Pharmacological studies of calmodulin-dependent tau factor phosphorylation in cultured cerebellar granule cells.

Ca ~（2+）/钙调素依赖性蛋白激酶II（CaM kinase II）是一种多功能蛋白激酶，在神经组织中高度富集，参与调节海马CA 1区神经递质的生物合成和释放以及长时程增强（LTP）的表达。最近，体外研究表明，CaM激酶II中Thr-286/287（α/β亚基）的自磷酸化可将其从依赖Ca^2+的形式转变为部分不依赖Ca^2+的形式。我们报道了在谷氨酸诱导的NMDA受体激活后，小脑颗粒细胞中形成非Ca^<2+>依赖性CaM激酶II的证据。在颗粒细胞中，CaM激酶II中Thr-287的自身磷酸化与非Ca^2+依赖性激酶活性的产生之间存在良好的时间相关性。然而，由于颗粒细胞短暂暴露于谷氨酸（30秒）导致了相对短暂（<5分钟）的非Ca^<2+>依赖性活性增加， 关于我们 f CaM激酶II。因此，我们将注意力集中在海马组织上，因为NMDA受体参与LTP已经在组织中清楚地显示。在培养的海马神经元中，谷氨酸确实像在小脑颗粒细胞中一样提高了钙调素激酶II的非钙依赖性活性。对Ca^<2+>-非依赖性的最大影响大于颗粒细胞中的影响，并且在持续存在谷氨酸的情况下，该活性在至少30分钟内更稳定。在实验中与_<32>P-标记的细胞，磷酸化的MAP 2以及自磷酸化的CaM激酶II被发现刺激暴露于谷氨酸。这些结果与以下假设一致：通过NMDA受体门控离子通道的突触后Ca^2+内流的增加将海马神经元中的CaM激酶II转化为组成型活性的、不依赖于Ca^2+的形式，并导致该酶的靶蛋白（如MAP 2）磷酸化的增加。少

Ca^<2+>/calmodulin-dependent protein kinase II(CaM kinase II), which is highly enriched in neural tissues, is a multifunctional protein kinase that is involved in regulation of biosynthesis and release of neurotransmitter, and expression of long-term potentiation(LTP)in CAl region of the hippocampus. Recently, it has been shown in vitro that autophosphorylation of Thr-286/287(alpha/beta subunits)in the CaM kinase II converts it from the Ca^<2+>-dependent form to a partially Ca^<2+>-independent species. We reported evidence for the formation of Ca^<2+>-independent CaM kinase II in cerebellar granule cells following the activation of the glutamate-induced NMDA receptor. In the granule cells, there was a good temporal correlation between autophosphorylation of Thr-287 in CaM kinase II and generation of Ca^<2+>-independent kinase activity. However, as brief exposure(30 sec)of granule cells to glutamate resulted in a relatively transient(<5 min)increase in the Ca^<2+>-independent activity o … More f CaM kinase II. Therefore, we have focused attention on the hippocampal tissue, since involvement of NMDA receptor in the LTP has been clearly shown in the tissue. In cultured hippocampal neurons, glutamate did elevated the Ca^<2+>-independent activity of CaM kinase II as in cerebellar granule cells. The maximal effect on the Ca^<2+>-independence was greater than that in the granule cells and the activity was more stable for at least 30 min in the continued presence of glutamate. In the experiments with _<32>P-labeled cells, the phosphorylation of MAP2 as well as autophosphorylation of CaM kinase II were found to be stimulated by the exposure to glutamate. These results were consistent with the hypothesis that the increase in the postsynaptic Ca^<2+> influx through the NMDA receptor-gated ion channel converts CaM kinase II to a constitutively active, Ca^<2+>-independent form in the hippocampal neurons, and results in an increase in the phosphorylation of target proteins such as MAP2 for the enzyme. Less