Identification of a novel CaMKK substrate
Identification of a novel CaMKK substrate
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DOI：
10.1016/j.bbrc.2011.05.102
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发表时间：
2011-06-24
影响因子：
3.1
通讯作者：
Tokumitsu, Hiroshi
中科院分区：
文献类型：
--
作者：
Fujimoto, Tomohito;Hatano, Naoya;Tokumitsu, Hiroshi
Ca（2+）/钙调蛋白依赖性蛋白激酶激酶（CaMKK）磷酸化并激活特异性下游蛋白激酶，包括CaMKI、CaMKIV和5 '-AMP-激活的蛋白激酶。为了研究CaMKK介导的信号通路的多样性，我们使用N（6）-（1-甲基丁基）-ATP和基因工程化的CaMKK α突变体CaMKK α（Phe（230）Gly）寻找新的CaMKK底物，所述突变体能够利用这种ATP类似物作为磷酸供体。在存在N（6）-（1-甲基丁基）-ATP和Ca（2+）/CaM的情况下，将大鼠脑提取物与重组CaMKK α（Phe（230）Gly）孵育，但不与野生型激酶孵育，诱导50 kDa蛋白的显著苏氨酸磷酸化以及Thr（177）处的CaMKI磷酸化。通过使用连续柱层析部分纯化50 kDa CaMKK底物，并通过LC-MS/MS分析鉴定为Syndapin I。我们证实了重组Syndapin I在体外被CaMKK α和β亚型在Thr（355）处磷酸化。在转染的HeLa细胞中，HA-Syndapin I在Thr（355）处的磷酸化被CaMKK同种型的组成型活性突变体的共表达显著诱导。这是首次报道CaMKK能够磷酸化非激酶底物，提示CaMKK介导的新的Ca（2+）信号通路的可能性，其独立于下游蛋白激酶。(C)2011 Elsevier Inc. All rights reserved.
Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates specific downstream protein kinases including CaMKI, CaMKIV and 5'-AMP-activated protein kinase. In order to examine the variety of CaMKK-mediated signaling pathways, we searched for novel CaMKK substrate(s) using N(6)-(1-methylbutyl)-ATP and genetically engineered CaMKK alpha mutant, CaMKK alpha (Phe(230)Gly), that was capable of utilizing this ATP analogue as a phosphate donor. Incubation of rat brain extracts with recombinant CaMKK alpha (Phe(230)Gly), but not with wild-type kinase, in the presence of N(6)-(1-methylbutyl)-ATP and Ca(2+)/CaM, induced significant threonine phosphorylation of a 50 kDa protein as well as CaMKI phosphorylation at Thr(177). The 50 kDa CaMKK substrate was partially purified by using serial column chromatography, and was identified as Syndapin I by LC-MS/MS analysis. We confirmed that recombinant Syndapin I was phosphorylated by CaMKK alpha and beta isoforms at Thr(355) in vitro. Phosphorylation of HA-Syndapin I at Thr(355) in transfected HeLa cells was significantly induced by co-expression of constitutively active mutants of CaMKK isoforms. This is the first report that CaMKK is capable of phosphorylating a non-kinase substrate suggesting the possibility of CaMKK-mediated novel Ca(2+)-signaling pathways that are independent of downstream protein kinases. (C) 2011 Elsevier Inc. All rights reserved.