Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase

• Published in: Biochemical and biophysical research communications 2022-01, Vol.587, p.160-165
• Main Authors: Fukumoto, Yusei, Harada, Yuhei, Ohtsuka, Satomi, Kanayama, Naoki, Magari, Masaki, Hatano, Naoya, Sakagami, Hiroyuki, Tokumitsu, Hiroshi
• Format: Article
• Language: English
• Subjects: Ca2+-signaling; CaM kinase cascade; CaMKK; Oligomerization; Phosphorylation
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2021.11.105

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. •CaMKK isoforms are capable of forming a multimeric complex in cells.•CaMKKα oligomerization is promoted by self-association, not mediated by scaffold proteins.•Promotion of homomeric oligomerization of CaMKKα is in a Ca2+/calmodulin-independent manner.•CaMKKα oligomer is catalytically active.