Identification of a Consensus Motif for Plk (Polo-like Kinase) Phosphorylation Reveals Myt1 as a Plk1 Substrate*

Plk1 (Polo-like kinase 1), an evolutionarily conserved serine/threonine kinase, is crucially involved in multiple events during the M phase. Here we have identified a consensus phosphorylation sequence for Plk1, by testing the ability of systematically mutated peptides derived from human Cdc25C to serve as a substrate for Plk1. The obtained results show that a hydrophobic amino acid at position +1 carboxyl-terminal of phosphorylated Ser/Thr and an acidic amino acid at position –2 are important for optimal phosphorylation by Plk1. We have then found that Myt1, an inhibitory kinase for MPF, has a number of putative phosphorylation sites for Plk1 in its COOH-terminal portion. While wild-type Myt1 (Myt1-WT) served as a good substrate for Plk1 in vitro, a mutant Myt1 (Myt1-4A), in which the four putative phosphorylation sites are replaced by alanines, did not. In nocodazole-treated cells, Myt1-WT, but not Myt1-4A, displayed its mobility shift in gel electrophoresis, due to phosphorylation. These results suggest that Plk1 phosphorylates Myt1 during M phase. Thus, this study identifies a novel substrate for Plk1 by determining a consensus phosphorylation sequence by Plk1. Plk1 (Polo-like kinase 1), an evolutionarily conserved serine/threonine kinase, is crucially involved in multiple events during the M phase. Here we have identified a consensus phosphorylation sequence for Plk1, by testing the ability of systematically mutated peptides derived from human Cdc25C to serve as a substrate for Plk1. The obtained results show that a hydrophobic amino acid at position +1 carboxyl-terminal of phosphorylated Ser/Thr and an acidic amino acid at position –2 are important for optimal phosphorylation by Plk1. We have then found that Myt1, an inhibitory kinase for MPF, has a number of putative phosphorylation sites for Plk1 in its COOH-terminal portion. While wild-type Myt1 (Myt1-WT) served as a good substrate for Plk1 in vitro, a mutant Myt1 (Myt1-4A), in which the four putative phosphorylation sites are replaced by alanines, did not. In nocodazole-treated cells, Myt1-WT, but not Myt1-4A, displayed its mobility shift in gel electrophoresis, due to phosphorylation. These results suggest that Plk1 phosphorylates Myt1 during M phase. Thus, this study identifies a novel substrate for Plk1 by determining a consensus phosphorylation sequence by Plk1. Plk1 1The 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.1The 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. (Polo-like kinase 1), a mammalian ortholog of Drosophila Polo, is an evolutionarily conserved serine/threonine kinase involved in multiple events during M phase (1Nigg E.A. Curr. Opin. Cell Biol. 1998; 10: 776-783Crossref PubMed Scopus (308) Google Scholar, 2Glover D.M. Hagan I.M. Travares A.A. Genes Dev. 1998; 12: 3777-3787Crossref PubMed Scopus (394) Google Scholar, 3Donaldson M.M. Tavares A.A. Hagan I.M. Nigg E.A. Glover D.M. 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A. 2001; 98: 9128-9132Crossref PubMed Scopus (103) Google Scholar). The activity of Plk1 peaks at M phase and is regulated by phosphorylation and cell cycle-specific changes in its abundance (19Hamanaka 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, 20Golsteyn R.M. Mundt K.E. Fry A.M. Nigg E.A. J. Cell Biol. 1995; 129: 1617-1628Crossref PubMed Scopus (394) Google Scholar, 21Qian Y.W. Erikson E. Maller J.L. Science. 1998; 282: 1701-1704Crossref PubMed Scopus (105) Google Scholar). This kinase undergoes remarkable intracellular redistribution through cell cycle (20Golsteyn R.M. Mundt K.E. Fry A.M. Nigg E.A. J. Cell Biol. 1995; 129: 1617-1628Crossref PubMed Scopus (394) Google Scholar, 22Golsteyn R.M Schultz S.J. Bartek J. Ziemiecki A. Ried T. Nigg E.A. J. Cell Sci. 1994; 107: 1509-1517Crossref PubMed Google Scholar, 23Arnaud L. Pines J. Nigg E.A. Chromosoma (Berl.). 1998; 10: 424-429Crossref Scopus (119) Google Scholar). Such multiple roles and dynamic behaviors of Plk1 suggest that Plk1 phosphorylates a number of substrates during M phase. A consensus sequence for Plk1 phosphorylation has not been determined, although several substrates have been identified (1Nigg E.A. Curr. Opin. Cell Biol. 1998; 10: 776-783Crossref PubMed Scopus (308) Google Scholar, 2Glover D.M. Hagan I.M. Travares A.A. Genes Dev. 1998; 12: 3777-3787Crossref PubMed Scopus (394) Google Scholar, 3Donaldson M.M. Tavares A.A. Hagan I.M. Nigg E.A. Glover D.M. J. Cell Sci. 2001; 114: 2357-2358Crossref PubMed Google Scholar, 7Toyoshima-Morimoto F. Taniguchi E. Shinya N. Iwamatsu A. Nishida E. Nature. 2001; 410: 215-220Crossref PubMed Scopus (311) Google Scholar, 9Toyoshima-Morimoto F. Taniguchi E. Nishida E. EMBO Rep. 2002; 3: 341-348Crossref PubMed Scopus (270) Google Scholar, 12Alexandru G. Uhlmann F. Mechtler K. Poupart M.A. Nasmyth K. Cell. 2001; 105: 459-472Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 17Lin C.Y. Madsen M.L. Yarm F.R. Jang Y.J. Liu X. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12589-12594Crossref PubMed Scopus (109) Google Scholar). The activity of MPF is regulated by phosphorylation and dephosphorylation of Cdc2 and accumulation of cyclin B protein (24Nurse P. Nature. 1990; 344: 503-508Crossref PubMed Scopus (2220) Google Scholar, 25Hunt T. Semin. Cell Biol. 1991; 2: 213-222PubMed Google Scholar). 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 (26Coleman T.R. Dunphy W.G. Curr. Opin. Cell Biol. 1994; 6: 877-882Crossref PubMed Scopus (326) Google Scholar, 27Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2925) Google Scholar, 28Nigg E.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 21-32Crossref PubMed Scopus (1244) Google Scholar). 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 (30McGowan C.H. Russell P. EMBO J. 1993; 12: 75-85Crossref PubMed Scopus (384) Google Scholar, 31Heald R. McLoughlin M. McKeon F. Cell. 1993; 74: 463-474Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 32Watanabe N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (356) Google Scholar, 33Baldin V. Ducommun B. J. Cell Sci. 1995; 108: 2425-2432PubMed Google Scholar). Myt1 is a membrane-associated, dual-specific protein kinase that phosphorylates both Thr-14 and Tyr-15 of Cdc2 (34Kornbluth S. Sebastian B. Hunter T. Newport J. Mol. Biol. Cell. 1994; 5: 273-282Crossref PubMed Scopus (100) Google Scholar, 35Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (535) Google Scholar, 36Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (264) Google Scholar, 37Booher R.N. Holman P.S. Fattaey A. J. Biol. Chem. 1997; 272: 22300-22306Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Myt1 is shown to be hyperphosphorylated during M phase, which is coincident with its inactivation (35Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (535) Google Scholar, 37Booher R.N. Holman P.S. Fattaey A. J. Biol. Chem. 1997; 272: 22300-22306Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). It is reported that p90rsk and Akt can phosphorylate and down-regulate Myt1 during miosis in Xenopus and Asterina oocytes, respectively (38Palmer A. Gavin A.C. Nebreda A.R. EMBO J. 1998; 17: 5037-5047Crossref PubMed Scopus (288) Google Scholar, 39Okumura E. Fukuhara T. Yoshida H. Hanada S. Kozutsumi R. Mori M. Tachibana K. Kishimoto T. Nat. Cell Biol. 2002; 4: 111-116Crossref PubMed Scopus (175) Google Scholar). 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. Mutagenesis and Protein Preparation—Mutagenesis was performed by using the QuikChange site-directed mutagenasis kit (Stratagene). Truncated GST-Myt1 and GST-Cdc25C-(173–206) proteins were prepared by using pGEX6P1 (Pharmacia Corp.), expressed in Escherichia coli, and purified by glutathione-Sepharose 4B (Pharmacia Corp.). His-Plk1 was prepared as described previously (40Toyoshima F. Moriguchi T. Wada A. Fukuda M. Nishida E. EMBO J. 1998; 17: 2728-2735Crossref PubMed Scopus (279) Google Scholar). Cell Culture and Synchronization—HeLa 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. Immunoprecipitation—Cells 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 × 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 Assays—In the kinase assay for His-tagged Plk1 or immunoprecipitates, 0.5–1 μg of His-tagged Plk1 or immunoprecipitates were mixed with substrate (0.1–3 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 γ-32PATP. The reactions were stopped by addition of Laemmli's sample buffer and boiling. Histone H1 kinase assay was conducted as described previously (40Toyoshima F. Moriguchi T. Wada A. Fukuda M. Nishida E. EMBO J. 1998; 17: 2728-2735Crossref PubMed Scopus (279) Google Scholar). siRNA—RNA oligonucleotides (21 nucleotides) homologous to human Plk1 were designed as described previously (15Liu X. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8672-8676Crossref PubMed Scopus (183) Google Scholar). Annealed siRNAs were transfected by the use of Oligofectamine (Invitrogen). Transfection and Immunoblotting—HeLa 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) (41Liu F. Rothblum-Oviatt C. Ryan C.E. Piwnica-Worms H. Mol. Cell. Biol. 1999; 19: 5113-5123Crossref PubMed Scopus (98) Google Scholar) and centrifuged at 20,000 × g for 15 min. The cell extracts were subjected to immunoblotting with anti-Myc (Santa Cruz) or anti-HA antibody (Santa Cruz). Identification of a Consensus Motif for Plk1 Phosphorylation—We tested the ability of systematically mutated peptides derived from human Cdc25C to serve as a substrate for Plk1 in vitro. We have recently shown that human Cdc25C is phosphorylated on Ser-198 by Plk1 (9Toyoshima-Morimoto F. Taniguchi E. Nishida E. EMBO Rep. 2002; 3: 341-348Crossref PubMed Scopus (270) Google Scholar). Then, we used GST-fused Cdc25C peptides (residues 173–206) as test substrates. We constructed various GST-fused Cdc25C peptides (173–206), in which amino acids in residues 192–203 are mutated, to examine which amino acids surrounding Ser198 are important for the phosphorylation by Plk1. 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. 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 195–197 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 199–201 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 was phosphorylated efficiently (Fig. that a hydrophobic amino acid should locate at position +1 for optimal phosphorylation. to examine the importance of an acidic amino acid at position we made four new peptide sequences in which residues were or as shown in an upper panel of Fig. The sequence was phosphorylated efficiently peptides (Fig. lower). Thus, when an acidic amino acid at position the sequence is optimal for phosphorylation by Plk1. results suggest a sequence amino a hydrophobic amino acid) as an optimal phosphorylation sequence by Plk1 (Fig. of Myt1 by Plk1 in that Myt1, a of MPF has multiple putative phosphorylation sites for Plk1 on its (Fig. Myt1 was reported to be phosphorylated during M phase (35Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (535) Google Scholar, 37Booher R.N. Holman P.S. Fattaey A. J. Biol. Chem. 1997; 272: 22300-22306Abstract Full Text Full Text PDF PubMed Scopus (223) Google we that Myt1 be a substrate of Plk1. We an (residues or a COOH-terminal (residues of Myt1 to and used shown in Fig. His-Plk1 efficiently phosphorylated the COOH-terminal of Myt1, but not the in 1 and 2 results from of the as a of did not phosphorylation with His-Plk1 3 and are four phosphorylation sites and for Plk1 in the COOH-terminal of Myt1 Fig. When a mutant of Myt1 in which the four putative phosphorylation sites were replaced by Ala, was tested for phosphorylation by Plk1, was not phosphorylated by Plk1 (Fig. HA-tagged Plk1, which was expressed in cells and purified by with anti-HA was able to phosphorylate WT, but not (Fig. A of Plk1 did not phosphorylate at (Fig. Thus, Plk1 phosphorylates Myt1 in on several or of four of Myt1 during 2 in Endogenous Plk1 was immunoprecipitated from synchronized cells with anti-Plk1 antibody and tested for the ability to phosphorylate of by immunoprecipitated Plk1 was increased during phase, h after from a thymidine (Fig. was not phosphorylated at (Fig. Thus, Plk1 in phase is able to phosphorylate To examine Plk1 is able to phosphorylate Myt1 in cells, we Myt1 with HA-tagged Plk1. We performed as Myt1 is shown to a mobility shift phosphorylation (35Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (535) Google Scholar, 37Booher R.N. Holman P.S. Fattaey A. J. Biol. Chem. 1997; 272: 22300-22306Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). A of Myt1 was when wild-type Plk1 was (Fig. When Plk1 was the mobility shift of Myt1 did not (Fig. the mobility shift of Myt1 was not when a mutant of Myt1, Myt1 was expressed (Fig. have shown that Myt1 is phosphorylated during and mobility (35Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (535) Google Scholar, 37Booher R.N. Holman P.S. Fattaey A. J. Biol. Chem. 1997; 272: 22300-22306Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 39Okumura E. Fukuhara T. Yoshida H. Hanada S. Kozutsumi R. Mori M. Tachibana K. Kishimoto T. Nat. Cell Biol. 2002; 4: 111-116Crossref PubMed Scopus (175) Google Scholar). To test or not phosphorylation of Myt1 on the putative Plk1 phosphorylation sites during phase, we expressed Myt1 or Myt1 in cells and treated the cells with thymidine or nocodazole to arrest cells in S phase or M phase, showed that in M cells, but not in S cells, Myt1 displayed whereas Myt1 did not (Fig. When Plk1 was in cells by the of in M cells were markedly reduced (Fig. These results suggest that of the phosphorylation of Myt1 during phase is by Plk1. Plk1 of examine on which sites Myt1 is phosphorylated by Plk1, we constructed several of Myt1, in which or of the four putative Plk1 sites was replaced by alanines, and with Plk1. showed that Myt1 Myt1 or Myt1 did not a markedly whereas Myt1 displayed the significantly and Myt1 slightly when with Plk1 (Fig. These results suggest that is a phosphorylation by Plk1, and is a When cells were arrested in M phase by nocodazole Myt1 Myt1 Myt1 or Myt1 did not markedly whereas Myt1 displayed the to the as Myt1 did (Fig. suggesting that phosphorylation of Myt1 by Plk1 on during M phase. We then tested the ability of mutant of Myt1 to serve as a substrate for Plk1 in vitro. of the (Fig. showed that is the phosphorylation by Plk1 in and the This is to that obtained from the (Fig. In this study, we have identified a consensus for Plk1 phosphorylation. 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 sites on Xenopus of M. Nigg E.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and the identified phosphorylation of by of G. Uhlmann F. Mechtler K. Poupart M.A. Nasmyth K. Cell. 2001; 105: 459-472Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) this optimal This consensus sequence can be used for of novel for Plk1. A showed that of by the of antibody the mobility shift of Myt1 in the of Xenopus extracts A. Brassac T. Galas S. Fisher D. Labbe J.C. Doree M. J. Cell Sci. 1998; 111: 1751-1757Crossref PubMed Google suggesting that the activity of is for phosphorylation of Myt1 in this we have shown that Plk1 is responsible for 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 with hyperphosphorylated of Myt1 (35Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (535) Google Scholar, 37Booher R.N. Holman P.S. Fattaey A. J. Biol. Chem. 1997; 272: 22300-22306Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Myt1 was shown to be phosphorylated by Cdc2, but this phosphorylation did not the kinase activity of Myt1 R.N. Holman P.S. Fattaey A. J. Biol. Chem. 1997; 272: 22300-22306Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). p90rsk and Akt are reported to phosphorylate and down-regulate Myt1 at the of in Xenopus and Asterina oocytes, respectively (38Palmer A. Gavin A.C. Nebreda A.R. EMBO J. 1998; 17: 5037-5047Crossref PubMed Scopus (288) Google Scholar, 39Okumura E. Fukuhara T. Yoshida H. Hanada S. Kozutsumi R. Mori M. Tachibana K. Kishimoto T. Nat. Cell Biol. 2002; 4: 111-116Crossref PubMed Scopus (175) Google Scholar). However, a kinase(s) responsible for the regulation of Myt1 during M phase in somatic cell has not been from phosphorylation of Myt1 should be in the We K. Todokoro for a Plk1 We T. and H. for and S. for