Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase

被引:5
|
作者
Fukumoto, Yusei [1 ]
Harada, Yuhei [1 ]
Ohtsuka, Satomi [1 ]
Kanayama, Naoki [1 ]
Magari, Masaki [1 ]
Hatano, Naoya [1 ]
Sakagami, Hiroyuki [2 ]
Tokumitsu, Hiroshi [1 ]
机构
[1] Okayama Univ, Grad Sch Interdisciplinary Sci & Engn Hlth Syst, Appl Cell Biol, Okayama 7008530, Japan
[2] Kitasato Univ, Dept Anat, Sch Med, Sagamihara, Kanagawa 2520374, Japan
基金
日本学术振兴会;
关键词
CaMKK; Oligomerization; Ca2+-signaling; Phosphorylation; CaM kinase cascade; MOLECULAR-CLONING; REGULATORY MECHANISM; CALMODULIN; CASCADE; BETA; LOCALIZATION; INHIBITION; EXPRESSION; UPSTREAM; COMPLEX;
D O I
10.1016/j.bbrc.2021.11.105
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:160 / 165
页数:6
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