Dong ◽

Liyi Dong ◽

Wei Li Wang ◽

...

Keyword(s):

Dna Binding ◽

Dna Interaction ◽

Kinase Domain ◽

Nonhomologous End Joining ◽

Globular Domain ◽

End Joining ◽

Large Protein ◽

Dependent Protein Kinase ◽

Cryo Electron Microscopy ◽

Dependent Protein

DNA-dependent protein kinase (DNA-PK) is a large protein complex central to the nonhomologous end joining (NHEJ) DNA-repair pathway. It comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer of DNA-binding proteins Ku70 and Ku80. Here, we report the cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs at 4.4-Å resolution and the DNA-PK holoenzyme at 5.8-Å resolution. The DNA-PKcs structure contains three distinct segments: the N-terminal region with an arm and a bridge, the circular cradle, and the head that includes the kinase domain. Two perpendicular apertures exist in the structure, which are sufficiently large for the passage of dsDNA. The DNA-PK holoenzyme cryo-EM map reveals density for the C-terminal globular domain of Ku80 that interacts with the arm of DNA-PKcs. The Ku80-binding site is adjacent to the previously identified density for the DNA-binding region of the Ku70/Ku80 complex, suggesting concerted DNA interaction by DNA-PKcs and the Ku complex.

Download Full-text

Influence of Lead Exposure in the Expression of Calmodulin – Related Genes: A Preliminary Study on Workers Working in Industry of Batteries, In Iraq

Baghdad Science Journal ◽

10.21123/bsj.15.2.145-149 ◽

2018 ◽

Vol 15 (2) ◽

pp. 145-149

Author(s):

Baghdad Science Journal

Keyword(s):

Gene Expression ◽

The Body ◽

Mitogen Activated Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Exposed Workers ◽

Mitogen Activated Protein ◽

Dependent Protein ◽

Preliminary Study ◽

Chemokine Receptor 4 ◽

Protein Kinase Kinase

Most pathological effects of lead on the body are due to ability of lead to bind with important cellular molecules of various tissues and organs leading to formation abnormal molecules and thus to emergence of pathological conditions. To evaluation the risk to the health status of Iraqi workers who work in the batteries industry, expression of three types of calmodulin related genes were examined. Blood samples were collected from worker working in Iraqi industry of batteries (located in Al-Waziriya), then RNAs extraction were done thereby gene expression for Calcium/Calmodulin- dependent protein kinase2 (CaMKK2), C-X-C Chemokine receptor 4 (CXCR4) and mitogen activated protein kinase kinase 6 (MAP2K6) was done for each sample by using RT-qPCR. The study showed that the expression of CXCR4 gene was significantly decrement in the lead exposed workers meanwhile the MAP2K6 gene insignificantly increment in those workers, but no effect appeared on their CaMKK2 gene expression. Conclusion obtained from this study is that lead has ability to impact on calmodulin related genes in the workers working in the batteries manufacture, but this ability is variable according to the type of gene.

Download Full-text

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

10.1101/2020.03.27.011866 ◽

2020 ◽

Author(s):

Alise R. Muok ◽

Teck Khiang Chua ◽

Madhur Srivastava ◽

Wen Yang ◽

Zach Maschmann ◽

...

Keyword(s):

Kinase Activity ◽

Kinase Domain ◽

X Ray ◽

X Ray Scattering ◽

Cryo Electron Microscopy ◽

Bacterial Chemoreceptors ◽

Coupling Protein ◽

Site Directed Spin Labeling ◽

Dynamics Simulations ◽

Receptor Dimers

AbstractBacterial chemoreceptors, the CheA histidine kinase, and the coupling protein CheW comprise transmembrane molecular arrays with remarkable sensing properties. An unanswered question concerns how receptors turn off CheA kinase activity. Chemoreceptor cytoplasmic regions engineered to assume a trimer-of-receptor-dimers configuration form well-defined complexes with CheA and CheW and promote a kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar ESR spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The kinase-off state is especially stable, has relatively low domain mobility and associates the histidine substrate domain P1 and docking domain P2 with the kinase core. Distances measured between spin-labeled ADP molecules bound to the P4 kinase domain provide evidence for a “dipped conformation” that has been previously proposed from molecular dynamics simulations. Taken together, the data provide an experimentally restrained model for the inhibited state of the core-signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

Download Full-text

Structural and binding studies of human CAMKK2 kinase domain bound to small molecule ligands

10.1101/538157 ◽

2019 ◽

Author(s):

Gerson S. Profeta ◽

Caio V. dos Reis ◽

Paulo H. C. Godoi ◽

Angela M. Fala ◽

Roger Sartori ◽

...

Keyword(s):

Protein Kinase ◽

Small Molecule ◽

Kinase Inhibitors ◽

Kinase Domain ◽

Titration Calorimetry ◽

Binding Studies ◽

Regulatory Pathways ◽

Calcium Calmodulin Dependent ◽

Small Molecule Ligands ◽

Dependent Protein

AbstractCalcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no specific small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homologue, CAMKK1, are thought to have overlapping biological roles. Here we present six novel co-structures of CAMKK2 bound to potent ligands identified from a library of ATP-competitive kinase inhibitors. Isothermal titration calorimetry (ITC) revealed that binding to some of these molecules is enthalpy driven. We expect our results to further advance current efforts to discover small molecule kinase inhibitors specific to each human CAMKK.

Download Full-text