Schook W J, Puszkin S
Proc Natl Acad Sci U S A. 1985 Dec;82(23):8039-43. doi: 10.1073/pnas.82.23.8039.
A protein kinase activity was observed in coated vesicles, prepared from bovine brain, that had clathrin-associated protein 2 (CAP2, also known as clathrin light chain 2) as its principal substrate. Coated vesicles were purified by sucrose density gradient centrifugation followed by Sephacryl S-1000 column chromatography, and all buffers utilized in these procedures contained a mixture of proteolysis inhibitors to maintain CAP2 kinase activity. Incubation of vesicles with [gamma-32P]ATP in the presence of 7 microM polylysine resulted in an overall increase in the incorporation of phosphate. NaDodSO4/PAGE revealed that the principal recipient of this additional phosphate was CAP2 (Mr 33,000), the faster-migrating component of the clathrin coat-associated proteins, whereas CAP1 (Mr 36,000) was not phosphorylated. A number of other proteins, in the Mr 140,000 and 100,000 regions, were phosphorylated to a lesser extent. Polyarginine and polyethylenimine also supported CAP2 phosphorylation, but arginine and lysine were ineffective. The phosphorylated protein was identified as CAP2 because addition of exogenous CAPs resulted in increased incorporation of label into Mr 33,000 polypeptides and because heat treatment of labeled vesicles followed by ultracentrifugation resulted in recovery of labeled Mr 33,000 protein in the supernatant. Phosphorylation of CAP2 may play a regulatory role in clathrin coat/coated vesicle functions.
在从牛脑中制备的被膜小泡中观察到一种蛋白激酶活性，其主要底物是网格蛋白相关蛋白2（CAP2，也称为网格蛋白轻链2）。通过蔗糖密度梯度离心，随后进行Sephacryl S - 1000柱色谱法纯化被膜小泡，并且在这些操作中使用的所有缓冲液都含有蛋白酶抑制剂混合物以维持CAP2激酶活性。在7 microM聚赖氨酸存在下，将小泡与[γ-32P]ATP一起孵育导致磷酸盐掺入总体增加。NaDodSO4/PAGE显示，这种额外磷酸盐的主要接受者是CAP2（分子量33,000），它是网格蛋白包被相关蛋白中迁移较快的组分，而CAP1（分子量36,000）未被磷酸化。在分子量140,000和100,000区域的许多其他蛋白质被磷酸化的程度较低。聚精氨酸和聚乙烯亚胺也支持CAP2磷酸化，但精氨酸和赖氨酸无效。磷酸化的蛋白质被鉴定为CAP2，因为添加外源CAPs导致标记物掺入分子量33,000多肽的量增加，并且因为对标记的小泡进行热处理，然后超速离心，导致上清液中回收标记的分子量33,000蛋白质。CAP2的磷酸化可能在网格蛋白包被/被膜小泡功能中起调节作用。
