Brown Bruce M, Carlson Brian L, Zhu Xuemei, Lolley Richard N, Craft Cheryl M
The Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, and Department of Cell and Neurobiology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90089-9112, USA.
Biochemistry. 2002 Nov 19;41(46):13526-38. doi: 10.1021/bi0204490.
In steps of protein purification of bovine retinal protein phosphatase 2A (PP2A), phosducin dephosphorylation activity peaks coelute with a PP2A enzyme complex, shown by peptide sequence analysis to contain a B' subunit, B56 epsilon. Other PP2A complexes with a slightly larger (56.5 kDa) B' subunit (sequenced to be B56 alpha) or with the B alpha regulatory subunit have no phosducin dephosphorylation activity. Upon exposure to light, a significant increase in the immunoreactive protein level of the A, C, and B56 epsilon PP2A subunits is observed in the cytosolic fraction of mouse retina, the phosducin dephosphorylation of which occurs rapidly. During dark exposure, these subunits translocate to the membrane fraction where rhodopsin is slowly dephosphorylated. This PP2A redistribution occurs in less than 1.5 min and is dependent upon light and not upon an intrinsic circadian rhythm. Forty times more of the A subunit (approximately 20 ng/mouse retina) and 9 times more of the C subunit (approximately 4 ng/mouse retina) than of the B56 epsilon subunit (approximately 0.45 ng/mouse retina) redistribute, which suggests that the predominant form of the PP2A enzyme complex on the membrane in the dark is a dimer, consisting of only A and C subunits. We observe that the dimer favors phosphorylated opsin as a substrate, while the trimer, particularly the enzyme complex with the B56 epsilon subunit, greatly prefers phosphorylated phosducin, with an activity several hundred times those of other substrates that were tested. This light-driven PP2A translocation provides a potential mechanism for efficient dephosphorylation of two critical photoreceptor transduction proteins, cytosolic phosducin and membrane-bound rhodopsin, by the same enzyme.
在牛视网膜蛋白磷酸酶2A（PP2A）的蛋白质纯化步骤中，视紫红质磷酸化脱磷酸化活性峰与一种PP2A酶复合物共洗脱，肽序列分析表明该复合物含有一个B'亚基，即B56ε。其他含有稍大（56.5 kDa）B'亚基（测序为B56α）或Bα调节亚基的PP2A复合物没有视紫红质磷酸化脱磷酸化活性。暴露于光下时，在小鼠视网膜的胞质部分观察到A、C和B56ε PP2A亚基的免疫反应性蛋白水平显著增加，其视紫红质磷酸化脱磷酸化迅速发生。在黑暗暴露期间，这些亚基转移到膜部分，视紫红质在那里缓慢脱磷酸化。这种PP2A重新分布在不到1.5分钟内发生，并且依赖于光而不是内在的昼夜节律。重新分布的A亚基（约20 ng/小鼠视网膜）和C亚基（约4 ng/小鼠视网膜）分别是B56ε亚基（约0.45 ng/小鼠视网膜）的40倍和9倍，这表明黑暗中膜上PP2A酶复合物的主要形式是二聚体，仅由A和C亚基组成。我们观察到二聚体有利于将磷酸化视蛋白作为底物，而三聚体，特别是含有B56ε亚基的酶复合物，非常偏好磷酸化视紫红质，其活性是测试的其他底物的数百倍。这种光驱动的PP2A易位为同一酶高效去磷酸化两种关键光感受器转导蛋白——胞质视紫红质和膜结合视蛋白提供了一种潜在机制。
