Yano S, Fukunaga K, Ushio Y, Miyamoto E
Department of Pharmacology, Kumamoto University School of Medicine, Japan.
J Biol Chem. 1994 Feb 18;269(7):5428-39.
We investigated the activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) via stimulation of glutamate receptors and subsequent phosphorylation of vimentin and glial fibrillary acidic protein (GFAP) in cultured rat cortical astrocytes. The indirect immunofluorescence analysis with the anti-CaM kinase II antibody revealed that the enzyme was detected diffusely in the cytoplasm and more intensely in the nucleus. Glutamate elevated the Ca(2+)-independent activity of CaM kinase II through autophosphorylation, and this response was blocked by both DL-2-amino-3-phosphonopropionate and 6-cyano-7-nitroquinoxaline-2,3-dione, but not by D-2-amino-5-phosphonovalerate. In the experiments using 32P-labeled astrocytes, the phosphorylation of vimentin and GFAP as well as autophosphorylation of CaM kinase II were found to be stimulated after the exposure to glutamate. It was concluded by two-dimensional phosphopeptide analysis that the increased phosphorylation of vimentin and GFAP observed in intact cells were due to the activation of CaM kinase II by glutamate. These results suggest that glutamate can activate CaM kinase II through stimulation of both the metabotropic and non-N-methyl-D-aspartate receptors, and that the concomitant phosphorylation of vimentin and GFAP may in turn regulate the functions of intermediate filament proteins in intact astrocytes.
我们研究了在培养的大鼠皮质星形胶质细胞中，通过刺激谷氨酸受体激活钙/钙调蛋白依赖性蛋白激酶II（CaM激酶II）以及随后波形蛋白和胶质纤维酸性蛋白（GFAP）的磷酸化情况。用抗CaM激酶II抗体进行的间接免疫荧光分析显示，该酶在细胞质中呈弥散性分布，在细胞核中分布更为密集。谷氨酸通过自身磷酸化提高了CaM激酶II的钙非依赖性活性，并且这种反应被DL-2-氨基-3-膦酰丙酸和6-氰基-7-硝基喹喔啉-2,3-二酮阻断，但不被D-2-氨基-5-膦酰戊酸阻断。在使用32P标记星形胶质细胞的实验中，发现暴露于谷氨酸后波形蛋白和GFAP的磷酸化以及CaM激酶II的自身磷酸化均受到刺激。二维磷酸肽分析得出结论，在完整细胞中观察到的波形蛋白和GFAP磷酸化增加是由于谷氨酸激活了CaM激酶II。这些结果表明，谷氨酸可通过刺激代谢型和非N-甲基-D-天冬氨酸受体激活CaM激酶II，并且波形蛋白和GFAP的伴随磷酸化可能反过来调节完整星形胶质细胞中中间丝蛋白的功能。
