Cysteine-rich regions of protein kinase C delta are functionally non-equivalent - Differences between cysteine-rich regions of non-calcium-dependent protein kinase C delta and calcium-dependent protein kinase C gamma
Cysteine-rich regions of protein kinase C delta are functionally non-equivalent - Differences between cysteine-rich regions of non-calcium-dependent protein kinase C delta and calcium-dependent protein kinase C gamma
复制标题
DOI：
10.1016/s0014-5793(96)01395-6
复制
发表时间：
1997-01-03
期刊：
影响因子：
3.5
通讯作者：
Quest, AFG
中科院分区：
文献类型：
--
作者：
Hunn, M;Quest, AFG
非钙依赖性蛋白激酶C δ (nPKC δ)的调控结构域元件，包括半胱氨酸富集区域Cys1（delta）和Cys2（delta）中的一个或两个，以谷胱甘肽- s -转移酶的融合蛋白表达，并使用脂质体或混合胶束磷酸酯结合试验进行表征。而仅含有Cys1（δ）的蛋白则没有明显的结合。同样，在混合胶束实验中，与Cys2（delta）融合的蛋白与PDBu的结合具有高亲和力（K-d: 14-37 nM）和显著的化学计量水平（0.23-0.66 mol/mol），而仅与Cys1（delta）融合的蛋白未检测到结合。PDBu结合Cys2（δ）的PS依赖性与Hill数高度配合，Hill数在2.5 ~ 5.2之间。这些结果表明，在nPKC δ的富含半胱氨酸的区域和钙依赖性亚型cPKC γ之间存在显著的功能差异，其中两个富含半胱氨酸的区域都代表功能性PDBu结合元件。
Regulatory domain elements of the non-calcium-dependent protein kinase C delta (nPKC delta), including either or both of the cysteine-rich regions Cys1(delta) and Cys2(delta), were expressed as fusion proteins with glutathione-S-transferase and characterized using Liposomal or mixed micellar phorbol ester binding assays, Fusion proteins containing Cys2(delta) bound phorbol-12,13-dibutyrate (PDBu) efficiently in the assay employing phosphatidylserine (PS) vesicles, while no significant binding was seen for proteins containing only Cys1(delta). Likewise, in mixed micellar assays, fusion proteins with Cys2(delta) bound PDBu with high affinity (K-d: 14-37 nM) and to significant stoichiometric levels (0.23-0.66 mol/mol), but no binding could be detected for proteins with Cys1(delta) only. The PS dependence of PDBu binding to Cys2(delta) was highly cooperative with Hill numbers lying in the range of 2.5-5.2. These results demonstrate the presence of striking functional differences between the cysteine-rich regions of nPKC delta and the calcium-dependent isoform, cPKC gamma, where both cysteine-rich regions represent functional PDBu binding elements.