Key points are not available for this paper at this time.
The mammalian polo-like kinase (Plk) plays a critical role in M-phase progression. Plk is phosphorylated and activated by an upstream kinase(s), which has not yet been identified in mammalian cells. Phosphopeptide mapping and phosphoamino acid analyses of Plk labeled in vivo and phosphorylated in vitro by Xenopus polo-like kinase kinase-1 (xPlkk1) or by lymphocyte-oriented kinase, its most closely related mammalian enzyme, indicate that Thr-210 is a major phosphorylation site in activated Plk from mitotic HeLa cells. Although the amino acid sequence surrounding Ser-137 is similar to that at Thr-210 and is conserved in Plk family members, Ser-137 is not detectably phosphorylated in mitotic mammalian cells or by xPlkk1 in vitro. Nevertheless, the substitution of either Thr-210 or Ser-137 with Asp (T210D or S137D) elevates the kinase activity of Plk. The kinase activity of the double mutant S137D/T210D is not significantly different from that of T210D or S137D, demonstrating that substitution of both residues does not have an additive effect on Plk activity. Expression of the S137D mutant construct arrested HeLa cells in early S-phase with slightly separated centrosomes, whereas cells expressing wild type and T210D were arrested or delayed in M-phase. These data indicate that the Ser-137 may have an unexpected and novel role in the function of Plk. The mammalian polo-like kinase (Plk) plays a critical role in M-phase progression. Plk is phosphorylated and activated by an upstream kinase(s), which has not yet been identified in mammalian cells. Phosphopeptide mapping and phosphoamino acid analyses of Plk labeled in vivo and phosphorylated in vitro by Xenopus polo-like kinase kinase-1 (xPlkk1) or by lymphocyte-oriented kinase, its most closely related mammalian enzyme, indicate that Thr-210 is a major phosphorylation site in activated Plk from mitotic HeLa cells. Although the amino acid sequence surrounding Ser-137 is similar to that at Thr-210 and is conserved in Plk family members, Ser-137 is not detectably phosphorylated in mitotic mammalian cells or by xPlkk1 in vitro. Nevertheless, the substitution of either Thr-210 or Ser-137 with Asp (T210D or S137D) elevates the kinase activity of Plk. The kinase activity of the double mutant S137D/T210D is not significantly different from that of T210D or S137D, demonstrating that substitution of both residues does not have an additive effect on Plk activity. Expression of the S137D mutant construct arrested HeLa cells in early S-phase with slightly separated centrosomes, whereas cells expressing wild type and T210D were arrested or delayed in M-phase. These data indicate that the Ser-137 may have an unexpected and novel role in the function of Plk. Progression through the mammalian cell cycle depends on the periodic control of various cyclin-dependent kinases (for reviews, see Refs. 1Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2935) Google Scholar and 2Nigg E.A. BioEssays. 1995; 17: 471-480Crossref PubMed Scopus (791) Google Scholar). In addition, members of the polo-like kinase (Plk) 1The abbreviations used are: Plk, polo-like kinase; Plx1, Xenopus Plk; xPlkk1, Xenopus Plk kinase-1; LOK, lymphocyte-oriented kinase; HA, hemagglutinin; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; PBS, phosphate-buffered saline; WT, wild type; HPLC, high pressure liquid chromatography family influence multiple events during cell division (3Glover D.M. Hagan I.M. Tavares A. Genes Dev. 1998; 12: 3777-3787Crossref PubMed Scopus (396) Google Scholar, 4Nigg E.A. Curr. Opin. Cell Biol. 1998; 10: 776-783Crossref PubMed Scopus (309) Google Scholar). Mammalian Plk is involved in bipolar spindle formation, chromosome separation (5Sunkel C.E. Glover D.M. J. Cell Sci. 1988; 89: 25-38Crossref PubMed Google Scholar, 6Ohkura H. Hagan I.M. Glover D.M. Genes Dev. 1995; 9: 1059-1073Crossref PubMed Scopus (328) Google Scholar, 7Tavares A. Glover D.M. Sunkel C.E. EMBO J. 1996; 15: 4873-4883Crossref PubMed Scopus (79) Google Scholar), centrosome maturation (8Lane R.J. Nigg E.A. J. Cell Biol. 1996; 135: 1701-1713Crossref PubMed Scopus (597) Google Scholar), and regulation of the anaphase-promoting complex (9Descombes P. Nigg E.A. EMBO J. 1998; 17: 1328-1335Crossref PubMed Scopus (195) Google Scholar, 10Shirayama M. Zachariae W. Ciosk R. Nasmyth K. EMBO J. 1998; 17: 1336-1349Crossref PubMed Scopus (439) Google Scholar). In Xenopus, Plk may play an important role at the onset of mitosis; Xenopus Plk (Plx1) phosphorylates and activates the phosphatase Cdc25C, a positive regulator of Cdc2-cyclin B (11Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (470) Google Scholar). In addition to proteolytic regulation, post-translational modifications such as phosphorylation and dephosphorylation are also a key regulatory mechanism of protein kinases in the cell cycle. Throughout late S and early G2 phases, Cdc2 is kept inactive as the result of phosphorylation of Thr-14 and Tyr-15 in the ATP-binding site. Phosphorylation at these sites is catalyzed by the protein kinases Wee1 and Myt1 (12Parker L.L. Atherton-Fessler S. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2917-2921Crossref PubMed Scopus (216) Google Scholar, 13Liu F. Stanton J.J., Wu, Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (265) Google Scholar). Activation of Cdc2-cyclin B at the G2/M transition requires dephosphorylation of both residues by Cdc25C (14Gautier J. Solomon M.J. Booher R.N. Bazan J.F. Kirschner M.W. Cell. 1991; 67: 197-211Abstract Full Text PDF PubMed Scopus (688) Google Scholar, 15Kumagai A. Dunphy W.G. Cell. 1991; 64: 903-914Abstract Full Text PDF PubMed Scopus (358) Google Scholar, 16Lee M.S. Ogg S., Xu, M. Parker L.L. Donoghue D.J. Maller J.L. Piwnica-Worms H. Mol. Biol. Cell. 1992; 3: 73-84Crossref PubMed Scopus (153) Google Scholar). Plk is also regulated by phosphorylation. During mitosis, the phosphorylated enzyme is detected as a more slowly migrating form. Phosphatase treatment increases the mobility and reduces the activity of Plk (17Hamanaka R. Smith M.R. O'Connor P.M. Maloid S. Mihalic K. Spivak J.L. Longo D.L. Ferris D.K. J. Biol. Chem. 1995; 270: 21086-21091Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 18Lee K.S. Yuan Y.L. Kuriyama R. Erikson R.L. Mol. Cell. Biol. 1995; 15: 7143-7151Crossref PubMed Scopus (244) Google Scholar, 19Mundt K.E. Golsteyn R.M. Lane H.A. Nigg E.A. Biochem. Biophys. Res. Comm. 1997; 239: 377-385Crossref PubMed Scopus (171) Google Scholar). The original studies on Thr-210 in Plk demonstrated that substitution of aspartate at this site significantly elevates protein kinase activity (20Lee K.S. Erikson R.L. Mol. Cell. Biol. 1997; 17: 3408-3417Crossref PubMed Scopus (164) Google Scholar). This residue is in the activation loop between protein kinase subdomains VII and VIII, and phosphorylation within this loop results in the activation of several kinases including Cdc2 and Mek1 (1Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2935) Google Scholar, 21Huang W. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8960-8963Crossref PubMed Scopus (133) Google Scholar). These data suggest that aspartate mimics the phosphorylation of serine or threonine in the activation loop and, moreover, raise the possibility that Thr-210 is a physiologically important phosphorylation site. xPlkk1 was identified in Xenopus egg extracts and was cloned as a major upstream kinase of Plx1 (22Qian Y.-W. Erikson E. Maller J.L. Science. 1998; 282: 1701-1704Crossref PubMed Scopus (107) Google Scholar). Microinjection of xPlkk1 into Xenopusoocytes accelerates the time of Plx1 activation as well as the transition from G2 to M phase of the cell cycle (23Qian Y.-W. Erikson E., Li, C. Maller J.L. Mol. Cell. Biol. 1998; 18: 4262-4271Crossref PubMed Scopus (213) Google Scholar). The upstream activating kinase of Plk in mammalian cells still remains to be identified, although there are enzymes closely related to xPlkk1, such as LOK (24Kuramochi S. Moriguchi T. Kuida K. Endo J. Semba K. Nishida E. Karasuyama H. J. Biol. Chem. 1997; 272: 22679-22684Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and STE20-like kinase (SLK) (25Ellinger-Ziegelbauer H. Karasuyama H. Yamada E. Tsujikawa K. Todokoro K. Nishida E. Genes Cells. 2000; 5: 491-498Crossref PubMed Scopus (80) Google Scholar), in mammalian cells. We have shown 2Y.-J. Jang, Y. Terada, and R. L. Erikson, unpublished data. that LOK functions in vitro in a fashion similar to xPlkk1; however, as yet, there are no data that indicate it is the relevant upstream activator of Plk in vivo. However, in previous studies, the site(s) of activating phosphorylation in Plk were not identified. In this study, we provide direct evidence that Thr-210 is a major phosphorylation site in mitotic Plk and that this site is important for Plk activation. In contrast, although substitution of Ser-137, which is embedded in a similar sequence as Thr-210, with aspartate also activates Plk, this residue is not phosphorylated in vitroby xPlkk1 or in vivo in mitotic cells and does not appear to be involved in activation of Plk during mitosis. Nevertheless, experiments with Ser-137 mutants suggest that this site has the potential to have a biologically significant role in regulating Plk activity during other stages of the cell cycle. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen). To synchronize cells at the G1/S transition, HeLa cells were treated with a double thymidine block (2.5 mm). To obtain mitotically synchronized cells, G1 cells, which were synchronized by treatment with mimosine (300 μm) for 16 h, were washed with fresh medium and were released into medium containing 200 ng/ml nocodazole for 18 h. To obtain metabolically labeled endogenous Plk, actively growing HeLa cells were arrested with 200 ng/ml nocodazole for 12 h. The mitotic cells were collected by shake-off and washed with labeling medium (phosphate-free Dulbecco's modified Eagle's medium plus 10% dialyzed serum, 10% normal Dulbecco's modified Eagle's medium, and 200 ng/ml nocodazole). Cells were labeled with 32Porthophosphate (ICN) at a final concentration of 0.7 mCi/ml for 4 h before washing and lysis. Transfection was performed by the standard calcium chloride method (26Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4821) Google Scholar) except that HEPES-buffered saline was used. Mutations at the indicated sites in the Plk construct were generated by PCR. All the PCR products were cloned into pGEX vector (Promega) and sequenced. The mutant proteins were expressed in Escherichia coli by isopropyl-1-thio-β-d-galactopyranoside induction and confirmed by Western blot with anti-Plk antibody. To generate recombinant baculoviruses, the pGEX constructs were cloned into pAcGHLT vector (Pharmingen) and were transfected into Sf9 cells with BaculoGoldTM (Pharmingen). For expression in mammalian cells, all constructs were cloned into pCMV-Tag2 vector (Stratagene), which encodes eight amino acids of FLAG (DYKDDDDK) epitope at the N terminus. Recombinant baculoviruses for expression of His-xPlkk1 were a kind gift of James Maller. Recombinant LOK or xPlkk1 proteins were prepared from Hi5 cells untreated or treated with 0.1 μm okadaic acid for 3 h with the use of TALON affinity resin and were further purified as described previously (22Qian Y.-W. Erikson E. Maller J.L. Science. 1998; 282: 1701-1704Crossref PubMed Scopus (107) Google Scholar). Fractions containing xPlkk1 proteins were stored in small portions at −80 °C. Cells were lysed with Nonidet P-40 buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 1 mmEGTA, 25 mm β-glycerophosphate, 25 mm NaF, 0.5 mm Na3VO4, 10 mm p-nitro phenylphosphate, and 1% Nonidet P-40) supplemented with protease inhibitors and centrifuged at 15,000 × gfor 20 min. The supernatant was incubated with antibody, anti-Plk monoclonal antibody (Zymed Laboratories Inc.) for metabolically labeled endogenous Plk or anti-FLAG monoclonal antibody (Sigma) for transfected Plk. Protein A/G- or protein G-PLUS Agarose (Zymed Laboratories Inc.) was added to isolate the immunocomplexes. Kinase reactions were carried out as described (20Lee K.S. Erikson R.L. Mol. Cell. Biol. 1997; 17: 3408-3417Crossref PubMed Scopus (164) Google Scholar). To examine the effect of wild type or T210D Plk on the activation of Cdc2-cyclin B, HeLa cells were transfected with plasmids encoding HA-Plk or HA-PlkT210D. About 60% of the total cell population expressed Plk1 under these conditions. At 12 h, mimosine was added to a final concentration of 0.3 mm, and cells were incubated for an 20 h. Cells were released from the mimosine block by washing with medium and incubated in medium before to Cdc2-cyclin B kinase activity in Cell extracts were incubated with antibody, and were performed as described previously (20Lee K.S. Erikson R.L. Mol. Cell. Biol. 1997; 17: 3408-3417Crossref PubMed Scopus (164) Google Scholar). phosphorylation was with a and The proteins were on a and to a For the of the was with 1 of in for at °C. The were washed with and with mm and incubated in of containing 1 mm and 20 of (Sigma) at The supernatant was in of and This washing was The final were a in the was performed at for in 1% containing 0.5 mm with the The was for h with For phosphoamino acid the in were and by at pH in the for 20 and pH in the for 16 with the Biochem. PubMed Scopus Google Scholar). Cells with Plk constructs and vector were from the by in at 4 for 16 h, cells were in supplemented with and for at of green and were with was to cells with a green fluorescent at cells. were collected for more events with the and Cell and W. T. 1998; Scopus Google Scholar, M. PubMed Scopus Google Scholar). For HeLa cells were and were transfected as described The cells were in by treatment with washing with PBS, the cells were incubated for h in supplemented with The were by and the cells were on the To the and activation of Plk during the cell HeLa cells were synchronized in G1/S phase with mimosine and into fresh medium with nocodazole to into M Cells were at various and by Plk from cells arrested in G1/S phase as a The migrating phosphorylated of Plk as cells into M phase and was at To the kinase activity of Plk at various endogenous Plk was with anti-Plk antibody from of G1/S phase and cells, and the phosphorylation of was The results shown in B previous studies that Plk activity from mitotic cells is that from G1/S cells (17Hamanaka R. Smith M.R. O'Connor P.M. Maloid S. Mihalic K. Spivak J.L. Longo D.L. Ferris D.K. J. Biol. Chem. 1995; 270: 21086-21091Abstract Full Text Full Text PDF PubMed Scopus (155) Google K.E. Golsteyn R.M. Lane H.A. Nigg E.A. Biochem. Biophys. Res. Comm. 1997; 239: 377-385Crossref PubMed Scopus (171) Google Scholar). of Plk phosphorylation and kinase activity was performed vitro kinase which has been as a kinase in Y.-W. Erikson E. Maller J.L. Science. 1998; 282: 1701-1704Crossref PubMed Scopus (107) Google Scholar), was purified from cells and used to mammalian Plk. Plk which activity was phosphorylated by xPlkk1 wild type was incubated and phosphorylated by purified xPlkk1 or LOK, activity was B, xPlkk1 not phosphorylation of These results that xPlkk1 and mammalian Plk in vitro. results have also been purified LOK, a mammalian enzyme closely related in sequence to xPlkk1 not shown for xPlkk1, purified wild type Plk has significant activity To the phosphorylation by an upstream kinase and on a of both phosphorylated by xPlkk1 and were Although the of phosphorylated that of Plk several that not with of phosphorylated and acid that the site(s) phosphorylated by xPlkk1 is threonine whereas the site(s) on serine These data indicate that phosphorylation by an upstream kinase and in Plk on different residues and that phosphorylation on threonine is to be important for Plk activation. To Plk phosphorylation during mitosis, HeLa cells were metabolically labeled with 32Porthophosphate during treatment with nocodazole as described under Plk was from cell to and to and into Plk was detected by 3 In the proteins from the and the cell were by Western blot with anti-Plk antibody and of endogenous Plk from cells was detected in this migrating that this to Plk phosphorylated during in HeLa cells. To in vivo and in on the the of Plk labeled in vivo was with that of Plk phosphorylated in vitro. The labeled protein from the 3 vitro labeled Plk and 3 1 for in vivo labeled was and to Plk labeled in major were these were in the as on the of Plk phosphorylated in vitro 3 B, acid analyses on that the major 1 and The 3 which are with the major in also 3 that threonine may be phosphorylated in vitro vivo. other 4 also whereas the 3 and not which is with the phosphoamino acid are several conserved serine and threonine residues in the of (20Lee K.S. Erikson R.L. Mol. Cell. Biol. 1997; 17: 3408-3417Crossref PubMed Scopus (164) Google Scholar, Y.-W. Erikson E. Maller J.L. Mol. Cell. Biol. PubMed Google Scholar). The of Thr-210 with Asp elevates the kinase activity of Plk in Sf9 cells and in (20Lee K.S. Erikson R.L. Mol. Cell. Biol. 1997; 17: 3408-3417Crossref PubMed Scopus (164) Google Scholar). In Xenopus, to Thr-210 in mammalian is for activation of Plx1 Y.-W. Erikson E. Maller J.L. Mol. Cell. Biol. PubMed Google Scholar). These results however, provide direct evidence that Thr-210 is a phosphorylation site in mammalian Plk. To the residue in Plk vitro and in vivo is Thr-210, we generated a mutant in which Thr-210 is with Asp in the phosphorylation in vitro with xPlkk1, the of was with that of the All which were detected in the of mutant and were in the of the mutant 4 that xPlkk1 phosphorylates We that the result from which is as the of the is not as as 1 and The of in which Ser-137 was with the in the of the mutant 4 3 and This although the surrounding Ser-137 and Thr-210 are this residue is not phosphorylated in vivo or in vitroby The that Thr-210 were on the of the mutant and at a To of Thr-210 or Ser-137 the activity of Plk, we and the kinase of these expressed in and from HeLa cells, were Thr-210 or Ser-137 was with Asp or Plk activity 4 and In contrast, of Thr-210 to its activity of Ser-137 to not its activity significantly from that of wild type Plk Plk, in which both Ser-137 and Thr-210 were with Asp was not more Plk with a that substitution of both Ser-137 and Thr-210 with Asp does not have an additive effect on the activity of Plk. This result is in with that with Plx1, in which the are additive Y.-W. Erikson E. Maller J.L. Mol. Cell. Biol. PubMed Google Scholar). We transfected the various mutant constructs into HeLa cells for h and both the by and the by as described under to the The G2/M population of HeLa cells transfected with wild type and Thr-210 mutant constructs slightly and The cells expressing the mutant a similar as the wild type and Thr-210 which in M-phase and in with a previous K.E. Golsteyn R.M. Lane H.A. Nigg E.A. Biochem. Biophys. Res. Comm. 1997; 239: 377-385Crossref PubMed Scopus (171) Google Scholar). This that expression of Plk does not the G2/M transition does mitotic progression. However, the S137D which has activity similar to that of was more of the transfected cells G1/S and B, 4 and and and The double in of a G1/S population as well that the block by S137D is not by Cells were transfected with the to as shown by with the and which as slightly separated To Plk T210D accelerates into HeLa cells were transfected with wild or T210D constructs for protein expression and at G1/S 12 h with for an 20 h, the cells were released from the mimosine and the activity of Cdc2-cyclin B was in as a The and of Cdc2-cyclin B activity were not as the result of Plk and cells expressing construct to G2/M be that 60% of the cells is transfected in this type of and that Cdc2-cyclin B activity is on the In experiments Xenopus egg the addition of which is to Plk accelerates Cdc2-cyclin B activation by as with wild type Plx1 or endogenous enzyme Y.-W. Erikson E. Maller J.L. Mol. Cell. Biol. PubMed Google Scholar). The Xenopus extracts are as with transfected cells, and the in a high of in an cell population it to in activation. In this study, we have demonstrated that Thr-210 is the vivo phosphorylation site of activated mammalian Plk during M Plk labeled with during M phase major B, both of which and several The major with in of Plk with In contrast, these are in of Plk double mutant other are generated by xPlkk1 under these in vitro are in endogenous Plk in vivo labeling with We that are not for activation of Plk in vivo. Thr-210 does not appear to be a major in vitro in this serine are However, activated M phase Plk that has been metabolically labeled does not major that is not a significant during activation of Plk during M Ser-137 in mammalian Plk is by amino acids and by a residue similar to the Thr-210 site of the residue to aspartate in Plx1 activates the enzyme Y.-W. Erikson E. Maller J.L. Mol. Cell. Biol. PubMed Google Scholar), that it may be a physiologically relevant phosphorylation site. of Ser-137 to in mammalian Plk also results in kinase activity and however, in to Plx1, Plk with both Ser-137 and Thr-210 to aspartate is not further activated Y.-W. Erikson E. Maller J.L. Mol. Cell. Biol. PubMed Google Scholar). we that Ser-137 is not phosphorylated in vitro by xPlkk1 is it phosphorylated in M phase cells. We have not however, or not Ser-137 is phosphorylated at in the cell cycle to M We have also used LOK, the mammalian enzyme closely related xPlkk1, to and Plk in vitro with the results shown for studies to however, not LOK as the upstream activating enzyme of Plk in To of Ser-137 has the potential to influence the cell S137D Plk was expressed in HeLa cells, and its influence on the cell cycle was shown in B, cells expressing S137D the novel and unexpected a G1 in to all other Plk in which expression in a high of cells with a G2 of cells expressing Plk S137D that and well with in the to centrosomes, which were separated slightly and were similar to of cells arrested in late G1 phase by treatment with mimosine not These data suggest that Ser-137 to M phase may be of in the cell cycle and that polo-like kinase kinase may be at that of the of synchronized cell it has not yet been to Plk is phosphorylated on Ser-137 at other in the cell cycle. expression of all other Plk including wild appear to result in cells in or We this is to be the result of the of the of Plk to as a which this the of the kinase S. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, K. E. Kuriyama R. T. K.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, HeLa cells are at G1/S with mimosine expression of Plk1 does not appear to the of Cdc2-cyclin B activation. This result is with the that Cdc2-cyclin B is activated in cells of Plk with small Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This direct evidence that Thr-210 is a site for an activating phosphorylation in vitro and the major phosphorylation site in vivo as In addition, we that Ser-137 is not phosphorylated in M The that Plk S137D cell cycle in a fashion for the of the polo-like kinase this was under M. Nigg E.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) studies on the phosphorylation of that phosphorylation of is an activating in in with the results however, that although xPlkk1 does Plx1, this phosphorylation does not Plx1 and does not on The for these are We Erikson for and critical of this
Jang et al. (Fri,) studied this question.