Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase

Ca2+/calmodulin-dependent protein kinase kinases (CaMKK alpha and beta) are regulatory kinases for multiple downstream kinases, including CaMKI, CaMKIV, PKB/Alt, 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 CaMKK alpha (His-CaMKK alpha) pulled down with FLAG-tagged CaMKK alpha (FLAG-CaMKK alpha) in transfected cells. The oligomerization of CaMKK alpha was confirmed by the fact that GST-CaMKK alpha/His-CaMKK alpha complex from transiently expressed COS-7 cells extracts was purified to near homogeneity by the sequential chromatography using glutathionesepharose/Ni-sepharose and was observed in a Ca2+/CaM-independent manner by reciprocal pulldown assay, suggesting the direct interaction between monomeric CaMKK alpha. Furthermore, the His-CaMKK alpha kinase-dead mutant (D293A) complexed with FLAG-CaMKK alpha exhibited significant CaMKK activity, indicating the active CaMKK alpha multimeric complex. Collectively, these results suggest that CaMKK alpha can self-associate in the cells, constituting a catalytically active oligomer that might be important for the efficient activation of CaMKK-mediated intracellular signaling. (C) 2021 Elsevier Inc. All rights reserved.