Sokolova M, Prüfer D, Tacke E, Rohde W
Max-Planck-Institut für Züchtungsforschung, Cologne, Germany.
FEBS Lett. 1997 Jan 3;400(2):201-5. doi: 10.1016/s0014-5793(96)01380-4.
The 17 kDa protein (pr17), the phloem-limited movement protein (MP) of potato leafroll luteovirus (PLRV), is associated with membranous structures and localized to plasmodesmata [Tacke et al. (1993) Virology 197, 274-282; Schmitz, J. (1995) Ph.D. Thesis, University of Cologne]. In planta the protein is predominantly present in its phosphorylated form, but it is rapidly dephosphorylated during isolation under native conditions. In an effort to examine the nature of the protein kinase(s) involved in the phosphorylation reaction, pr17 deletion mutants were expressed as fusion proteins in a bacterial expression vector system and tested for their ability to be phosphorylated by potato membrane preparations as well as by commercially available kinases. A fusion protein containing the nucleic acid-binding, basic, C-proximal domain (pr17C1) was identified to be phosphorylated by a Ca2+- and phospholipid-dependent, membrane-associated protein kinase. This protein kinase activity was inhibited by the addition of (19-36) protein kinase C (PKC) inhibitory peptide, known to be a highly specific inhibitor of mammalian PKC. Moreover, also the mammalian PKC from rat was able to phosphorylate pr17 in vitro. The results suggest that phosphorylation of pr17 takes place at membranous structures, possibly at the deltoid plasmodesmata connecting the sieve cell-companion cell complex of the phloem, by the activity of PKC-related, membrane-associated protein kinase activity.
17 kDa蛋白（pr17）是马铃薯卷叶黄化病毒（PLRV）的韧皮部限制运动蛋白（MP），与膜结构相关并定位于胞间连丝[Tacke等人（1993年），《病毒学》197卷，274 - 282页；Schmitz，J.（1995年），博士论文，科隆大学]。在植物体内，该蛋白主要以磷酸化形式存在，但在天然条件下分离过程中会迅速去磷酸化。为了研究参与磷酸化反应的蛋白激酶的性质，pr17缺失突变体在细菌表达载体系统中作为融合蛋白表达，并测试它们被马铃薯膜制剂以及市售激酶磷酸化的能力。一种包含核酸结合、碱性、C端近端结构域的融合蛋白（pr17C1）被鉴定可被一种Ca2 +和磷脂依赖性的膜相关蛋白激酶磷酸化。这种蛋白激酶活性可被添加的（19 - 36）蛋白激酶C（PKC）抑制肽抑制，已知该抑制肽是哺乳动物PKC的高度特异性抑制剂。此外，来自大鼠的哺乳动物PKC在体外也能够使pr17磷酸化。结果表明，pr17的磷酸化发生在膜结构上，可能是在连接韧皮部筛管细胞 - 伴胞复合体的三角形胞间连丝处，通过PKC相关的膜相关蛋白激酶活性实现。
