Gorelick F S, Wang J K, Lai Y, Nairn A C, Greengard P
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
J Biol Chem. 1988 Nov 25;263(33):17209-12.
The autophosphorylation of purified Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) on a threonine-containing phosphopeptide common to both the alpha and beta subunits was previously shown to convert this enzyme into a catalytically active Ca2+-independent species. We now have examined the phosphorylation and activation of Ca2+/CaM kinase II in synaptosomes, a Ca2+-dependent neurosecretory system consisting of isolated nerve terminals. Synaptosomes were prelabeled with 32Pi and the alpha subunit of Ca2+/CaM kinase II was immunoprecipitated. Under basal incubation conditions the alpha subunit was phosphorylated. Depolarization of synaptosomes produced a rapid (2-5 s) Ca2+-dependent increase of about 50% in the state of phosphorylation of the alpha subunit. This was followed by a slower increase in the 32P content of the alpha subunit over the next 5 min of depolarization. The enhanced phosphorylation was characterized by an initial rise (2 s) and subsequent decrease (30 s) in the phosphothreonine content of the alpha subunit. In contrast, the phosphoserine content of the alpha subunit slowly increased during the course of depolarization. Thermolytic two-dimensional phosphopeptide maps of the alpha subunit demonstrated that depolarization stimulated the labeling of a phosphopeptide associated with autoactivation. In parallel experiments, unlabeled synaptosomes were depolarized, and lysates of these synaptosomes were assayed for Ca2+/CaM kinase II activity. Depolarization produced a rapid (less than or equal to 2 s) increase in Ca2+-independent Ca2+/CaM kinase II activity. This activity returned to basal levels by 60 s. Thus, depolarization of intact synaptosomes is associated with the transient phosphorylation of Ca2+/CaM kinase II on threonine residues, presumably involving an autophosphorylation mechanism and concomitantly the transient generation of the Ca2+-independent form of Ca2+/CaM kinase II.
先前的研究表明，纯化的钙调蛋白依赖性蛋白激酶II（Ca2+/CaM激酶II）在α和β亚基共有的含苏氨酸磷酸肽上进行自磷酸化后，会转变为一种催化活性的不依赖Ca2+的形式。我们现在研究了突触体（一种由分离的神经末梢组成的依赖Ca2+的神经分泌系统）中Ca2+/CaM激酶II的磷酸化和激活情况。突触体先用32Pi进行预标记，然后免疫沉淀Ca2+/CaM激酶II的α亚基。在基础孵育条件下，α亚基发生了磷酸化。突触体去极化导致α亚基磷酸化状态迅速（2 - 5秒）且依赖Ca2+地增加约50%。随后在接下来5分钟的去极化过程中，α亚基的32P含量缓慢增加。增强的磷酸化表现为α亚基磷酸苏氨酸含量先升高（2秒），随后下降（30秒）。相比之下，α亚基的磷酸丝氨酸含量在去极化过程中缓慢增加。α亚基的热解二维磷酸肽图谱表明，去极化刺激了与自激活相关的磷酸肽的标记。在平行实验中，未标记的突触体去极化，然后测定这些突触体裂解物中的Ca2+/CaM激酶II活性。去极化导致不依赖Ca2+的Ca2+/CaM激酶II活性迅速（小于或等于2秒）增加。该活性在60秒时恢复到基础水平。因此，完整突触体的去极化与Ca2+/CaM激酶II在苏氨酸残基上的瞬时磷酸化有关，推测涉及自磷酸化机制，并伴随着不依赖Ca2+形式的Ca2+/CaM激酶II的瞬时产生。
