Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin-dependent protein kinase II self-association
Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin-dependent protein kinase II self-association
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DOI：
10.1046/j.1471-4159.2001.00119.x
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发表时间：
2001-03-01
影响因子：
4.7
通讯作者：
Waxham, MN
中科院分区：
文献类型：
--
作者：
Hudmon, A;Kim, SA;Waxham, MN
使用光散射分析由α或β亚基组成的钙调蛋白（CaM）-激酶II全酶，以确定自缔合的机制。在相同的反应条件下，只有α-钙调素激酶II全酶自我关联。在非常低的酶浓度（0.14 μ M或7 μ g/mL）下检测到自缔合。光散射揭示了两个阶段的自关联：迅速上升，达到峰值，然后由一个缓慢的下降，稳定后2-3分钟。电子显微镜确定，散射的快速上升是由于形成松散的簇的全酶，经历进一步的协会成几个微米的直径随着时间的推移大复合物。自缔合需要激活的Ca 2 +/CaM和强烈依赖于pH值。自缔合没有检测到在pH值7.5，但是，这个过程的程度增加，反应pH值降低到7.0以下。一种肽底物（autocamtide-2）和抑制剂（AIP）的设计，从钙调蛋白激酶II的自动调节结构域有力地防止自协会，而肽底物合成肽-2没有。因此，CaM-激酶II自缔合是同种型特异性的，受活化条件调节，并且被可能通过其自身调节样序列结合至催化结构域的肽抑制。钙调蛋白激酶II自关联的模型，其中在一个全酶的催化结构域与相邻的全酶的监管结构域相互作用。这些intersubunit-interholoenzyme自抑制相互作用可能有助于易位和失活的钙调蛋白激酶II先前报道的模型缺血。
Calmodulin (CaM)-kinase II holoenzymes composed of either alpha or beta subunits were analyzed using light scattering to determine a mechanism for self-association. Under identical reaction conditions, only alpha CaM-kinase II holoenzymes self-associated. Self-association was detected at a remarkably low enzyme concentration (0.14 muM or 7 mug/mL). Light scattering revealed two phases of self-association: a rapid rise that peaked, followed by a slower decrease that stabilized after 2-3 min. Electron microscopy identified that the rapid rise in scattering was due to the formation of loosely packed clusters of holoenzymes that undergo further association into large complexes of several microns in diameter over time. Self-association required activation by Ca2+/CaM and was strongly dependent on pH. Self-association was not detected at pH 7.5, however, the extent of this process increased as reaction pH decreased below 7.0. A peptide substrate (autocamtide-2) and inhibitor (AIP) designed from the autoregulatory domain of CaM-kinase II potently prevented self-association, whereas the peptide substrate syntide-2 did not. Thus, CaM-kinase II self-association is isoform specific, regulated by the conditions of activation, and is inhibited by peptides that bind to the catalytic domain likely via their autoregulatory-like sequence. A model for CaM-kinase II self-association is presented whereby catalytic domains in one holoenzyme interact with the regulatory domains in neighboring holoenzymes. These intersubunit-interholoenzyme autoinhibitory interactions could contribute to both the translocation and inactivation of CaM-kinase II previously reported in models of ischemia.