Caudell E G, Caudell J J, Tang C H, Yu T K, Frederick M J, Grimm E A
Departments of Molecular and Cellular Oncology, Laboratory Medicine, and Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Biochemistry. 2000 Oct 24;39(42):13034-43. doi: 10.1021/bi001250v.
The copines, first described by Creutz et al. [(1998) J. Biol. Chem. 273, 1393-1402], comprise a two C2 domain-containing protein family and are known to aggregate phosphatidylserine membranes in a calcium-dependent manner. No enzymatic function has been attributed to copines yet. Due to a cross-reacting activity of Mikbeta1, an antibody to the IL-2Rbeta chain, we were able to serendipitously purify, partially microsequence, and clone human copine III. The 5 kb copine III transcript is expressed ubiquitously as determined by a multitissue Northern blot analysis. Phosphoamino acid analysis revealed phosphorylation of copine III on serine and threonine residues. In vitro kinase assays were performed with immunoprecipitated endogenous copine III, chromatography-purified endogenous copine III, and recombinant copine III expressed in Saccharomyces cerevisiae. The exogenous substrate myelin basic protein was phosphorylated in all in vitro kinase assays containing copine III immunoprecipitate or purified copine III. A 60-kDa band was observed in corresponding in gel kinase assays with staurosporine-activated cells. Cell lines expressing high levels of copine III protein had correspondingly high kinase activity in copine III antiserum immunoprecipitate. However, the copine amino acid sequences lack the traditional kinase catalytic domain. Therefore, the data suggest copine III may possess an intrinsic kinase activity and represent a novel unconventional kinase family.
柯平蛋白最初由克鲁茨等人描述[（1998年）《生物化学杂志》273卷，1393 - 1402页]，它包含一个含有两个C2结构域的蛋白家族，已知能以钙依赖的方式聚集磷脂酰丝氨酸膜。目前尚未发现柯平蛋白具有酶促功能。由于抗白细胞介素 - 2β链抗体Mikbeta1的交叉反应活性，我们意外地纯化了人柯平蛋白III，对其进行了部分微测序并克隆。通过多组织Northern印迹分析确定，5kb的柯平蛋白III转录本在全身广泛表达。磷酸氨基酸分析显示柯平蛋白III的丝氨酸和苏氨酸残基发生了磷酸化。我们用免疫沉淀的内源性柯平蛋白III、色谱纯化的内源性柯平蛋白III以及在酿酒酵母中表达的重组柯平蛋白III进行了体外激酶测定。在所有含有柯平蛋白III免疫沉淀或纯化的柯平蛋白III的体外激酶测定中，外源性底物髓鞘碱性蛋白都被磷酸化。在用星形孢菌素激活的细胞进行的相应凝胶内激酶测定中观察到一条60kDa的条带。表达高水平柯平蛋白III的细胞系在柯平蛋白III抗血清免疫沉淀中相应地具有高激酶活性。然而，柯平蛋白的氨基酸序列缺乏传统的激酶催化结构域。因此，数据表明柯平蛋白III可能具有内在激酶活性，代表一个新的非传统激酶家族。
