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

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.

Serum amyloid A induces lipolysis by downregulating perilipin through ERK1/2 and PKA signaling pathways.

Serum amyloid A (SAA) is not only an apolipoprotein, but also a member of the adipokine family with potential to enhance lipolysis. The purpose of this study was to explore how SAA facilitates lipolysis in porcine adipocytes. We found that SAA increased the phosphorylation of perilipin and hormone-sensitive lipase (HSL) after 12-h treatment and decreased perilipin expression after 24-h treatment, and these effects were prevented by extracellular signal-regulated kinase (ERK) or protein kinase A (PKA) inhibitors in primary adipocyte cell culture. SAA treatment decreased HSL and adipose triglyceride lipase (ATGL) expression. SAA treatment also activated ERK and PKA by increasing the phosphorylation of these kinases. Moreover, SAA significantly increased porcine adipocyte glycerol release and lipase activity, which was inhibited by either ERK (PD98059) or PKA (H89) inhibitors, suggesting that ERK and PKA were involved in mediating SAA enhanced lipolysis. SAA downregulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) mRNA, which was reversed by the ERK inhibitor. We performed a porcine perilipin promoter assay in differentiated 3T3-L1 adipocytes and found that SAA reduced the porcine perilipin promoter specifically through the function of its PPAR response element (PPRE), and this effect was reversed by the ERK inhibitor. These findings demonstrate that SAA-induced lipolysis is a result of downregulation of perilipin and activation of HSL via ERK/PPARγ and PKA signaling pathways. The finding could lead to developing new strategies for reducing human obesity.