Kakkar R, Raju R V, Sharma R K
College of Medicine, University of Saskatchewan, Saskatoon, S7N 4H4, Canada.
Arch Biochem Biophys. 1998 Oct 15;358(2):320-8. doi: 10.1006/abbi.1998.0858.
In the present study we have shown that bovine brain 60-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme (CaMPDE - PDE1A2) is proteolyzed by a Ca2+-dependent cysteine protease, m-calpain. The proteolysis of PDE1A2 by m-calpain results in its conversion to a totally calmodulin (CaM)-independent form accompanied by degradation of PDE1A2 into a 45-kDa catalytic fragment and a 15-kDa fragment. The activity of PDE1A2 is unaffected by the presence or absence of CaM during cleavage, suggesting that the interaction between CaM and PDE1A2 does not alter substrate recognition by calpain. Furthermore, we provide evidence, based on the studies of CaM overlay and phosphorylation, that the cleavage site is not present either in the CaM-binding domain or phosphorylation site. N-terminal sequence analysis of the 45-kDa fragment indicated that cleavage occurs between residues 126Gln and 127Ala, and eliminates the CaM-dependent activity of carboxy termini PDE1A2. The present findings suggest that limited proteolysis in the brain through calpains could be an alternate mechanism for activating CaMPDE(s) and for regulating intracellular levels of cAMP.
在本研究中，我们已表明牛脑60 kDa钙调蛋白依赖性环核苷酸磷酸二酯酶同工酶（CaMPDE - PDE1A2）被一种Ca2+依赖性半胱氨酸蛋白酶m-钙蛋白酶水解。m-钙蛋白酶对PDE1A2的蛋白水解导致其转变为完全不依赖钙调蛋白（CaM）的形式，同时PDE1A2降解为一个45 kDa的催化片段和一个15 kDa的片段。在裂解过程中，PDE1A2的活性不受CaM存在与否的影响，这表明CaM与PDE1A2之间的相互作用不会改变钙蛋白酶对底物的识别。此外，基于CaM覆盖和磷酸化研究，我们提供的证据表明裂解位点既不存在于CaM结合域也不存在于磷酸化位点。对45 kDa片段的N端序列分析表明，裂解发生在第126位谷氨酰胺和第127位丙氨酸残基之间，并消除了羧基末端PDE1A2的CaM依赖性活性。目前的研究结果表明，通过钙蛋白酶在脑中进行的有限蛋白水解可能是激活CaMPDE和调节细胞内cAMP水平的另一种机制。
