German Cancer Research Center (DKFZ); Research Group Cellular Senescence; DKFZ-ZMBH Alliance; Heidelberg, Germany.
The European Molecular Biology Laboratory (EMBL); Proteomics Core Facility; Heidelberg, Germany.
Cell Cycle. 2014;13(1):115-25. doi: 10.4161/cc.26857. Epub 2013 Oct 25.
Non-receptor tyrosine kinase Src is a master regulator of cell proliferation. Hyperactive Src is a potent oncogene and a driver of cellular transformation and carcinogenesis. Homeodomain-interacting protein kinase 2 (HIPK2) is a tumor suppressor mediating growth suppression and apoptosis upon genotoxic stress through phosphorylation of p53 at Ser46. Here we show that Src phosphorylates HIPK2 and changes its subcellular localization. Using mass spectrometry we identified 9 Src-mediated Tyr-phosphorylation sites within HIPK2, 5 of them positioned in the kinase domain. By means of a phosphorylation-specific antibody we confirm that Src mediates phosphorylation of HIPK2 at Tyr354. We demonstrate that ectopic expression of Src increases the half-life of HIPK2 by interfering with Siah-1-mediated HIPK2 degradation. Moreover, we find that hyperactive Src binds HIPK2 and redistributes HIPK2 from the cell nucleus to the cytoplasm, where both kinases partially colocalize. Accordingly, we find that hyperactive Src decreases chemotherapeutic drug-induced p53 Ser46 phosphorylation and apoptosis activation. Together, our results suggest that Src kinase suppresses the apoptotic p53 pathway by phosphorylating HIPK2 and relocalizing the kinase to the cytoplasm.
非受体酪氨酸激酶Src 是细胞增殖的主要调节因子。高活性 Src 是一种有效的致癌基因，是细胞转化和癌变的驱动因素。同源结构域相互作用蛋白激酶 2（HIPK2）是一种肿瘤抑制因子，通过磷酸化 p53 的丝氨酸 46 介导细胞生长抑制和凋亡。在这里，我们表明 Src 磷酸化 HIPK2 并改变其亚细胞定位。通过质谱分析，我们在 HIPK2 内鉴定了 9 个 Src 介导的 Tyr 磷酸化位点，其中 5 个位于激酶结构域内。通过一种磷酸化特异性抗体，我们证实 Src 介导 HIPK2 在 Tyr354 上的磷酸化。我们证明，Src 的异位表达通过干扰 Siah-1 介导的 HIPK2 降解来增加 HIPK2 的半衰期。此外，我们发现高活性 Src 与 HIPK2 结合并将 HIPK2 从细胞核重新分配到细胞质，两个激酶部分共定位。因此，我们发现高活性 Src 降低了化疗药物诱导的 p53 Ser46 磷酸化和凋亡激活。总之，我们的结果表明，Src 激酶通过磷酸化 HIPK2 和将激酶重新定位到细胞质来抑制凋亡 p53 途径。
