Related Experiment Video
Updated: Jun 1, 2026
11:23
Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
The CaMKII D135N mutation blocks kinase activity and reduces GluN2B binding.
Matthew E Larsen1, C Madison Barker2, Raul Satoshi Vargas2
1Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado; Program in Neuroscience, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
Molecular Pharmacology
|May 30, 2026
Summary
The Ca2+/calmodulin-dependent protein kinase II (CaMKII) D135N mutant abolishes enzymatic activity but also reduces GluN2B binding, impacting its use in studying synaptic potentiation. This suggests a structural role for CaMKII in long-term potentiation.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Recent studies suggest Ca2+/calmodulin-dependent protein kinase II (CaMKII) functions structurally, not enzymatically, in synaptic potentiation.
- The CaMKII D135N mutation was proposed to selectively impair enzymatic activity while preserving structural binding to GluN2B.
Purpose of the Study:
- To characterize the CaMKII D135N mutant's enzymatic activity and binding to GluN2B.
- To evaluate the utility of the D135N mutant in distinguishing between enzymatic and structural roles of CaMKII in long-term potentiation (LTP).
Main Methods:
- In vitro biochemical assays to assess kinase activity and autophosphorylation.
- Co-condensation assays to evaluate CaMKII D135N binding and persistence with GluN2B.
- Experiments using AS283, an inhibitor enhancing GluN2B binding, to probe mutant behavior.
Main Results:
- The CaMKII D135N mutant demonstrated abolished kinase activity and autophosphorylation at T286.
- D135N mutation reduced binding to GluN2B and prevented co-condensation with GluN2B.
- AS283 partially rescued GluN2B binding of the D135N mutant, similar to the T286A mutant.
Conclusions:
- The D135N mutant's reduced GluN2B binding necessitates careful interpretation when used to differentiate CaMKII's enzymatic versus structural functions.
- Findings support a structural role for CaMKII in LTP induction, but with caveats regarding the D135N mutant's specific effects.
- This study provides crucial insights for utilizing the D135N mutant as a tool in CaMKII research.
Related Concept Videos
02:34Inhibition of Cdk Activity
The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be restricted when undergoing abnormal changes. Most cancers correlate to the deregulated cell cycle, and since Cdks are a central component of the cell cycle, Cdk inhibitors are extensively studied to develop anticancer agents. For instance, cyclin D associates with several Cdks, such as Cdk 4/6, to form an active complex. The cyclin D-Cdk4/6 complex...
01:25cAMP-dependent Protein Kinase Pathways
Cyclic Adenosine Monophosphate (cAMP) is an essential second messenger that activates protein kinase A (PKA) and regulates various biological processes. A single epinephrine molecule binds to GPCR and activates several heterotrimeric G proteins, each stimulating multiple adenylyl cyclase, amplifying the signal, and synthesizing large numbers of cAMP molecules. Small changes in cAMP concentration affect PKA activity. The binding of four cAMP molecules induces a conformational change in PKA,...
01:22PI3K/mTOR/AKT Signaling Pathway
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast, mTORC2 consists of a rapamycin-insensitive companion...
01:16Calmodulin-dependent Signaling
Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
01:20The JAK-STAT Signaling Pathway
Several cytokine receptors have tightly bound Janus kinase or JAK proteins attached at their cytosolic tail. Small signaling molecules such as cytokines, growth hormones, or prolactins bind to the cytokine receptors and initiate their dimerization. The dimerization brings the cytosolic JAKs together that trans-phosphorylate and activates each other. The activated JAKs now phosphorylate cytosolic tails of the cytokine receptors, which serve as binding sites for adaptor proteins such as SH2...