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Self-regulated mechanism of Plk1 localization to kinetochores: lessons from the Plk1-PBIP1 interaction
© Lee et al; licensee BioMed Central Ltd. 2008
Received: 07 January 2008
Accepted: 23 January 2008
Published: 23 January 2008
Mammalian polo-like kinase 1 (Plk1) has been studied extensively as a critical element in regulating various mitotic events during M-phase progression. Plk1 function is spatially regulated through the targeting activity of the conserved polo-box domain (PBD) present in the C-terminal non-catalytic region. Recent progress in our understanding of Plk1 localization to the centromeres shows that Plk1 self-regulates its initial recruitment by phosphorylating a centromeric component PBIP1 and generating its own PBD-binding site. Paradoxically, Plk1 also induces PBIP1 delocalization and degradation from the mitotic kinetochores late in the cell cycle, consequently permitting itself to bind to other kinetochore components. Thus, PBIP1-dependent self-recruitment of Plk1 to the interphase centromeres serves as a prelude to the efficient delivery of Plk1 itself to other kinetochore components whose interactions with Plk1 are vital for proper mitotic progression.
PBD, a phospho-binding module critical for subcellular localization of Plk1
Studies in cultured mammalian cells revealed that Plk1 localizes to the centrosomes as early as late S and to the centromeres in early G2. These localizations become most prominent during early mitosis and persist until late anaphase ([7–9], and Fig. 1B). In anaphase, likely due to changes in Plk1-binding proteins, Plk1 delocalizes from the centrosomes and kinetochores and relocalizes to the spindle midzone (later it becomes midbody). However, the mechanisms underlying Plk1 relocalization to specific subcellular locations and the elements critical for these events are largely elusive. A growing body of evidence suggests that PBD plays a pivotal role in targeting the catalytic activity of Plk1 to specific subcellular structures. The initial finding that demonstrated the importance of the PBD for Plk1 localization came from the analysis of PBD mutants in a genetically-amenable heterologous organism, budding yeast. A single point mutation of the conserved Trp414 in the PB1 of PBD (Fig. 1A) was sufficient to disrupt both the localization and the ability of Plk1 to functionally complement a mitotic defect associated with a mutation in the budding yeast Plk1 homolog CDC5 . Subsequent analyses with analogous mutations in the PBD show that PBD is critical for the localization and mitotic functions of various Plks in their native organisms [9, 11, 12]. Consistent with these observations, a single W414F mutation is sufficient to disrupt the ability of PBD to bind to its binding target peptide .
Recently, PBD has been shown to bind to a phosphorylated motif . Determination of the co-crystal structure of the Plk1 PBD bound to its optimal phosphopeptide provided molecular insights as to how the PBD interacts with its binding target. Results show that PBD is composed of PB1 (aa 411–489 in Plk1) and PB2 (aa 511–592 in Plk1) motifs that have identical folds of β6α (a six-stranded anti-parallel β-sheet and an α-helix). PB1 and PB2 form a hetero-dimeric phosphorecognition module of a zipper-like structure that can accommodate a phosphorylated peptide at a cleft between the two motifs [15, 16]. The Trp414 and Leu490 residues from PB1 appear to be crucial for non-polar interactions with the neighboring residues of the phospho-peptide, whereas the His538 and Lys540 residues from PB2 are vital for electrostatic interactions with the negative charges of the phospho-Ser/Thr residue of the phospho-peptide. Subsequent studies show that the PBD of Plk1 binds to phosphorylated optimal peptide sequences of Ser-pThr/pSer-Pro/X (pThr/pSer, pThr or pSer; X, any amino acid) with the critical requirement for Ser at the pThr-1 position and a loose selectivity for Pro at the pThr+1 position . These findings suggest that PBD likely binds to a phosphorylated target that is primarily primed by cyclin-dependent kinases (Cdks) or other Pro-directed kinases [14, 16]. Since priming phosphorylation of PBD-docking proteins by another kinase promotes Plk1 interaction with these proteins or targets Plk1 in proximity with other substrates, the priming step provides an additional layer of regulation to ensure ordered Plk1 functions during M-phase progression.
Inverse correlation between Plk1 and its PBD-binding protein, PBIP1
PBIP1/MLF1IP/KLIP1/CENP-50/CENP-U (PBIP1 hereafter) was isolated as a PBD-interacting protein from a yeast two-hybrid screening  and has been shown to be critical for proper recruitment of Plk1 to the interphase and mitotic centromeres . PBIP1 also interacts with latent nuclear antigen (LNA) of Kaposi's sarcoma-associated herpes virus (KSHV)  and with MDS/myeloid leukemia factor 1 (MLF1) , although the physiological significance of these interactions are largely elusive. In cultured cells, PBIP1 colocalizes with kinetochore-specific CREST antigens (Fig. 1C). In addition, PBIP1 has been shown to be a centromere component important for proper chromosome segregation [17, 20–22]. Since PBIP1 directly interacts and colocalizes with Plk1 at the centromeres, one simple possibility is that the PBIP1-Plk1 interaction at the centromeres targets Plk1 to PBIP1 itself and/or other centromere substrates whose phosphorylation by Plk1 is critical for interphase and mitotic events (Fig. 1D).
PBIP1 delocalization vs. degradation
Paradoxical nature of the Plk1-PBIP1 interaction
The inverse nature of PBIP1 and Plk1 localization to the centromeres suggests that these two proteins function either antagonistically or independently for different cellular processes. However, depletion of PBIP1 disrupts Plk1 localization to the interphase centromeres and greatly impairs Plk1 localization to the mitotic kinetochores , suggesting that PBIP1 is critical for proper Plk1 localization to these locations during interphase and early mitosis. Further investigation on the mechanism underlying Plk1 recruitment to the centromeres revealed that, contrary to the widespread model that Plk1 binds to the phospho-epitope generated by Pro-directed kinases, Plk1 phosphorylates PBIP1 at T78 and binds to the resulting S77-p-T78 motif through the PBD . These findings demonstrate that Plk1 phosphorylates and generates a self-docking site on PBIP1 to induce a stable interaction between Plk1 and PBIP1. Thus, recruitment of Plk1 to the kinetochores requires both the localized PBIP1 scaffold and the S77-p-T78 motif-dependent PBIP1-PBD interaction. This view predicts that Plk1 should bind to PBIP1 with a low affinity prior to PBIP1 phosphorylation at T78. Intriguingly, only Plk1, but not the two closely related Plk2 or Plk3, generates and binds to the S77-p-T78 motif (J. E. Park and K. S. Lee, unpublished), demonstrating the specificity of the p-T78-dependent PBIP1-Plk1 interaction.
Ironically, Plk1 also induces PBIP1 degradation in a S77-p-T78 motif-dependent manner , suggesting that both S77 and T78 residues that are central for PBD binding are also required for proper PBIP1 degradation. The current hypothesis is that Plk1-dependent PBIP1 phosphorylation at multiple sites after binding to the S77-p-T78 motif induces disassembly of the Plk1-PBIP1 complex and dissociation of PBIP1 from the mitotic kinetochores that ultimately leads to PBIP1 degradation during mitosis. Thus, Plk1-dependent PBIP1 phosphorylation not only promotes its own recruitment to the interphase centromeres but also triggers its own liberation from the PBIP1 scaffold at the mitotic kinetochores (Fig. 4). Then, how can Plk1 self-regulate these seemingly contradictory events at distinct stages of the cell cycle? One possibility is that each event requires different levels of Plk1 activities. A low level of Plk1 activity during interphase could be sufficient to generate the p-T78 tether for binding, but not sufficient to induce the disassembly of the Plk1-PBIP1 complex and degradation of PBIP1. Following its activation at the onset of mitosis, Plk1 may hyperphosphorylate PBIP1 to disassemble the Plk1-PBIP1 complex and delocalize PBIP1 for degradation. Alternatively, a positive factor accumulated in mitosis may bind to the Plk1-PBIP1 complex to induce PBIP1 delocalization and degradation. Since the level of the p-T78 epitope reaches a maximum level prior to PBIP1 delocalization/degradation, Plk1-dependent PBIP1 phosphorylation may serve as a signature for recruiting component(s) important for this event. In either case, these arguments suggest that a well-controlled balance between the Plk1 activity and the PBIP1 level is critical for proper Plk1 recruitment and timely elimination of PBIP1 from the mitotic kinetochores.
PBIP1, a temporary Plk1 scaffold at the interphase and mitotic centromeres
Although it is clear that PBIP1 is critical for Plk1 recruitment to the interphase and early mitotic centromeres, Plk1 still localizes to the mitotic kinetochores even after delocalization/degradation of PBIP1 from these structures (Fig. 2). This finding suggests that the mechanism of Plk1 localization at the kinetochores likely involves a dynamic exchange of various Plk1-binding proteins. Consistent with this view, Plk1 is shown to interact with INCENP, Bub1, and BubR1 [23–26], whose localization to the kinetochores are most prominent at the prometaphase. These observations suggest that the Plk1 population freed from the Plk1-PBIP1 complex following PBIP1 delocalization/degradation interacts with INCENP, Bub1, and BubR1 and stably localizes to the mitotic kinetochores. However, it should be noted that INCENP, Bub1, and BubR1 do not localize to the interphase kinetochores, suggesting that they are not responsible for the early recruitment of Plk1 to the interphase centromeres. Thus, PBIP1 functions as a temporary scaffold crucial for the early recruitment of the centromeric Plk1 population until its delocalization/degradation allows Plk1 to interact with other kinetochore components during early mitosis. The interaction between Plk1 and INCENP or BubR1 has shown to be required for normal mitotic progression [24, 25], supporting the notion that timely release of Plk1 from the PBIP1 scaffold is important for proper interaction with other kinetochore components, and therefore normal mitotic progression. In this regard, it will be interesting to examine whether a prolonged Plk1-PBIP1 interaction by a Plk1-binding competent, yet stable, PBIP1 mutant restrains the kinetochore Plk1 from interacting with other components or its substrates and interferes with its function during M-phase progression.
Summary and perspective
In addition to the much-studied cyclin-dependent protein kinases, it is now widely appreciated that Plk1 functions as an integral component for various mitotic events. Plk1 is tightly regulated both temporally and spatially throughout the cell cycle. Timely recruitment of Plk1 to specific subcellular structures is likely important for proper function of Plk1 at these locations. A centromeric protein, PBIP1, binds to Plk1 with a high affinity and plays an important role in recruiting Plk1 to the interphase and early mitotic centromeres. However, contrary to the simple view that the Plk1-PBIP1 complex formation is critical for mitotic functions of Plk1 (as illustrated in Fig. 1D), PBIP1 appears to function as a temporary scaffold to tightly build up and sequester Plk1 at the interphase centromeres in a manner that requires Plk1-dependent PBIP1 phosphorylation at T78. Paradoxically, Plk1 also induces PBIP1 delocalization/degradation from the early mitotic kinetochores, thus allowing a swift conveyance of Plk1 itself to other kinetochore components critical for proper M-phase progression (see Fig. 4). These findings suggest that PBIP1 functions as a conveyor belt that Plk1 can hop onto at its own self-determined pace and bring itself to other mitotic kinetochore components accumulated at the other end of the belt.
Proper recruitment and function of Plk1 at the mitotic kinetochores appear to be critical for normal chromosome congression and segregation. Failure in this process ultimately leads to the development of aneuploidy . The Plk1-PBIP1 interaction exemplifies a unique mechanism involving both temporal and spatial regulation of PBIP1 at the centromeres. Proper regulation of Plk1 function at the centromeres appears to require multiple components that orchestrate timely recruitment, sequestration, and delivery of Plk1 to the right components at the mitotic kinetochores. Adding more complexity to the already complicated Plk1-PBIP1 interaction, other studies suggest that PBIP1 also forms a complex with other centromeric components such as CENP-O, CENP-P, CENP-Q, and CENP-R [21, 22]. Further investigation on the regulation of PBIP1 modification, delocalization, and degradation, as well as the determination of additional components critical for PBIP1 function will be necessary to better comprehend the functions of the Plk1-PBIP1 interaction during M-phase progression.
We are grateful to Rachel H. Lee for her help in preparing the manuscript, and Yasuhiko Terada and Susan Garfield for critical comments and suggestions. We are also indebted to the present and past members of the Lee laboratory for their great work and stimulating discussions, and our collaborators for generously sharing their ideas. We apologize to all authors whose work could not be cited due to space limitations.
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