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Oligomerization of Ca 2+ /calmodulin-dependent protein kinase kinase.
- Source :
-
Biochemical and biophysical research communications [Biochem Biophys Res Commun] 2022 Jan 08; Vol. 587, pp. 160-165. Date of Electronic Publication: 2021 Dec 01. - Publication Year :
- 2022
-
Abstract
- Ca <superscript>2+</superscript> /calmodulin-dependent protein kinase kinases (CaMKKα and β) are regulatory kinases for multiple downstream kinases, including CaMKI, CaMKIV, PKB/Akt, and AMP-activated protein kinase (AMPK) through phosphorylation of each activation-loop Thr residue. In this report, we biochemically characterize the oligomeric structure of CaMKK isoforms through a heterologous expression system using COS-7 cells. Oligomerization of CaMKK isoforms was readily observed by treating CaMKK transfected cells with cell membrane permeable crosslinkers. In addition, His-tagged CaMKKα (His-CaMKKα) pulled down with FLAG-tagged CaMKKα (FLAG-CaMKKα) in transfected cells. The oligomerization of CaMKKα was confirmed by the fact that GST-CaMKKα/His-CaMKKα complex from transiently expressed COS-7 cells extracts was purified to near homogeneity by the sequential chromatography using glutathione-sepharose/Ni-sepharose and was observed in a Ca <superscript>2+</superscript> /CaM-independent manner by reciprocal pulldown assay, suggesting the direct interaction between monomeric CaMKKα. Furthermore, the His-CaMKKα kinase-dead mutant (D293A) complexed with FLAG-CaMKKα exhibited significant CaMKK activity, indicating the active CaMKKα multimeric complex. Collectively, these results suggest that CaMKKα can self-associate in the cells, constituting a catalytically active oligomer that might be important for the efficient activation of CaMKK-mediated intracellular signaling.<br />Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest.<br /> (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Subjects :
- Animals
Binding Sites
COS Cells
Calcium-Calmodulin-Dependent Protein Kinase Kinase genetics
Calcium-Calmodulin-Dependent Protein Kinase Kinase metabolism
Calcium-Calmodulin-Dependent Protein Kinase Type 1 genetics
Calcium-Calmodulin-Dependent Protein Kinase Type 1 metabolism
Chlorocebus aethiops
Cloning, Molecular
Escherichia coli genetics
Escherichia coli metabolism
Gene Expression
Gene Expression Regulation
Genetic Vectors chemistry
Genetic Vectors metabolism
Glutathione Transferase genetics
Glutathione Transferase metabolism
Isoenzymes chemistry
Isoenzymes genetics
Isoenzymes metabolism
Phosphorylation
Protein Binding
Protein Multimerization
Rats
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins metabolism
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Signal Transduction
Calcium-Calmodulin-Dependent Protein Kinase Kinase chemistry
Calcium-Calmodulin-Dependent Protein Kinase Type 1 chemistry
Glutathione Transferase chemistry
Recombinant Fusion Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1090-2104
- Volume :
- 587
- Database :
- MEDLINE
- Journal :
- Biochemical and biophysical research communications
- Publication Type :
- Academic Journal
- Accession number :
- 34875535
- Full Text :
- https://doi.org/10.1016/j.bbrc.2021.11.105