Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase
2022
Fukumoto, Yusei | Harada, Yuhei | Ohtsuka, Satomi | Kanayama, Naoki | Magari, Masaki | Hatano, Naoya | Sakagami, Hiroyuki | Tokumitsu, Hiroshi
Ca²⁺/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 Ca²⁺/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.
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