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Proteomic identification of Hsp70 as a new Plk1 substrate in arsenic trioxide-induced mitotically arrested cells
Yu J Chen1, Ya-Ping Lin, Lu-Ping Chow
1Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
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.
Insights
Polo-like kinase 1 (Plk1) phosphorylates heat shock protein 70 (Hsp70) at Ser631 in arsenic trioxide-treated cells, preventing mitotic death and attenuating abnormalities.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Arsenic trioxide (ATO) induces mitotic arrest in HeLa S3 cells (AIMACs).
- Staurosporine (SSP) treatment causes rapid mitotic exit in AIMACs.
- SSP alters the phosphoproteome of AIMACs, suggesting key regulatory events.
Purpose of the Study:
- To identify cellular targets and mechanisms underlying mitotic exit in AIMACs.
- To investigate the role of polo-like kinase 1 (Plk1) in regulating protein phosphorylation during mitotic arrest.
- To elucidate the function of Hsp70 phosphorylation in response to ATO treatment.
Main Methods:
- 2-D DIGE and LC-MS/MS analysis to profile phosphoproteome changes.
- TiO(2) phospho-peptide enrichment and MS/MS for precise phosphorylation site identification.
- Antibody-based detection, kinase-dead Plk1 expression, and pharmacological inhibition to confirm kinase-substrate interactions.
Main Results:
- Hsp70, Rad 23B, and eIF4B identified as potential Plk1 substrates.
- Serine 631 (Ser631) on Hsp70 was confirmed as a Plk1 phosphorylation site in AIMACs.
- Plk1-mediated Hsp70 phosphorylation localized Hsp70 and Plk1 and prevented mitotic cell death.
Conclusions:
- Hsp70 is a novel substrate of Plk1.
- Plk1-mediated phosphorylation of Hsp70 attenuates ATO-induced mitotic abnormalities.
- This phosphorylation event plays a crucial role in cellular response to genotoxic stress during mitosis.
