A Significant but Rather Mild Contribution of T286 Autophosphorylation to Ca2+/CaM-Stimulated CaMKII Activity
A Significant but Rather Mild Contribution of T286 Autophosphorylation to Ca2+/CaM-Stimulated CaMKII Activity
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DOI：
10.1371/journal.pone.0037176
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发表时间：
2012-05-16
期刊：
影响因子：
3.7
通讯作者：
Bayer, K. Ulrich
中科院分区：
文献类型：
--
作者：
Coultrap, Steven J.;Barcomb, Kelsey;Bayer, K. Ulrich
背景资料：T286处的Ca 2 +/钙调素（CaM）依赖性蛋白激酶II（CaMKII）的自磷酸化产生部分Ca 2 +/CaM非依赖性的“自主”活性，这被认为是长时程增强（LTP）所必需的，LTP是一种突触可塑性形式，被认为是学习和记忆的基础。T286自磷酸化也为有效的钙/钙调素刺激的CaMKII活性的要求已被描述，但仍然存在争议。方法/主要发现：为了确定T286自磷酸化的贡献钙2 +/钙调素刺激的CaMKII活性，CaMKII野生型和其磷酸化无能T286 A突变体的活性进行了比较。由于单个激酶制剂之间的绝对活性可能不同，因此在六种不同提取物中测量每种激酶（在HEK-293细胞中表达）的活性。与对纯化的激酶（来自杆状病毒/Sf 9细胞表达系统）的测量一致，CaMKII T286 A对于两种不同的肽底物显示出轻度但显著降低的Ca 2 +/CaM刺激的磷酸化速率（与野生型的75-84%相似）。在T305/306处的额外较慢的CaMKII自磷酸化抑制由Ca 2 +/CaM引起的刺激，但在CaM刺激活性测定期间仅最低限度地发生于CaMKII野生型。因此，我们测试了T286 A突变体是否可以显示更广泛的抑制性自磷酸化，这可以解释其降低的刺激活性。相反，在我们的测定条件下，发现T286 A突变体的抑制性自磷酸化甚至进一步降低。另一方面，磷酸化-T305抗体也显示出与非磷酸化CaMKII的一些基础背景免疫反应性，如T305/306 A突变体所示。这些结果表明，Ca 2 +/CaM刺激的CaMKII活性是温和的，（类似于1.2- 1.3倍）通过额外的T286自磷酸化进一步增加，但这种自磷酸化不是受刺激活性的主要部分所必需的。这表明CaMKII T286 A突变小鼠的表型确实是由于缺乏自主活动，因为T286 A突变体在刺激活动中没有显示出显著降低。
Background: Autophosphorylation of the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) at T286 generates partially Ca2+/CaM-independent "autonomous" activity, which is thought to be required for long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. A requirement for T286 autophosphorylation also for efficient Ca2+/CaM-stimulated CaMKII activity has been described, but remains controversial.Methodology/Principal Findings: In order to determine the contribution of T286 autophosphorylation to Ca2+/CaM-stimulated CaMKII activity, the activity of CaMKII wild type and its phosphorylation-incompetent T286A mutant was compared. As the absolute activity can vary between individual kinase preparations, the activity was measured in six different extracts for each kinase (expressed in HEK-293 cells). Consistent with measurements on purified kinase (from a baculovirus/Sf9 cell expression system), CaMKII T286A showed a mildly but significantly reduced rate of Ca2+/CaM-stimulated phosphorylation for two different peptide substrates (to similar to 75-84% of wild type). Additional slower CaMKII autophosphorylation at T305/306 inhibits stimulation by Ca2+/CaM, but occurs only minimally for CaMKII wild type during CaM-stimulated activity assays. Thus, we tested if the T286A mutant may show more extensive inhibitory autophosphorylation, which could explain its reduced stimulated activity. By contrast, inhibitory autophosphorylation was instead found to be even further reduced for the T286A mutant under our assay conditions. On a side note, the phospho-T305 antibody showed some basal background immuno-reactivity also with non-phosphorylated CaMKII, as indicated by T305/306A mutants.Conclusions/Significance: These results indicate that Ca2+/CaM-stimulated CaMKII activity is mildly (similar to 1.2-1.3fold) further increased by additional T286 autophosphorylation, but that this autophosphorylation is not required for the major part of the stimulated activity. This indicates that the phenotype of CaMKII T286A mutant mice is indeed due to the lack of autonomous activity, as the T286A mutant showed no dramatic reduction in stimulated activity.