Department of Environmental Medicine and Pharmacology, New York University School of Medicine, Tuxedo, New York 10987, USA.
J Biol Chem. 2010 Dec 17;285(51):39935-42. doi: 10.1074/jbc.M110.166462. Epub 2010 Oct 12.
By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3 null MEFs contain a reduced level of phosphatase and tensin homolog (PTEN) and increased Akt1 activation coupled with decreased GSK3β activation under normoxia and hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, efficiently phosphorylates PTEN in vitro. Mass spectrometry identifies threonine 366 and serine 370 as two putative residues that are phosphorylated by Plk3. Immunoblotting using a phosphospecific antibody confirms these sites as Plk3 phosphorylation sites. Moreover, treatment of MEFs with LiCl, an inhibitor of GSK3β and CK2, only partially suppresses the phosphorylation, suggesting Plk3 as an additional kinase that phosphorylates these sites in vivo. Plk3-targeting mutants of PTEN are expressed at a reduced level in comparison with the wild-type counterpart, which is associated with an enhanced activity of PDK1, an upstream activator of Akt1. Furthermore, the reduced level of PTEN in PLK3 null MEFs is stabilized by treatment with MG132, a proteosome inhibitor. Combined, our study identifies Plk3 as a new player in the regulation of the PI3K/PDK1/Akt signaling axis by phosphorylation and stabilization of PTEN.
通过研究原代同基因鼠胚胎成纤维细胞（MEFs），我们发现 PLK3 缺失的 MEFs 在常氧和低氧条件下，PTEN 水平降低，磷酸化 Akt1 增加，同时 GSK3β 激活减少。纯化的重组 Plk3，但不是激酶缺陷突变体，能够有效地在体外磷酸化 PTEN。质谱分析鉴定出丝氨酸 366 和苏氨酸 370 是两个可能被 Plk3 磷酸化的残基。使用磷酸化特异性抗体进行免疫印迹证实了这些位点是 Plk3 的磷酸化位点。此外，用 LiCl（GSK3β 和 CK2 的抑制剂）处理 MEFs 只能部分抑制磷酸化，表明 Plk3 是体内磷酸化这些位点的另一种激酶。与野生型相比，PTEN 的 Plk3 靶向突变体的表达水平降低，这与 Akt1 的上游激活物 PDK1 的活性增强有关。此外，PLK3 缺失的 MEFs 中 PTEN 水平降低，这一现象通过蛋白酶体抑制剂 MG132 的处理得到稳定。综上所述，我们的研究表明 Plk3 通过磷酸化和稳定 PTEN，成为调节 PI3K/PDK1/Akt 信号通路的新成员。
