From the Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8503, Japan
Received for publication, March 20, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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The activity of MPF is regulated by phosphorylation and dephosphorylation of Cdc2 and accumulation of cyclin B protein (24, 25). Until the end of G2 phase, Cdc2, in higher eukaryotes, remains inactive through inhibitory phosphorylation on Thr-14 and Tyr-15. At M phase entry, the Cdc25 phosphatase dephosphorylates Thr-14 and Tyr-15, thereby activating MPF (2628). Wee1 and Myt1 are responsible for such inhibitory phosphorylation of Cdc2. Wee1, a nuclear protein, is capable of phosphorylating Tyr-15 of Cdc2, but not Thr-14 (3033). Myt1 is a membrane-associated, dual-specific protein kinase that phosphorylates both Thr-14 and Tyr-15 of Cdc2 (3437). Myt1 is shown to be hyperphosphorylated during M phase, which is coincident with its inactivation (35, 37). It is reported that p90rsk and Akt can phosphorylate and down-regulate Myt1 during miosis in Xenopus and Asterina oocytes, respectively (38, 39). However, a kinase(s) responsible for the regulation of Myt1 in the somatic cell cycle has been unknown.
In this study, we have identified a consensus sequence for Plk1 phosphorylation and found Myt1 a Plk1 substrate.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and SynchronizationHeLa cells were cultured in Dul-becco's modified Eagle's medium with 10% bovine calf serum. Cells were synchronized with a double-thymidine block. Exponentially growing cells were arrested in S phase by treatment with thymidine (2 mM) for 17 h and were released from the arrest by washing twice with fresh medium. Cells were grown in fresh medium for 9 h and then re-treated with thymidine (2 mM) for 15 h.
ImmunoprecipitationCells were lysed in buffer A (20 mM Hepes (pH 7.4), 25 mM 2-glycerophosphate, 150 mM NaCl, 1.5 mM MgCl2,2mM EGTA, 0.5% Triton X-100, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 2 µg/ml aprotinin, and 1 µM okadaic acid) and centrifuged at 20,000 x g for 15 min. Endogenous Plk1 was immunoprecipitated with anti-Plk1 antibody (Zymed Laboratories Inc.) coupled to protein A-Sepharose (Pharmacia Corp.). HA-tagged Plk1 was immunoprecipitated with anti-HA antibody (Santa Cruz). The immunoprecipitates were further washed with buffer A and subjected to kinase assays as described below.
Kinase AssaysIn the kinase assay for His-tagged Plk1 or
immunoprecipitates, 0.51 µg of His-tagged Plk1 or immunoprecipitates
were mixed with substrate (0.13 µg), 50 µM ATP, and 15
mM MgCl2 in a final volume of 15 µl and incubated for
20 min at 30 °C in the presence of 3 µCi of
[-32P]ATP. The reactions were stopped by addition of
Laemmli's sample buffer and boiling. Histone H1 kinase assay was conducted as
described previously (40).
siRNARNA oligonucleotides (21 nucleotides) homologous to human Plk1 were designed as described previously (15). Annealed siRNAs were transfected by the use of Oligofectamine (Invitrogen).
Transfection and ImmunoblottingHeLa cells were transiently transfected by the use of FuGENE6 according to the manufacturer's instructions. To arrest cells, cells were treated with 250 ng/ml nocodazole or 2 mM thymidine for 18 h at 20 h after transfection. Cells were lysed in buffer B (50 mM Tris (pH 8.0), 100 mM NaCl, 5 mM EDTA, and 0.5% Nonidet P-40, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 2 µg/ml aprotinin) (41) and centrifuged at 20,000 x g for 15 min. The cell extracts were subjected to immunoblotting with anti-Myc (Santa Cruz) or anti-HA antibody (Santa Cruz).
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RESULTS AND DISCUSSION |
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First, we replaced each amino acid surrounding Ser-198 by Ala or Gly. The obtained results showed that the ability of the peptides to serve as a substrate for Plk1 was markedly reduced when Glu-196, Leu-199, or Asp-201 was replaced by Ala or Gly (Fig. 1A). When Lys-200 was mutated, the resultant peptides were phosphorylated more efficiently (Fig. 1A). These results suggest that amino acid residues at positions 2 to +3 of the phosphorylated residue (Ser-198) are primarily important for phosphorylation by Plk1.
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When we performed a single amino acid exchange at Glu-196, a peptide with Asp-196 was phosphorylated as efficiently as the original peptide, whereas other peptides with Leu-, Gln-, Lys-, Ala-, or Gly-196 were poorly phosphorylated (Fig. 1B). This suggests that an acidic amino acid at position 2 is important for optimal phosphorylation. Replacement of Phe-197 by Ala, Glu, Leu, Arg, or Lys did not affect significantly the efficiency of phosphorylation, while replacement by Gly reduced the efficiency of phosphorylation (Fig. 1C). Thus, Gly at position 1 is inhibitory for the phosphorylation. Replacement of Leu-199 by a hydrophobic amino acid such as Val, Ile, Phe, Trp, or Met did not decrease, or rather increase, the phosphorylation, whereas replacement by Pro, Arg, Glu, Gln, Ala, Gly, or Lys significantly decreased the phosphorylation (Fig. 1D). This indicates the significance of a hydrophobic amino acid at position +1. Replacement of Lys-200 by Ala or Glu significantly increased the phosphorylation, while replacement by Arg or Gly did not significantly affect the phosphorylation efficiency (Fig. 1E), suggesting that a basic amino acid at position +2 is slightly inhibitory. Replacement of Asp-201 by Glu did not affect the phosphorylation, while replacement by Ala or Gly decreased the phosphorylation (Fig. 1F). Thus, an acidic amino acid at position +3 is important for optimal phosphorylation. When the target Ser-198 was mutated into Thr, the efficiency of phosphorylation did not change markedly (Fig. 1G), suggesting that Thr as well as Ser is able to be phosphorylated by Plk1.
To examine the importance of an acidic amino acid at position 2 in
more detail, we made four new peptide sequences in which residues
195197 were Ala-Ala-Ala, Glu-Ala-Ala, Ala-Glu-Ala, or Ala-Ala-Glu as
shown in an upper panel of Fig.
2A. Only the sequence Ala-Glu-Ala was phosphorylated
efficiently (Fig. 2A,
lower). Therefore, an acidic amino acid should locate at position
2 for optimal phosphorylation. To examine the importance of a
hydrophobic amino acid at position +1, we made four new peptide sequences in
which residues 199201 were Ala-Ala-Ala, Leu-Ala-Ala, Ala-Leu-Ala, or
Ala-Ala-Leu as shown in an upper panel of
Fig. 2B. Only the
sequence Leu-Ala-Ala was phosphorylated efficiently
(Fig. 2B,
lower), showing that a hydrophobic amino acid should locate at
position +1 for optimal phosphorylation. Finally, to examine the importance of
an acidic amino acid at position +3, we made four new peptide sequences in
which residues 199202 were Leu-Ala-Ala-Ala, Leu-Asp-Ala-Ala,
Leu-Ala-Asp-Ala, or Leu-Ala-Ala-Asp as shown in an upper panel of
Fig. 2C. The sequence
Leu-Ala-Asp-Ala was phosphorylated most efficiently among these peptides
(Fig. 2C,
lower). Thus, when an acidic amino acid locates at position +3, the
sequence is optimal for phosphorylation by Plk1. All these results taken
together suggest a sequence
D/E-X-S/T--X-D/E (X,
any amino acid;
, a hydrophobic amino acid) as an optimal phosphorylation
sequence by Plk1 (Fig.
2D).
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Phosphorylation of Myt1 by Plk1 in VitroWe noted that Myt1, a negative regulator of MPF (cdc2/cyclin B), has multiple putative phosphorylation sites for Plk1 on its C-terminal region (Fig. 3A). Because Myt1 was reported to be highly phosphorylated during M phase (35, 37), we hypothesized that Myt1 could be a substrate of Plk1. We fused an NH2-terminal region (residues 1377) or a COOH-terminal region (residues 401499) of Myt1 to GST and used these bacterially produced fusion proteins. As shown in Fig. 3B, recombinant His-Plk1 efficiently phosphorylated the COOH-terminal region of Myt1, but not the NH2-terminal region. Phosphorylation seen in lanes 1 and 2 results from autophosphorylation of the GST-Myt1(N), as a kinase-dead form of GST-Myt1(N), GST-Myt1(N,KD), did not undergo phosphorylation upon incubation with His-Plk1 (lanes 3 and 4). There are four possible phosphorylation sites (Ser-426, Ser-435, Ser-469, and Thr-495) for Plk1 in the COOH-terminal region of Myt1 (see Fig. 3A). When a mutant form of Myt1 (GST-Myt1(C) 4A), in which the four putative phosphorylation sites were replaced by Ala, was tested for phosphorylation by Plk1, it was not phosphorylated by Plk1 (Fig. 3C). HA-tagged Plk1, which was expressed in HeLa cells and purified by immunoprecipitation with anti-HA antibody, was also able to phosphorylate GST-Myt1(C) WT, but not GST-Myt1(C) 4A (Fig. 3D). A kinase-dead form of Plk1 (HA-Plk1 KD) did not phosphorylate GST-Myt1(C) WT at all (Fig. 3D). Thus, Plk1 phosphorylates Myt1 in vitro on several or all of these four residues.
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Phosphorylation of Myt1 during G2-M Phase in HeLa Cells Endogenous Plk1 was immunoprecipitated from synchronized HeLa cells with anti-Plk1 antibody and tested for the ability to phosphorylate GST-Myt1(C). Phosphorylation of Myt1(C) WT by immunoprecipitated Plk1 was increased during G2-M phase, i.e. 911 h after release from a double thymidine block (Fig. 4A). GST-Myt1(C) 4A was not phosphorylated at all (Fig. 4A). Thus, Plk1 in G2-M phase is able to phosphorylate Myt1.
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To examine whether Plk1 is able to phosphorylate Myt1 in intact cells, we co-expressed Myc-tagged full-length Myt1 with HA-tagged Plk1. We performed immnunoblotting analysis, as Myt1 is shown to display a mobility shift upon phosphorylation (35, 37). A mobility-shifted band of Myt1 was detected when wild-type Plk1 was co-expressed (Fig. 4B, Myc-Myt1 WT/HA-Plk1 WT). When kinase-dead Plk1 was co-expressed, the mobility shift of Myt1 did not occur (Fig. 4B, HA-Plk1 KD). Importantly, the mobility shift of Myt1 was not observed when a mutant form of Myt1, Myt1 4A, was expressed (Fig. 4B, Myc-Myt1 4A).
Previous reports have shown that Myt1 is phosphorylated during G2-M and displays mobility shifts (35, 37, 39). To test whether or not phosphorylation of Myt1 on the putative Plk1 phosphorylation sites occurs during G2-M phase, we expressed Myc-tagged Myt1 WT or Myt1 4A in HeLa cells and treated the cells with thymidine or nocodazole to arrest cells in S phase or M phase, respectively. Immunoblotting showed that in M phase-arrested cells, but not in S phase-arrested cells, Myt1 WT displayed mobility-shifted bands, whereas Myt1 4A did not (Fig. 4C). When Plk1 was specifically depleted in cells by siR-NAs, the mobility-shifted bands of Myc-Myt1 WT seen in M phase-arrested cells were markedly reduced (Fig. 4D). These results suggest that part of the phosphorylation of Myt1 during G2-M phase is mediated by Plk1.
Plk1 Phosphorylation Sites of Myt1To examine on which sites Myt1 is phosphorylated by Plk1, we constructed several forms of Myt1, in which one, two, or three of the four putative Plk1 sites was replaced by alanines, and co-expressed them with Plk1. Immunoblotting showed that Myt1 4A, Myt1 426A-469A-495A, or Myt1 426A-495A did not display a markedly shifted band, whereas Myt1 495A displayed the shifted band significantly and Myt1 426A slightly when co-expressed with wild type Plk1 (Fig. 5A). These results suggest that Ser-426 is a major phosphorylation site by Plk1, and Thr-495 is a second major site. When HeLa cells expressing these constructs were arrested in M phase by nocodazole treatment, Myt1 4A, Myt1 426A-469A-495A, Myt1 426A-495A, or Myt1 426A did not display markedly shifted band, whereas Myt1 495A displayed the shifted band to almost the same extent as Myt1 WT did (Fig. 5B), suggesting that phosphorylation of Myt1 by Plk1 on Ser-426 occurs during M phase. We then tested the ability of these mutant forms of Myt1 (GST fusion proteins), to serve as a substrate for Plk1 in vitro. Quantification of the result (Fig. 5C) showed that Ser-426 is the major phosphorylation site by Plk1 in vitro and Thr-495 the second major site. This conclusion is identical to that obtained from the co-expression experiment (Fig. 5A).
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In this study, we have identified a consensus motif for Plk1 phosphorylation. Our results show that a hydrophobic amino acid at position +1 and an acidic amino acid at position 2 are important for optimal phosphorylation. The reported autophosphorylation sites on Plx1 (a Xenopus homolog of Plk1) (42) and the identified phosphorylation site of Scc1 by Cdc5 (a yeast homolog of Plk1) (12) match this optimal motif. This consensus sequence can be used for identification of novel targets for Plk1.
A previous report showed that inhibition of Plx1 by the injection of anti-Plx1 antibody impaired the mobility shift of Myt1 in the system of Xenopus cycling extracts (6), suggesting that the activity of Plx1 is required for phosphorylation of Myt1 in this system. Here, we have shown that Plk1 is responsible for part of the phosphorylation of Myt1 during M phase. The kinase activity of human Myt1 is reported to be decreased during M phase, and the decreased activity correlates with hyperphosphorylated forms of Myt1 (35, 37). Previously, Myt1 was shown to be phosphorylated by Cdc2, but this phosphorylation did not decrease the kinase activity of Myt1 (37). Most recently, p90rsk and Akt are reported to phosphorylate and down-regulate Myt1 at the onset of meiosis in Xenopus and Asterina oocytes, respectively (38, 39). However, a kinase(s) responsible for the regulation of Myt1 during M phase in somatic cell cycles has not been fully identified. Functional consequences resulting from Plk1-mediated phosphorylation of Myt1 should be elucidated in the future studies.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Cell and Developmental
Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto
606-8502, Japan. Tel.: 81-75-753-4230; Fax: 81-75-753-4235; E-mail:
L50174{at}sakura.kudpc.kyoto-u.ac.jp.
1 The abbreviations used are: Plk1, Polo-like kinase 1; MPF, M-phase
promoting factor; Myt1, membrane-associated tyrosine-and threonine-specific
cdc2-inhibitory kinase; GST, glutathione S-transferase; HA,
hemagglutinin; siRNA, small interfering RNA; WT, wild-type; KD,
kinase-dead.
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ACKNOWLEDGMENTS |
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REFERENCES |
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