School of Biological Sciences, University of California, Irvine, California 92717-4550.
Mol Cell Neurosci. 1993 Dec;4(6):562-70. doi: 10.1006/mcne.1993.1069.
Following incubation of rat brain membranes with the catalytic subunit of cAMP-dependent protein kinase and [(32)P]ATP, a previously unreported phosphoprotein, pp59, was found to be enriched in cerebellar synaptic plasma membrane preparations, but not in those prepared from cerebral cortex, hippocampus, olfactory bulb, or striatum. This protein, which has an M(r) of 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is not phosphorylated by the cGMP-dependent protein kinase. While pp59 was consistently detected in cerebellar membranes from adult Sprague-Dawley rats, it was not detected in bovine or rabbit cerebellar membranes. Moreover, pp59 did not comigrate with any of the autophosphorylated subunits of the Ca(2+)/calmodulindependent protein kinase in rat cerebellar membranes. Extraction of pp59 from these membranes could be accomplished with 6 M urea, but not with 0.4 M NaCl or 0.5% (v/v) Triton X-100. The urea solubility suggests that pp59 is not an integral membrane protein. Acid hydrolysis of the protein phosphorylated in vitro yielded phosphoserine but no significant amount of phosphothreonine or phosphotyrosine. Further analysis of pp59 may provide new insights into the role of cAMP in modulation of synaptic function in the cerebellum.
用 cAMP 依赖性蛋白激酶的催化亚基和 [(32)P]ATP 孵育大鼠脑组织膜后，发现一种以前未报道的磷酸蛋白 pp59 在小脑突触质膜制剂中丰富，但不在大脑皮层、海马体、嗅球或纹状体中。这种蛋白的 M(r) 为 59000，用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，它不是 cGMP 依赖性蛋白激酶磷酸化的。虽然 pp59 在成年 Sprague-Dawley 大鼠的小脑膜中始终被检测到，但在牛或兔的小脑膜中未被检测到。此外，pp59 没有与大鼠小脑膜中的任何钙/钙调蛋白依赖性蛋白激酶的自身磷酸化亚基共迁移。可以用 6 M 尿素从这些膜中提取 pp59，但不能用 0.4 M NaCl 或 0.5%（v/v）Triton X-100 提取。尿素的溶解性表明 pp59 不是膜内在蛋白。体外磷酸化的蛋白质经酸水解后产生磷酸丝氨酸，但没有显著量的磷酸苏氨酸或磷酸酪氨酸。进一步分析 pp59 可能为 cAMP 在小脑突触功能调节中的作用提供新的见解。
