CAMKK2 regulates mitochondrial function by controlling succinate dehydrogenase expression, post-translational modification, megacomplex assembly, and activity in a cell-type-specific manner.
Calcium-Calmodulin-Dependent Protein Kinase Kinase
/ genetics
Electron Transport
/ genetics
Gene Expression Regulation, Enzymologic
/ genetics
HEK293 Cells
Hep G2 Cells
Homeostasis
/ genetics
Humans
Mitochondria
/ genetics
Multiprotein Complexes
/ genetics
Oxygen Consumption
/ genetics
Protein Processing, Post-Translational
/ genetics
Proteomics
Succinate Dehydrogenase
/ genetics
CAMKK2
Oxidative phosphorylation
Respiration
Respiratory supercomplex
Succinate dehydrogenase
Journal
Cell communication and signaling : CCS
ISSN: 1478-811X
Titre abrégé: Cell Commun Signal
Pays: England
ID NLM: 101170464
Informations de publication
Date de publication:
25 09 2021
25 09 2021
Historique:
received:
11
06
2021
accepted:
14
08
2021
entrez:
26
9
2021
pubmed:
27
9
2021
medline:
25
2
2022
Statut:
epublish
Résumé
The calcium (Ca2+)/calmodulin (CAM)-activated kinase kinase 2 (CAMKK2)-signaling regulates several physiological processes, for example, glucose metabolism and energy homeostasis, underlying the pathogenesis of metabolic diseases. CAMKK2 exerts its biological function through several downstream kinases, therefore, it is expected that depending on the cell-type-specific kinome profile, the metabolic effects of CAMKK2 and its underlying mechanism may differ. Identification of the cell-type-specific differences in CAMKK2-mediated glucose metabolism will lead to unravelling the organ/tissue-specific role of CAMKK2 in energy metabolism. Therefore, the objective of this study was to understand the cell-type-specific regulation of glucose metabolism, specifically, respiration under CAMKK2 deleted conditions in transformed human embryonic kidney-derived HEK293 and hepatoma-derived HepG2 cells. Cellular respiration was measured in terms of oxygen consumption rate (OCR). OCR and succinate dehydrogenase (SDH) enzyme activity were measured following the addition of substrates. In addition, transcription and proteomic and analyses of the electron transport system (ETS)-associated proteins, including mitochondrial SDH protein complex (complex-II: CII) subunits, specifically SDH subunit B (SDHB), were performed using standard molecular biology techniques. The metabolic effect of the altered SDHB protein content in the mitochondria was further evaluated by cell-type-specific knockdown or overexpression of SDHB. CAMKK2 deletion suppressed cellular respiration in both cell types, shifting metabolic phenotype to aerobic glycolysis causing the Warburg effect. However, isolated mitochondria exhibited a cell-type-specific enhancement or dampening of the respiratory kinetics under CAMKK2 deletion conditions. This was mediated in part by the cell-type-specific effect of CAMKK2 loss-of-function on transcription, translation, post-translational modification (PTM), and megacomplex assembly of nuclear-encoded mitochondrial SDH enzyme complex subunits, specifically SDHB. The cell-type-specific increase or decrease in SDHs protein levels, specifically SDHB, under CAMKK2 deletion condition resulted in an increased or decreased enzymatic activity and CII-mediated respiration. This metabolic phenotype was reversed by cell-type-specific knockdown or overexpression of SDHB in respective CAMKK2 deleted cell types. CAMKK2 loss-of-function also affected the overall assembly of mitochondrial supercomplex involving ETS-associated proteins in a cell-type-specific manner, which correlated with differences in mitochondrial bioenergetics. This study provided novel insight into CAMKK2-mediated cell-type-specific differential regulation of mitochondrial function, facilitated by the differential expression, PTMs, and assembly of SDHs into megacomplex structures. Video Abstract.
Sections du résumé
BACKGROUND
The calcium (Ca2+)/calmodulin (CAM)-activated kinase kinase 2 (CAMKK2)-signaling regulates several physiological processes, for example, glucose metabolism and energy homeostasis, underlying the pathogenesis of metabolic diseases. CAMKK2 exerts its biological function through several downstream kinases, therefore, it is expected that depending on the cell-type-specific kinome profile, the metabolic effects of CAMKK2 and its underlying mechanism may differ. Identification of the cell-type-specific differences in CAMKK2-mediated glucose metabolism will lead to unravelling the organ/tissue-specific role of CAMKK2 in energy metabolism. Therefore, the objective of this study was to understand the cell-type-specific regulation of glucose metabolism, specifically, respiration under CAMKK2 deleted conditions in transformed human embryonic kidney-derived HEK293 and hepatoma-derived HepG2 cells.
METHODS
Cellular respiration was measured in terms of oxygen consumption rate (OCR). OCR and succinate dehydrogenase (SDH) enzyme activity were measured following the addition of substrates. In addition, transcription and proteomic and analyses of the electron transport system (ETS)-associated proteins, including mitochondrial SDH protein complex (complex-II: CII) subunits, specifically SDH subunit B (SDHB), were performed using standard molecular biology techniques. The metabolic effect of the altered SDHB protein content in the mitochondria was further evaluated by cell-type-specific knockdown or overexpression of SDHB.
RESULTS
CAMKK2 deletion suppressed cellular respiration in both cell types, shifting metabolic phenotype to aerobic glycolysis causing the Warburg effect. However, isolated mitochondria exhibited a cell-type-specific enhancement or dampening of the respiratory kinetics under CAMKK2 deletion conditions. This was mediated in part by the cell-type-specific effect of CAMKK2 loss-of-function on transcription, translation, post-translational modification (PTM), and megacomplex assembly of nuclear-encoded mitochondrial SDH enzyme complex subunits, specifically SDHB. The cell-type-specific increase or decrease in SDHs protein levels, specifically SDHB, under CAMKK2 deletion condition resulted in an increased or decreased enzymatic activity and CII-mediated respiration. This metabolic phenotype was reversed by cell-type-specific knockdown or overexpression of SDHB in respective CAMKK2 deleted cell types. CAMKK2 loss-of-function also affected the overall assembly of mitochondrial supercomplex involving ETS-associated proteins in a cell-type-specific manner, which correlated with differences in mitochondrial bioenergetics.
CONCLUSION
This study provided novel insight into CAMKK2-mediated cell-type-specific differential regulation of mitochondrial function, facilitated by the differential expression, PTMs, and assembly of SDHs into megacomplex structures. Video Abstract.
Identifiants
pubmed: 34563205
doi: 10.1186/s12964-021-00778-z
pii: 10.1186/s12964-021-00778-z
pmc: PMC8466908
doi:
Substances chimiques
Multiprotein Complexes
0
SDHD protein, human
0
Succinate Dehydrogenase
EC 1.3.99.1
CAMKK2 protein, human
EC 2.7.11.17
Calcium-Calmodulin-Dependent Protein Kinase Kinase
EC 2.7.11.17
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
98Informations de copyright
© 2021. The Author(s).
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