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Related Concept Videos

Calmodulin-dependent Signaling01:16

Calmodulin-dependent Signaling

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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,...
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Related Experiment Video

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Imaging Local Ca2+ Signals in Cultured Mammalian Cells
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Real-time single-molecule imaging of CaMKII-calmodulin interactions.

Shahid Khan1, Justin E Molloy2, Henry Puhl3

  • 1Molecular Biology Consortium at Lawrence Berkeley National Laboratory, Berkeley, California.

Biophysical Journal
|February 28, 2024
PubMed
Summary
This summary is machine-generated.

Calcium/calmodulin (CAM) binding to calcium/calmodulin-dependent protein kinase II (CaMKII) is ATP-dependent, influencing its affinity and function. This study reveals CaMKII

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Area of Science:

  • Biochemistry and Molecular Biology
  • Neuroscience
  • Cell Signaling

Background:

  • Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in cellular signaling pathways.
  • CaMKII activation involves calcium/calmodulin (CAM) binding and ATP-dependent autophosphorylation, which enhances CAM affinity.
  • Understanding the precise mechanisms of CAM association with CaMKII, particularly its ATP dependence, is vital for deciphering its regulatory functions.

Purpose of the Study:

  • To investigate the ATP dependence of CAM association with the CaMKIIβ isoform using single-molecule imaging.
  • To elucidate the role of CaMKII dimerization and specific phosphorylation sites in regulating CAM binding affinity.
  • To compare the behavior of wild-type CaMKII with various mutants under different ATP conditions.

Main Methods:

  • Single-molecule total internal reflection fluorescence microscopy (TIRFm) to observe Rhodamine-CAM association with Venus-CaMKIIβ.
  • High-resolution (0.5 s) tracking of CAM binding and dissociation events.
  • Spot-photobleaching to determine CaMKII holoenzyme stoichiometry simultaneously with CAM occupancy.
  • Utilized phosphorylation-defective, ATP-binding impaired, and phosphomimetic CaMKII mutants.

Main Results:

  • ATP-dependent CAM association with CaMKII requires dimer formation for both α and β isoforms.
  • CaMKII mutants revealed distinct low- and high-affinity states for CAM binding, modulated by ATP and phosphorylation.
  • Wild-type CaMKII exhibited a bimodal distribution of low- and high-affinity states in ATP, unlike previously published in vitro assays.
  • The phosphomimetic mutant (T287D) showed significantly higher CAM affinity, while ATP-binding impaired mutants displayed reduced affinity.

Conclusions:

  • CaMKII holoenzyme structure, dimerization, and phosphorylation status critically regulate CAM binding affinity in an ATP-dependent manner.
  • The observed bimodal distribution in wild-type CaMKII suggests complex in vivo regulation of CAM association.
  • Findings highlight how assay conditions can influence the perceived balance of CaMKII activation and inhibition, impacting kinase function in vivo.