Characterization of PBIP1, a novel polo-box-binding protein at the kinetochores

丝氨酸/苏氨酸蛋白激酶的Polo亚家族成员在多种真核生物的有丝分裂过程中起着关键作用。我们先前已经证明，在进化上保守的Polo-box结构域(PBD)对于将Polo激酶的催化活性靶向特定的亚细胞结构是必不可少的。为了鉴定Polo-Box结合蛋白，我们以哺乳动物Polo-like kinasePlk1的PBD为诱饵，进行酵母双杂交筛选，分离到一种新的动粒结合蛋白PBIP1(Polo-Box-Interaction Protein 1)。在生理条件下，PBIP1在体内与内源性Plk1相互作用，似乎是一种动粒特异性有丝分裂抑制物，在有丝分裂过程中被Plk1磷酸化并降解。缺乏Plk1依赖的磷酸化位点的不可降解的PBIP1的表达诱导有丝分裂停止，表明PBIP1的降解是正常有丝分裂进行所必需的。PBIP1与髓系白血病因子1(MLF1)结合，其与核磷蛋白(NPM)融合的去调节作用促进了急性髓系白血病(AML)从骨髓发育不良(MDS)的发展。因此，MLF1下调PBIP1的表达可能是AML发生发展的重要一步。根据这些观察，我们在白血病细胞系的PLK1 ORF中发现了多个突变。这些发现提出了Plk1基因失控导致PBIP1功能异常和白血病发生的可能性，并为Polo激酶在白血病和其他肿瘤再发生中的作用提供了新的观点。

Members of the polo subfamily of Ser/Thr protein kinases have been shown to play critical roles for mitotic progression in various eukaryotic organisms. We have previously demonstrated that the evolutionarily conserved polo-box domain (PBD) of polo kinases is essential in targeting the catalytic activity of the polo kinases to specific subcellular structures. In an effort to identify the polo-box-binding proteins, we carried out yeast two-hybrid screening using the PBD of the mammalian polo-like kinase Plk1 as a bait and isolated a novel kinetochore-associating protein that we termed PBIP1 (for Polo-Box-Interacting Protein 1). PBIP1 interacts with endogenous Plk1 in vivo under physiological conditions and appears to be a kinetochore-specific mitotic inhibitor that is phosphorylated and degraded by Plk1 in mitosis. Expression of the non-degradable PBIP1 lacking the Plk1-dependent phosphorylation sites induces a mitotic arrest, indicating that degradation of PBIP1 is required for proper mitotic progression. PBIP1 binds to myeloid leukemia factor 1 (MLF1), whose deregulation by fusing with nuclear phosmin (NPM) promotes the development of acute myeloid leukemia (AML) from myelodysplasia (MDS). Thus, down-regulation of PBIP1 by MLF1 could be an important step for the development of AML. In line with these observations, we identified multiple mutations in the PLK1 ORF of leukemia cell lines. These findings raise the possibility that deregulation of Plk1 leads to improper PBIP1 function and the genesis of leukemia, and provide new views on the role of polo kinase in the development of leukemia and other tumoregenesis.