Lin W J, Chang Y F, Wang W L, Huang C Y
Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, 112, Taiwan, Republic of China.
Biochem J. 2001 Mar 15;354(Pt 3):635-43. doi: 10.1042/0264-6021:3540635.
TIS21 is induced transiently by PMA and a number of extracellular stimuli. Yeast two-hybrid screening has identified three TIS21 interacting clones from a rat cDNA library [Lin, Gary, Yang, Clarke and Herschman (1996) J. Biol. Chem 271, 15034-15044]. The amino acid sequence deduced from clone 5A shows 96.9% identity with the murine PICK1, a protein kinase Calpha (PKCalpha)-binding protein postulated to act as an intracellular receptor for PKC. A fusion protein of glutathione S-transferase and rPICK1 associates with the TIS21 translated in vitro, suggesting a direct physical interaction between these two proteins. TIS21 and rPICK1 are co-immunoprecipitated from NIH 3T3 cells overexpressing these two proteins. This indicates that the interaction also occurs in mammalian cells. Deletion of the PDZ domain at the N-terminus of rPICK1 abolishes its interaction with TIS21. A putative carboxylate-binding loop required for PICK1 to bind PKCalpha [Staudinger, Lu and Olson (1997) J. Biol. Chem 272, 32019-32024] is within this deleted region. Our results suggest a potential competition between TIS21 and PKC for binding to PICK1. We show that recombinant TIS21 is phosphorylated by PKC in vitro. The catalytic activity of PKC towards TIS21 is significantly decreased in the presence of rPICK1, whereas phosphorylation of histone by PKC is not affected. rPICK1 seems to modulate the phosphorylation of TIS21 through specific interactions between these two proteins. TIS21 might have a role in PKC-mediated extracellular signal transduction through its interaction with rPICK1.
TIS21 可被佛波酯（PMA）和多种细胞外刺激瞬时诱导。酵母双杂交筛选从大鼠 cDNA 文库中鉴定出三个与 TIS21 相互作用的克隆[林、加里、杨、克拉克和赫希曼（1996 年）《生物化学杂志》271 卷，15034 - 15044 页]。从克隆 5A 推导的氨基酸序列与小鼠 PICK1 有 96.9%的同一性，PICK1 是一种蛋白激酶 Cα（PKCα）结合蛋白，被推测作为 PKC 的细胞内受体发挥作用。谷胱甘肽 S - 转移酶与 rPICK1 的融合蛋白与体外翻译的 TIS21 相关联，表明这两种蛋白之间存在直接的物理相互作用。TIS21 和 rPICK1 可从过表达这两种蛋白的 NIH 3T3 细胞中共免疫沉淀。这表明这种相互作用也发生在哺乳动物细胞中。rPICK1 N 端 PDZ 结构域的缺失消除了它与 TIS21 的相互作用。PICK1 结合 PKCα 所需的一个假定的羧酸盐结合环[施陶丁格、卢和奥尔森（1997 年）《生物化学杂志》272 卷，32019 - 32024 页]就在这个缺失区域内。我们的结果表明 TIS21 和 PKC 在与 PICK1 结合方面可能存在潜在竞争。我们发现重组 TIS21 在体外可被 PKC 磷酸化。在存在 rPICK1 的情况下，PKC 对 TIS21 的催化活性显著降低，而 PKC 对组蛋白的磷酸化不受影响。rPICK1 似乎通过这两种蛋白之间的特异性相互作用调节 TIS21 的磷酸化。TIS21 可能通过与 rPICK1 的相互作用在 PKC 介导的细胞外信号转导中发挥作用。
