title: 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.
authors: Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors: Yu J
authors: YJ
authors: Chen
authors: Ya-Ping
authors: YP
authors: Lin
authors: Lu-Ping
authors: LP
authors: Chow
authors: Te-Chang
authors: TC
authors: Lee
mesh_terms: D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008938:Mitosis / Q000187:drug effects / Q000502:physiology; D010087:Oxides / Q000494:pharmacology*; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D000097869:Polo-Like Kinase 1
chemical_list: D000077237:Arsenic Trioxide; D001152:Arsenicals; D018797:Cell Cycle Proteins; D018840:HSP70 Heat-Shock Proteins; D010087:Oxides; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D020543:Proteome; D011518:Proto-Oncogene Proteins; D019311:Staurosporine; D000097869:Polo-Like Kinase 1