Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, Japan.
Mol Brain. 2013 Aug 19;6:37. doi: 10.1186/1756-6606-6-37.
αCaMKII plays central and essential roles in long-term potentiation (LTP), learning and memory. αCaMKII is activated via binding with Ca²⁺/CaM in response to elevated Ca²⁺ concentration. Furthermore, prolonged increase in Ca²⁺ concentration leads to the auto-phosphorylation of αCaMKII at T286, maintaining the activation of αCaMKII even after Ca²⁺/CaM dissociation. Importantly, the active form of αCaMKII is thought to exhibit conformational change. In order to elucidate the relationships between the interaction of αCaMKII with CaM and the conformational change of αCaMKII, we generated molecular probes (YFP-αCaMKII with CFP-CaM and YFP-αCaMKII-CFP) and performed time-lapse imaging of the interaction with CaM and the conformational change, respectively, in living cells using FRET.
The interaction of YFP-αCaMKII with CFP-CaM and the conformational change of YFP-αCaMKII-CFP were induced simultaneously in response to increased concentrations of Ca²⁺. Consistent with previous predictions, high levels of Ca²⁺ signaling maintained the conformational change of YFP-αCaMKII-CFP at the time when CFP-CaM was released from YFP-αCaMKII. These observations indicated the transfer of αCaMKII conformational change from CaM-dependence to CaM-independence. Furthermore, analyses using αCaMKII mutants showed that phosphorylation at T286 and T305/306 played positive and negative roles, respectively, during in vivo interaction with CaM and further suggested that CaM-dependent and CaM-independent conformational changed forms displays similar but distinct structures.
Importantly, these structual differences between CaM-dependent and -independent forms of αCaMKII may exhibit differential functions for αCaMKII, such as interactions with other molecules required for LTP and memory. Our molecular probes could thus be used to identify therapeutic targets for cognitive disorders that are associated with the misregulation of αCaMKII.
αCaMKII 在长时程增强（LTP）、学习和记忆中发挥着核心和关键作用。αCaMKII 通过与 Ca²⁺/CaM 结合而被激活，以响应升高的 Ca²⁺浓度。此外，Ca²⁺浓度的持续增加导致 αCaMKII 在 T286 处发生自动磷酸化，即使在 Ca²⁺/CaM 解离后，αCaMKII 的激活也能维持。重要的是，活性形式的 αCaMKII 被认为会发生构象变化。为了阐明 αCaMKII 与 CaM 的相互作用与 αCaMKII 构象变化之间的关系，我们生成了分子探针（带有 CFP-CaM 的 YFP-αCaMKII 和 YFP-αCaMKII-CFP），并使用 FRET 分别在活细胞中进行了与 CaM 的相互作用和构象变化的延时成像。
YFP-αCaMKII 与 CFP-CaM 的相互作用和 YFP-αCaMKII-CFP 的构象变化同时在 Ca²⁺浓度增加时被诱导。与之前的预测一致，高水平的 Ca²⁺信号在 CFP-CaM 从 YFP-αCaMKII 释放时维持 YFP-αCaMKII-CFP 的构象变化。这些观察结果表明，αCaMKII 构象变化从 CaM 依赖性向 CaM 独立性的转移。此外，使用 αCaMKII 突变体进行的分析表明，T286 和 T305/306 的磷酸化分别发挥了正、负作用，这在与 CaM 的体内相互作用中进一步表明，CaM 依赖性和 CaM 独立性构象变化形式显示出相似但不同的结构。
重要的是，αCaMKII 的 CaM 依赖性和独立性形式之间的这些结构差异可能表现出不同的功能，例如与 LTP 和记忆所需的其他分子相互作用。因此，我们的分子探针可用于鉴定与 αCaMKII 调节失常相关的认知障碍的治疗靶标。
