Abstract
Polo-like kinases (Plks) define a highly conserved family of Ser/Thr kinases with crucial roles in the regulation of cell division. Here we show that Plk1 is cleaved by caspase 3, but not by other caspases in different hematopoietic cell lines treated with competitive inhibitors of the ATP-binding pocket of Plk1. Intriguingly, Plk1 was not cleaved in cells treated with Rigosertib, a non-competitive inhibitor of Plk1, suggesting that binding of the inhibitor to the ATP binding pocket of Plk1 triggers a conformational change and unmasks a cryptic caspase 3 cleavage site on the protein. Cleavage occurs after Asp-404 in a DYSD/K sequence and separates the kinase domain from the two PBDs of Plk1. All Plk1 inhibitors triggered G2/M arrest, activation of caspases 2 and 3, polyploidy, multiple nuclei and mitotic catastrophe, albeit at higher concentrations in the case of Rigosertib. Upon BI-2536 treatment, Plk1 cleavage occurred only in the cytosolic fraction and cleaved Plk1 accumulated in this subcellular compartment. Importantly, the cleaved N-Terminal fragment of Plk1 exhibited a higher enzymatic activity than its non-cleaved counterpart and accumulated into the cytoplasm conversely to the full length and the C-Terminal Plk1 fragments that were found essentially into the nucleus. Finally, the DYSD/K cleavage site was highly conserved during evolution from c. elegans to human. In conclusion, we described herein for the first time a specific cleavage of Plk1 by caspase 3 following treatment of cancer cells with ATP-competitive inhibitors of Plk1.
Highlights
Caspases are cysteine proteases that play an important role in the regulation of cellular homeostasis including cell death, differentiation and inflammation
We investigated the effect of three Plk1 inhibitors (BI-2536, GSK-461363 and Rigosertib) on Plk1 expression in the chronic myelogenous cell line (CML) K562
This result confirms that the cleavage of Plk1 induced by ATP-competitive inhibitors of Plk1 may occur in different hematopoietic cell lines
Summary
Caspases are cysteine proteases that play an important role in the regulation of cellular homeostasis including cell death, differentiation and inflammation. In some circumstances, cleavage occurs between distinctive constitutive domains of the protein substrate leading to modulation of its activity [4] This is true regarding transcription factors such as serum responsive factor, www.impactjournals.com/oncotarget for which caspase 3-dependent cleavage separates the DNA binding domain from the transcription activation domain, modulating its function [5]. This is exemplified by protein kinases, for which caspase 3 cleavage between different structural domains, may lead to deregulation of the enzymatic kinase activity. This type of cleavage has been noticeably reported for the Src tyrosine kinases Lyn and Fyn, in which cleavage after Asp-18 or Asp-19 respectively by caspase 3 eliminates the N-Terminal anchoring domain, thereby generating a more active form kinase that delocalizes into the cytoplasm [6,7,8]
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