Wei J, Wayman G, Storm D R
Department of Pharmacology, University of Washington, Seattle, Washington 98195-7820, USA.
J Biol Chem. 1996 Sep 27;271(39):24231-5. doi: 10.1074/jbc.271.39.24231.
Inhibition of type III adenylyl cyclase (III-AC) by intracellular Ca2+ in vivo provides a mechanism for attenuation of hormone-stimulated cAMP signals in olfactory epithelium, heart, and other tissues (Wayman, G. A., Impey, S., and Storm, D. R. (1995) J. Biol. Chem. 270, 21480-21486). Although the mechanism for Ca2+ inhibition of III-AC in vivo has not been defined, inhibition is not mediated by Gi, cAMP-dependent protein kinase, or protein kinase C. However, Ca2+ inhibition of III-AC is antagonized by KN-62, a CaM-dependent kinase inhibitor. In addition, constitutively activated CaM kinase II inhibits the enzyme. These data suggest that CaM kinase II regulates the activity of III-AC by direct phosphorylation or by an indirect mechanism involving phosphorylation of a protein that inhibits III-AC. Here we report that III-AC is phosphorylated in vivo when intracellular Ca2+ is increased and that phosphorylation is prevented by CaM-dependent kinase inhibitors. Site-directed mutagenesis of a CaM kinase II consensus site (Ser-1076 to Ala-1076) in III-AC greatly reduced Ca2+-stimulated phosphorylation and inhibition of III-AC in vivo. These data support the hypothesis that Ca2+ inhibition of III-AC is due to direct phosphorylation of the enzyme by CaM kinase II in vivo.
体内细胞内钙离子对III型腺苷酸环化酶（III-AC）的抑制作用为嗅觉上皮、心脏及其他组织中激素刺激的cAMP信号减弱提供了一种机制（Wayman, G. A., Impey, S., and Storm, D. R. (1995) J. Biol. Chem. 270, 21480 - 21486）。尽管体内钙离子对III-AC的抑制机制尚未明确，但这种抑制作用不是由Gi、cAMP依赖性蛋白激酶或蛋白激酶C介导的。然而，钙离子对III-AC的抑制作用可被CaM依赖性激酶抑制剂KN-62拮抗。此外，组成型激活的CaM激酶II可抑制该酶。这些数据表明，CaM激酶II通过直接磷酸化或通过涉及对抑制III-AC的蛋白质进行磷酸化的间接机制来调节III-AC的活性。在此我们报告，当细胞内钙离子增加时，III-AC在体内会发生磷酸化，且这种磷酸化可被CaM依赖性激酶抑制剂阻止。对III-AC中CaM激酶II共有位点（Ser-1076突变为Ala-1076）进行定点诱变，大大降低了体内钙离子刺激的磷酸化作用及对III-AC的抑制作用。这些数据支持了以下假说：体内钙离子对III-AC的抑制作用是由于CaM激酶II对该酶的直接磷酸化所致。
