Department of Biomedical Sciences, University of Padova, Padova, Italy.
Department of Molecular Medicine, University of Padova, Padova, Italy.
PLoS One. 2018 Mar 1;13(3):e0193479. doi: 10.1371/journal.pone.0193479. eCollection 2018.
CK2 is a pleiotropic S/T protein kinase (formerly known as casein kinase 2) which is attracting increasing interest as therapeutic target, and the identification of its substrates is a crucial step in determining its involvement in different pathological conditions. We recently found that S131 of Akt2 (homologous to the well established CK2 target S129 of Akt1) is not phosphorylated by CK2 either in vitro or in vivo, although the consensus sequence recognized by CK2 (S/T-x-x-E/D/pS/pT) is conserved in it. Here, by exploiting synthetic peptides, in cell transfection experiments, and computational analysis, we show that a single sequence element, a T at position n+1, hampers phosphorylation, causing an α-helix structure organization which prevents the recognition of its own consensus by CK2. Our results highlight the role of negative determinants as crucial modulators of CK2 targeting and corroborate the concept that Akt1 and Akt2 display isoform specific features. Experiments with synthetic peptides suggest that Akt2 S131 could be phosphorylated by kinases of the Plk (Polo-like kinase) family, which are insensitive to the presence of the n+1 T. The low phylogenetic conservation of the Akt2 sequence around S131, as opposed to the extremely well-conserved Akt1 homologous sequence, would indicate a dominant positive role in the selective pressure only for the Akt1 phosphoacceptor site committed to undergo phosphorylation by CK2. By contrast, Akt2 S131 may mediate the response to specific physio/pathological conditions, being consequently shielded against basal CK2 targeting.
CK2 是一种多功能 S/T 蛋白激酶（以前称为酪蛋白激酶 2），作为治疗靶点越来越受到关注，鉴定其底物是确定其在不同病理条件下参与的关键步骤。我们最近发现，Akt2 的 S131（与 Akt1 的成熟 CK2 靶标 S129 同源）既不在体内也不在体外被 CK2 磷酸化，尽管 CK2 识别的保守序列（S/T-x-x-E/D/pS/pT）在其中保守。在这里，我们通过利用合成肽、细胞转染实验和计算分析，表明一个单一的序列元素，即位置 n+1 的 T，会阻碍磷酸化，导致其自身的保守序列无法被 CK2 识别的α-螺旋结构组织。我们的结果强调了负决定因素作为 CK2 靶向的关键调节剂的作用，并证实了 Akt1 和 Akt2 显示同工型特异性特征的概念。合成肽实验表明，Akt2 的 S131 可以被 Plk（Polo 样激酶）家族的激酶磷酸化，而这些激酶对 n+1 T 的存在不敏感。与 Akt1 同源序列极其保守相比，Akt2 序列在 S131 周围的进化保守性较低，这表明在 CK2 磷酸化的选择性压力下，Akt1 的磷酸接受位点具有主导的积极作用。相比之下，Akt2 的 S131 可能介导对特定生理/病理条件的反应，因此免受基础 CK2 靶向的影响。
