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Updated: Apr 28, 2026

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Cdk5 phosphorylation of EFhd2 at S74 affects its calcium binding activity
Edwin Vázquez-Rosa1, Eva N Rodríguez-Cruz, Sybelle Serrano
1Department of Chemistry, College of Natural Sciences, University of Puerto Rico-Río Piedras Campus, San Juan, Puerto Rico, 00931; Protein Mass Spectrometry Core Facility, College of Natural Sciences, University of Puerto Rico - Río Piedras Campus, San Juan, Puerto Rico, 00931.
Cyclin-dependent kinase 5 (Cdk5) phosphorylates the calcium-binding protein EFhd2 at S74, impacting its function. This phosphorylation occurs in Alzheimer
Area of Science:
- Neuroscience
- Biochemistry
- Cell Biology
Background:
- EFhd2 is a calcium-binding protein highly expressed in the central nervous system.
- EFhd2 is associated with pathological tau proteins in tauopathies.
- Previous studies suggest EFhd2 may be a target for phosphorylation.
Purpose of the Study:
- To investigate if Cyclin-dependent kinase 5 (Cdk5) phosphorylates EFhd2.
- To determine the effect of Cdk5-mediated phosphorylation on EFhd2's molecular activities.
- To analyze the in vivo relevance of EFhd2 phosphorylation in neurodegenerative diseases.
Main Methods:
- In vitro kinase assays using Cdk5 and GSK3β.
- Biomass and tandem mass spectrometry for site identification.
- Site-directed mutagenesis to confirm phosphorylation sites.
- Generation and use of a phospho-specific antibody for detection in postmortem brain tissue.
Main Results:
- Cdk5 directly phosphorylates EFhd2 at serine 74 (S74), not S76.
- Cdk5-mediated phosphorylation of EFhd2 alters its calcium-binding activity.
- EFhd2 phosphorylation at S74 was detected in postmortem brain tissue from Alzheimer's disease patients and controls.
Conclusions:
- Cdk5 phosphorylates EFhd2 in vitro and in vivo.
- EFhd2 phosphorylation at S74 influences its calcium-binding function.
- EFhd2 phosphorylation state may regulate its physiological and pathological roles, particularly in tauopathies and Alzheimer's disease.
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