Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase

被引：8

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

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 2022年 / 587卷

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

页码：160 / 165

页数：6

共 36 条

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