Schechtman Deborah, Craske Madeleine L, Kheifets Viktoria, Meyer Tobias, Schechtman Jack, Mochly-Rosen Daria
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305, USA.
J Biol Chem. 2004 Apr 16;279(16):15831-40. doi: 10.1074/jbc.M310696200. Epub 2004 Jan 22.
Disruption of intramolecular interactions, translocation from one intracellular compartment to another, and binding to isozyme-specific anchoring proteins termed RACKs, accompany protein kinase C (PKC) activation. We hypothesized that in inactive epsilonPKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed psiepsilonRACK. An amino acid difference between the psiepsilonRACK sequence in epsilonPKC and its homologous sequence in epsilonRACK constitutes a change from a polar non-charged amino acid (asparagine) in epsilonRACK to a polar charged amino acid (aspartate) in epsilonPKC. Here we show that mutating the aspartate to asparagine in epsilonPKC increased intramolecular interaction as indicated by increased resistance to proteolysis, and slower hormone- or PMA-induced translocation in cells. Substituting aspartate for a non-polar amino acid (alanine) resulted in binding to epsilonRACK without activators, in vitro, and increased translocation rate upon activation in cells. Mathematical modeling suggests that translocation is at least a two-step process. Together our data suggest that intramolecular interaction between the psiepsilonRACK site and RACK-binding site within epsilonPKC is critical and rate limiting in the process of PKC translocation.
蛋白激酶C（PKC）激活伴随着分子内相互作用的破坏、从一个细胞内区室向另一个区室的转位以及与称为RACKs的同工酶特异性锚定蛋白的结合。我们推测，在无活性的εPKC中，RACK结合位点与一个类似于其RACK的序列（称为ψεRACK）发生分子内相互作用。εPKC中ψεRACK序列与其在εRACK中的同源序列之间的氨基酸差异构成了从εRACK中极性不带电氨基酸（天冬酰胺）到εPKC中极性带电氨基酸（天冬氨酸）的变化。在此我们表明，在εPKC中将天冬氨酸突变为天冬酰胺会增加分子内相互作用，这表现为对蛋白水解的抗性增加以及细胞中激素或佛波酯诱导的转位变慢。用天冬氨酸替代非极性氨基酸（丙氨酸）会导致在无激活剂的情况下体外与εRACK结合，并在细胞激活后增加转位速率。数学建模表明，转位至少是一个两步过程。我们的数据共同表明，εPKC内ψεRACK位点与RACK结合位点之间的分子内相互作用在PKC转位过程中至关重要且是限速的。
