Polo-Like Kinase 3 Is Required for Entry Into S Phase
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Polo-like kinase 3 is required for entry into S phase Wendy C. Zimmerman and Raymond L. Erikson* Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 Contributed by Raymond L. Erikson, December 11, 2006 (sent for review November 30, 2006) The polo-like kinase, Plk1, which is expressed and active in mitosis, localization throughout the cell cycle should shed light on its is involved in regulation of mitotic entry, spindle pole assembly, functions. We initially examined its localization by using the mitotic exit, and cytokinesis [Donaldson MM, Tavares AA, Hagan antibody and conditions reported in refs. 8 and 9. However, in IM, Nigg EA, Glover DM (2001) J Cell Sci 114:2357–2358]. In mam- contrast to their results, we were unable to detect any specific mals, there are two other members of the polo-like kinase family immunofluorescent localization (data not shown). We next that are less well understood, Plk2 and Plk3. Plk3 first was iden- screened other commercially available antibodies for effective tified and cloned as an immediate early gene. Here, we report Plk3 immunofluorescent staining. One affinity-purified rabbit poly- localizes to the nucleolus and is involved in regulation of the G1/S clonal antibody, Fnk140 (Santa Cruz Biotechnology, Santa Cruz, phase transition. We demonstrate that the level of Plk3 protein CA), appeared effective. Unexpectedly, in U2OS cells, the is cell cycle regulated, peaking in G1. We have delivered Plk3- staining of endogenous Plk3 appeared as discrete nuclear inclu- interfering RNA with lentivirus to serum-starved cells and found sions, morphologically similar to nucleoli (Fig. 1b). Identification that, upon serum stimulation, Plk3 is required for cyclin E expres- of these nuclear compartments as nucleoli was confirmed by sion and entry into S phase. Plk3-interfering RNA-induced Plk3 colocalization of Plk3 with nucleophosmin and nucleolin, pro- depletion resulted in a large fraction of asynchronously prolifer- teins that are known to localize to the nucleolus (Fig. 1c). Similar ating cells to become quiescent. We propose the Plk3 requirement colocalization was observed in HeLa, MCF10A, h-tert-RPE, in the cell cycle is fulfilled in G1, and that once cells pass this point, T98G, and 293T cells (data not shown). Plk3 localization was they are able to complete cell division, whereas in the absence of detectable only within the nucleolus; we were unable to detect Plk3, they fail to reenter the cell cycle. Additional data suggest that endogenous Plk3 at the centrosome, spindle pole, or any other Plk3 may regulate entry into S phase in part through interaction cytoplasmic location (Fig. 1c). with the phosphatase Cdc25A, because its depletion also resulted Human Plk3 is predicted to migrate as a 72-kDa protein. To CELL BIOLOGY in attenuation of cyclin E expression. confirm that the observed immunolocalization represented en- dogenous Plk3 and not a cross-reactive protein, asynchronous Cdc25A ͉ cyclin E ͉ nucleolus HeLa cells were fractionated according to the method of Andersen et al. (14). The Fnk140 antibody recognized a band of n yeast and Drosophila, polo kinases are master mitotic approximately the expected size that was significantly enriched Iregulators, involved in the regulation of mitotic entry, the in nucleolar fractions but undetectable in cytoplasmic and metaphase-to-anaphase transition, and mitotic exit (1, 2). soluble nuclear fractions (Fig. 1d). This Ϸ72-kDa band was Whereas only one polo kinase is found in yeast and Drosophila, specifically depleted by three different lentiviruses expressing three polo-like kinases have been identified in mammals: Plk1, Plk3-RNAi targeting different sequences in Plk3 mRNA, as Plk2 (Snk), and Plk3 (Prk and Fnk). Polo kinases are charac- discussed below (Figs. 3 and 4). These same RNAi vectors also terized by an N-terminal serine/threonine kinase domain and a significantly reduced the immunofluorescence signal at the conserved C-terminal substrate-binding domain, termed the nucleolus (see below). Moreover, we expressed full-length strep- Polo box domain (Fig. 1). All three mammalian polo-like kinases tagged mouse Plk3 and evaluated immunolocalization by using have predicted nuclear localization signals (NLSs), suggesting anti-strep-tag antibodies. At 20–24 h after transfection, ectopi- that all three may pass through the nucleus. However, to date, cally expressed Plk3 formed nuclear inclusions in both HeLa and only one NLS (in Plk1) has been shown to be functionally active MCF10A cells (Fig. 1 f and g). Taken together, these data show (3). A fourth related mammalian kinase, Sak (Plk4) shows that Plk3 is a nucleolar protein. significant homology to Plk1, Plk2, and Plk3 in its kinase domain but lacks conservation of the typical bipartite polo box domain The Level of Plk3 Protein Is Cell Cycle Regulated. Plk3 was initially (4). Plk1 is the best-studied enzyme of this family. Like yeast and cloned as an immediate early gene (11, 12). To shed further light Drosophila polo kinases, Plk1 regulates multiple aspects of on Plk3 function, we chose to investigate the protein level of Plk3 mitotic entry and exit (5). The functions of Plk2 and Plk3 are not throughout the cell cycle. As is the case for other predominantly well understood. nucleolar proteins, endogenous Plk3 is resistant to detergent Polo kinases colocalize with their substrates through interac- extraction and is not readily solubilized by standard isolation tions with the polo box domain (6, 7). There are several protocols for protein kinases. But it can be effectively solubilized conflicting reports of Plk3 localization in the literature. Plk3 has from either isolated nucleoli (data not shown) or detergent- been reported to colocalize with the centrosome, spindle pole extracted cell pellets (Fig. 1e) with a buffer containing high salt, and spindle microtubules (8), with Golgi apparatus (9), and with magnesium, and glycerol (15). Therefore, to follow the level of actin-containing plaques (10). In contrast to previous reports, we Plk3, we analyzed total protein by boiling the entire sample, not demonstrate here that Plk3 localizes in the nucleolus, based on just the soluble fraction, in SDS sample buffer. both immunofluorescence and subcellular fractionation. We analyzed the protein level of Plk3 throughout the cell cycle We show that the level of Plk3 protein is cell cycle regulated; in MCF10A cells synchronized by serum starvation (Fig. 2 a and unlike Plk1, the level of Plk3 peaks in G1, concurrent with the b) and in HeLa cells synchronized in early S phase by double reported peak in the level of Plk3 message (11–13). We further demonstrate that Plk3 is required for expression of cyclin E and for cells to pass from G1 to S phase. Author contributions: W.C.Z. and R.L.E. designed research; W.C.Z. performed research; W.C.Z. and R.L.E. analyzed data; and W.C.Z. wrote the paper. Results The authors declare no conflict of interest. Plk3 Localizes to the Nucleolus. Because Plks colocalize with their *To whom correspondence should be addressed. E-mail: [email protected]. substrates, we reasoned that a clear understanding of Plk3 © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610856104 PNAS ͉ February 6, 2007 ͉ vol. 104 ͉ no. 6 ͉ 1847–1852 Downloaded by guest on October 1, 2021 Fig. 2. Plk3 is expressed in G1.(a and b) MCF10A cells were synchronized by 72 h of serum starvation. (a) Serum and hormones then were added to the cells, and the cells analyzed at 4-h intervals by FACS. (b) Total cellular protein was analyzed from duplicate plates by Western blot. (a) Under these condi- Fig. 1. Plk3 localizes to the nucleolus. (a) Comparison of human polo-like tions, G2/M occurred between 24–32 h. (b) The level of Plk3 protein peaked in kinases. Polo kinases are characterized by a conserved serine-threonine kinase G1 near 8 h, whereas the level of Plk1 peaked as expected during G2/M. Levels domain (S-Tkc) and a bipartite polo box domain (polo box). All three mam- of the G1 cyclins, cyclin D1 and cyclin E1, were analyzed for comparison. malian polo-like kinases contain predicted nuclear localization signals (NLS), Nucleolin and Erk2 loading controls are included. (c and d) HeLa cells were although, to date, only one has been experimentally tested and proven to be synchronized by double thymidine block. Cells were analyzed at 2-h intervals functional (proven NLS) (3). Plk2 and Plk3 both contain predicted proline- by FACS (c), and total cellular protein was analyzed from duplicate plates by glutamate-serine-threonine (PEST) signals involved with targeting of proteins Western blot (d). (c) Double thymidine block synchronizes cells in early S phase for degradation by the proteosome. (b) Endogenous Plk3 (red) appears as with slightly more than 2N DNA content (0 h). M phase cyclin B1 expression nuclear inclusions in interphase U2OS cells. Endogenous Plk3 was visualized by peaked at 10 h after release and again at 24 h. The level of Plk3 protein peaked immunofluorescence microscopy using an affinity-purified rabbit polyclonal in G1 at 14–16 h (c and d), concurrent with the peak expression of the G1 phase antibody (Fnk140; Santa Cruz Biotechnology). Nuclear DNA is visualized with cyclin D1. Erk2 loading control is also shown. DAPI (green). (c) Endogenous Plk3 (green) colocalizes with nucleolar marker proteins nucleophosmin (NucP., red; Top) and nucleolin (Nuc., red; Middle), in U2OS cells, but has no detectable colocalization with the centrosome and on the proportion of cells with 4N DNA content, concurrent with spindle pole marker ␥-tubulin (␥Tub., red; Bottom). (Scale bar: 10 m.) (d) Plk3 is enriched in nucleoli isolated from asynchronous HeLa cells. HeLa cells were peak expression of Plk1. This pattern of Plk3 protein expression fractionated and nucleoli isolated by the method of Andersen et al.