Related Experiment Video

Updated: Aug 7, 2026

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

Phosphorylation of CaMKII at Thr253 occurs in vivo and enhances binding to isolated postsynaptic densities

Paola V Migues1, Ingo T Lehmann, Lisa Fluechter

  • 1School of Biomedical Sciences and Hunter Medical Research Institute, Faculty of Health, The University of Newcastle, Callaghan, New South Wales, Australia.

Journal of Neurochemistry
|June 30, 2006
PubMed

Insights

Calcium-calmodulin dependent protein kinase II (CaMKII) is phosphorylated at Thr253 in vivo, influencing its subcellular location. This dynamic phosphorylation regulates CaMKII function by altering its cellular distribution.

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biochemistry

Background:

  • Ca(2+)-calmodulin stimulated protein kinase II (CaMKII) autophosphorylation at Thr286 and Thr305/306 regulates its in vivo activity and localization.
  • CaMKII autophosphorylation at Thr253 is known in vitro, but its in vivo relevance and functional impact remain unclear.

Purpose of the Study:

  • To investigate the in vivo occurrence and functional consequences of CaMKII phosphorylation at Thr253.
  • To determine if Thr253 phosphorylation affects CaMKII kinase activity and subcellular localization.

Main Methods:

  • Utilized specific antibodies for phospho-Thr253 CaMKII and FLAG-tagged CaMKII mutants (wild type, phospho- and dephospho-mimic).
  • Assessed in vitro kinase activity and binding to subcellular fractions, including post-synaptic densities (PSDs).
  • Examined dynamic Thr253 phosphorylation in intact hippocampal slices following KCl-induced depolarization.

Main Results:

  • Thr253 phosphorylation did not alter CaMKII's in vitro kinase activity (Ca(2+)-calmodulin dependent or autonomous).
  • The Thr253Asp mutation increased CaMKII binding to PSD-enriched fractions, similar to and additive with Thr286 phosphorylation.
  • KCl depolarization dynamically increased Thr253 phosphorylation in hippocampal slices, with phospho-Thr253-CaMKII localized to PSD fractions.

Conclusions:

  • Identified Thr253 as a novel in vivo phosphorylation site for CaMKII.
  • Suggests that dynamic Thr253 phosphorylation regulates CaMKII function by modulating its subcellular distribution, particularly within PSDs.

More Related Videos

Oligopeptide Competition Assay for Phosphorylation Site Determination09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy10:41

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Published on: June 7, 2019

Related Concept Videos

Calmodulin-dependent Signaling01:16

Calmodulin-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,...
Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
IP3/DAG Signaling Pathway01:11

IP3/DAG Signaling Pathway

Membrane lipids such as phosphatidylinositol (PI) are precursors for several membrane-bound and soluble second messengers. Specific kinases phosphorylate PI and produce phosphorylated inositol phospholipids. One such inositol phospholipids are the  phosphatidylinositol-4,5 bisphosphate [PI(4,5)P2], present in the inner half of the lipid bilayer. Upon ligand binding, GPCR stimulates Gq proteins to turn on phospholipase Cꞵ. Activated phospholipase Cꞵ cleaves PI(4,5)P2 and produces two-second...
cAMP-dependent Protein Kinase Pathways01:25

cAMP-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,...
Amplifying Signals via Second Messengers01:15

Amplifying Signals via Second Messengers

Many receptor binding ligands are hydrophilic; they do not cross the cell membrane but bind to cell-surface receptors. Thus, their message must be relayed by second messengers present in the cell cytoplasm. There are several second messenger pathways, each with its own way of relaying information. For example, the G protein-coupled receptors can activate both phosphoinositol and cyclic AMP (cAMP) second messenger pathways. The phosphoinositol pathway is active when the receptor induces...