Functional studies on the role of the C-terminal domain of mammalian polo-like kinase

Abstract

Mammalian polo-like kinase (Plk) acts at various stages in early and late mitosis. Plk is phosphorylated and activated in mitosis, and the proper subcellular localization of Plk is essential for mitotic regulation. We have observed that overexpression of the C-terminal domain of Plk is more effective than wild-type or kinase-defective Plk in causing mitotic delay or arrest. The specific activity of Plk with C-terminal deletions or substitution of aspartate for threonine-210 is increased severalfold relative to wild type. We show in this communication that the C-terminal domain can bind to fulllength or the catalytic domain of Plk and inhibit its kinase activity, and that this binding is disrupted when threonine-210 is substituted with an aspartic acid residue. The C-terminal domain binds unphosphorylated Plk from G2 arrested cells, but not phosphorylated Plk from mitotic cells. Green fluorescent protein-C-terminal Plk is localized at the centrosome and the midbody of transfected cells as shown previously for full-length enzyme. These and other data indicate that although the C terminus serves to regulate Plk kinase activity, the localization of the C terminus at the centrosome and other sites in transfected cells may block the correct localization of endogenous Plk. intramolecular binding ͉ inhibition ͉ mitosis ͉ mammalian cells

Molecular and structural basis of polo-like kinase 1 substrate recognition: Implications in centrosomal localization

“…Previous work has shown that the PBD cannot interact with the kinase domain upon Plk1 Thr-210 phosphorylation (10). Interestingly, upon Plk1 activation in the presence of a mitotic extract, the kinase seems to bind both primed and unprimed peptides in a similar manner as the PBD (Fig.…”

“…2a). This behavior suggests that once Plk1 is activated, a conformational change may disrupt contacts between the kinase and the PBD (10). In this conformation, the PBD in Plk1 will resemble the properties of the PBD alone.…”

“…This motif is supposed to be involved in an autoregulatory mechanism or in targeting the kinase to its substrates (9). The intramolecular interactions between the catalytic and noncatalytic domain [PB domain (PBD)], as well as the phosphorylation of Plk1, regulate the activation of its protein kinase activity (10). Phosphorylation represents an important mechanism for Plk1 activation.…”

Plk1 Regulates Activation of the Anaphase Promoting Complex by Phosphorylating and Triggering SCFβTrCP-dependent Destruction of the APC Inhibitor Emi1

“…Conversely, we detected no Emi1 coprecipitating with Plk1's N-terminal kinase domain (Plk1-NT), indicating that the PBD is required for stable complex formation with Emi1. Despite the absence of the PBD, Plk1-NT reduced Emi1 levels in cell lysates as effectively as did full-length Plk1, presumably because overexpression obviates the need for stable association and because of the higher specific activity of Plk1's kinase domain in the absence of its C terminus (Mundt et al, 1997;Jang et al, 2002). We also found that the N terminus of Emi1, which contains its degron, was sufficient to associate with Plk1, whereas the region of Emi1 containing the F-box and zinc-binding motif was dispensable for this interaction ( Figure 4C).…”

Protein kinases controlling the onset of mitosis

“…Considering that the consensus for PBD binding fulfills the requirements for phosphorylation by Proline-directed kinases such as Cdks and MAP kinases, it has been suggested that the latter may act as priming factors for Plk1 targeting [70]. The PBD has two established functions: it constitutes an autoinhibitory domain [72] whose negative role is relieved through phosphorylation at the T-loop site T 210 [73] (see below), and it plays an important role in Plk1 subcellular localization [74]. The latter was elucidated in complementation studies conducted on the temperaturesensitive cdc5-1 mutant yeast strain, where mutation of conserved residues in the PBD disrupted localization and mitotic functions of human Plk1, without nonetheless altering catalytic activity [75].…”