Proteomic identification of Hsp70 as a new Plk1 substrate in arsenic trioxide‐induced mitotically arrested cells

Abstract

We previously demonstrated that when arsenic trioxide (ATO)-induced mitotically arrested HeLa S3 cells (AIMACs) were treated with staurosporine (SSP) the cells rapidly exited mitosis. To better define the cellular targets and the underlying mechanisms of AIMACs, we applied 2-D DIGE followed by LC-MS/MS analysis and showed that SSP induced a significant change in the phosphoproteome of AIMACs. Among the proteins whose phosphorylation was modulated by SSP, we identified Hsp70, Rad 23B, and eukaryotic translation initiation factor 4B as potentially new substrates of polo-like kinase 1 (Plk1), an essential serine/threonine kinase with versatile mitotic functions. Since Hsp70 is a stress protein responsible for ATO treatment, we further identified Thr(13) , Ser(362) , Ser(631) , and Ser(633) on Hsp70 intracellularly phosphorylated in AIMACs by combining TiO(2) phospho-peptides enrichment and MS/MS analysis. Using antibody specifically against phosph-Ser(631) Hsp70 and further aided by expression of kinase-dead Plk1 and pharmacological inhibition of Plk1, we concluded that Ser(631) on Hsp70 is phosphorylated by Plk1 in AIMACs. By immnuofluorescent staining, we found the colocalization of Hsp70 and Plk1 in AIMACs but not in interphase cells. In addition, Plk1-mediated phosphorylation of Hsp70 prevented AIMACs from mitotic death. Our results reveal that Hsp70 is a novel substrate of Plk1 and that its phosphorylation contributes to attenuation of ATO-induced mitotic abnormalities.

Interleukin-1 Receptor Type 2 Acts with c-Fos to Enhance the Expression of Interleukin-6 and Vascular Endothelial Growth Factor A in Colon Cancer Cells and Induce Angiogenesis

“…Immunoprecipitation-Western Blot Assay-The immunoprecipitation-Western blot assay was performed as previously described (35). Logarithmically growing cells were resuspended in 1 ml of radioimmune precipitation assay lysis buffer.…”

Regulatory functional territory of PLK-1 and their substrates beyond mitosis

“…Furthermore, PLK-1 was also reported to phosphorylate eukaryotic translation initiation factor 4B (eIF4B) in response to arsenic tetroxide treatment in HeLa cells. eIF4B is required for the formation of the pre-initiation complex to initiate translation [21]. Although the study showed that PLK-1 phosphorylates eIF4B, no further detailed mechanism was delineated [21].…”

“…eIF4B is required for the formation of the pre-initiation complex to initiate translation [21]. Although the study showed that PLK-1 phosphorylates eIF4B, no further detailed mechanism was delineated [21]. It will require additional biochemical studies to clarify the role of PLK-1 in the regulation of translation.…”

Low-dose arsenic-mediated metabolic shift is associated with activation of Polo-like kinase 1 (Plk1)

“…Considering that Plk1 phosphorylation of HSP70 contributes to attenuation of high-dose arsenic trioxide (5 mM for 24 hours)-induced mitotic abnormalities, such as formation of elongated spindles, 5,10 and that Plk1 elevation leads to aerobic glycolysis, 13 we then asked whether low-dose arsenic affects the level of Plk1. Toward that end, proteins were prepared from liver, kidney, lung and bladder tissues of mice as described above.…”

“…9 It was recently shown that HSP70 is a substrate of pololike kinase 1 (Plk1) in high-dose (5 mM for 24 h) arsenic trioxide-induced mitotically arrested cell and that its phosphorylation contributes to attenuation of arsenicinduced mitotic abnormalities. 10 Whether Plk1 also plays a critical role in low-dose (lower than 1 mM) arsenic-mediated phenotypes is not known. Plk1 has well documented roles in many mitotic-related events, such as centrosome maturation, bipolar spindle formation, sister chromatid segregation and cytokinesis.…”