PKS1, a substrate phosphorylated by phytochrome that modulates light signaling in Arabidopsis

Science

Volumn 284, Issue 5419, 1999, Pages 1539-1541

  Fankhauser, Christian ^a   Yeh, Kuo Chen ^b   Lagarias, J Clark ^b   Zhang, Hong ^c   Elich, Tedd D ^a,e   Chory, Joanne ^a,d  

^a SALK INSTITUTE FOR BIOLOGICAL STUDIES   (United States)

^b UNIVERSITY OF CALIFORNIA   (United States)

^c TEXAS TECH UNIVERSITY   (United States)

^d HOWARD HUGHES MEDICAL INSTITUTE   (United States)

^e MONSANTO COMPANY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BINDING PROTEIN; PHYTOCHROME; PHYTOCHROME KINASE SUBSTRATE 1; PROTEIN SERINE THREONINE KINASE; UNCLASSIFIED DRUG;

AMINO ACID SEQUENCE; ARABIDOPSIS; ARTICLE; CONTROLLED STUDY; ENZYME SUBSTRATE; LIGHT ADAPTATION; NONHUMAN; NUCLEOTIDE SEQUENCE; PHYTOCHEMISTRY; PLANT GROWTH; PLANT SEED; PRIORITY JOURNAL; PROTEIN DETERMINATION; PROTEIN PHOSPHORYLATION;

AMINO ACID SEQUENCE; ARABIDOPSIS; ARABIDOPSIS PROTEINS; CARRIER PROTEINS; GENES, PLANT; INTRACELLULAR SIGNALING PEPTIDES AND PROTEINS; LIGHT; MOLECULAR SEQUENCE DATA; MUTATION; PHOSPHOPROTEINS; PHOSPHORYLATION; PHOTORECEPTORS; PHYTOCHROME; PHYTOCHROME A; PHYTOCHROME B; PLANT PROTEINS; PROTEIN KINASES; PROTEIN-SERINE-THREONINE KINASES; RECOMBINANT FUSION PROTEINS; SIGNAL TRANSDUCTION; TRANSCRIPTION FACTORS;

EID: 0033612143     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5419.1539     Document Type: Article

References (39)

1. H. Smith, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 289 (1995).
2. P. H. Quail, Plant Cell Environ. 20, 657 (1997).
3. K. Sakamoto and A. Nagatani, Plant J. 10, 859 (1996).
4. P. F. Devlin, S. R. Patel, G. C. Whitetam, Plant Cell 10, 1479 (1998); G. C. Whitelam and P. F. Devlin, Plant Cell Environ. 20, 752 (1997); M. J. Aukerman et al., Plant Cell 9, 1317 (1997).
5. P. F. Devlin, S. R. Patel, G. C. Whitetam, Plant Cell 10, 1479 (1998); G. C. Whitelam and P. F. Devlin, Plant Cell Environ. 20, 752 (1997); M. J. Aukerman et al., Plant Cell 9, 1317 (1997).
6. P. F. Devlin, S. R. Patel, G. C. Whitetam, Plant Cell 10, 1479 (1998); G. C. Whitelam and P. F. Devlin, Plant Cell Environ. 20, 752 (1997); M. J. Aukerman et al., Plant Cell 9, 1317 (1997).
7. K. C. Yeh, S. H. Wu, J. T. Murphy, J. C. Lagarias, Science 277, 1505 (1997).
8. H. A. W. Schneider-Poetsch, Photochem. Photobiol. 56, 839 (1992); T. D. Elich and J. Chory, Cell 91, 713 (1997).
9. H. A. W. Schneider-Poetsch, Photochem. Photobiol. 56, 839 (1992); T. D. Elich and J. Chory, Cell 91, 713 (1997).
10. K. C. Yeh and J. C. Lagarias, Proc. Natl. Acad. Sci. U.S.A. 95, 13976 (1998).
11. R. A. Harris et al., Adv. Enzyme Regul. 37, 271 (1997).
12. A bait coding for the last 160 amino acids of Arabidopsis PHYA was cloned into pEG202 and used to screen an Arabidopsis cDNA library [H. Zhang, J. Wang, H. M. Goodman, Biochim. Biophys. Acta 1353, 199 (1997)]. Positive clones were tested against a bicoid bait for specificity [J. Gyuris, E. Golemis, H. Chertkov, R. Brent, Cell 75, 791 (1993)] and against a bait corresponding to the last 175 amino acids of Arabidopsis PHYB.
13. A bait coding for the last 160 amino acids of Arabidopsis PHYA was cloned into pEG202 and used to screen an Arabidopsis cDNA library [H. Zhang, J. Wang, H. M. Goodman, Biochim. Biophys. Acta 1353, 199 (1997)]. Positive clones were tested against a bicoid bait for specificity [J. Gyuris, E. Golemis, H. Chertkov, R. Brent, Cell 75, 791 (1993)] and against a bait corresponding to the last 175 amino acids of Arabidopsis PHYB.
14. C. Fankhauser, K. C. Yeh, J. C. Lagarias, J. Chory, data not shown.
15. A full-length PKS1 clone was obtained from an Arabidopsis cDNA library [J. Kieber, M. Rothenberg, G. Roman, K. Feldmann, J. Ecker, Cell 72, 427 (1993)]. This cDNA was polymerase chain reaction-amplified with Bam HI adapters and cloned into pCMX-PL1. In vitro transcription/translation was performed with a Promega TnT kit. A PHYA-GST fusion protein was created by cloning the Arabidopsis PHYA cDNA with a Bam HI site at the 5′ end and a Not I site after the last amino acid into pDS472a [S. L. Forsburg and D. A. Sherman, Gene 191, 191 (1997)] and expressed in Schizosaccharomyces pombe as described [S. L. Forsburg, Nucleic Acids Res. 21, 2955 (1993)]. A PHYB-GST fusion protein was created by adding a Not I site after the last amino acid of PHYB in clone p41 and adding an in-frame GST cDNA with Not I and Xba I adapters. GST, PHYA-GST, or PHYB-GST (1 μg) were bound onto glutathione-agarose beads (G4510, Sigma). The beads were washed three times with extraction buffer (EB). [T. D. Elich and J. Chory, Plant Cell 9, 2271 (1997)]; in vitro transcribed and translated PKS1 (diluted 1:10 in EB) was added to the beads and incubated for 60 min at 4°C with gentle shaking. The beads were washed five times with EB; the bound proteins were eluted with EB containing 10 mM glutathione, then separated by SDS-polyacrylamide gel electrophoresis (PAGE) [U. K. Laemmli, Nature 227, 680 (1970)], stained, and exposed.
16. A full-length PKS1 clone was obtained from an Arabidopsis cDNA library [J. Kieber, M. Rothenberg, G. Roman, K. Feldmann, J. Ecker, Cell 72, 427 (1993)]. This cDNA was polymerase chain reaction-amplified with Bam HI adapters and cloned into pCMX-PL1. In vitro transcription/translation was performed with a Promega TnT kit. A PHYA-GST fusion protein was created by cloning the Arabidopsis PHYA cDNA with a Bam HI site at the 5′ end and a Not I site after the last amino acid into pDS472a [S. L. Forsburg and D. A. Sherman, Gene 191, 191 (1997)] and expressed in Schizosaccharomyces pombe as described [S. L. Forsburg, Nucleic Acids Res. 21, 2955 (1993)]. A PHYB-GST fusion protein was created by adding a Not I site after the last amino acid of PHYB in clone p41 and adding an in-frame GST cDNA with Not I and Xba I adapters. GST, PHYA-GST, or PHYB-GST (1 μg) were bound onto glutathione-agarose beads (G4510, Sigma). The beads were washed three times with extraction buffer (EB). [T. D. Elich and J. Chory, Plant Cell 9, 2271 (1997)]; in vitro transcribed and translated PKS1 (diluted 1:10 in EB) was added to the beads and incubated for 60 min at 4°C with gentle shaking. The beads were washed five times with EB; the bound proteins were eluted with EB containing 10 mM glutathione, then separated by SDS-polyacrylamide gel electrophoresis (PAGE) [U. K. Laemmli, Nature 227, 680 (1970)], stained, and exposed.
17. A full-length PKS1 clone was obtained from an Arabidopsis cDNA library [J. Kieber, M. Rothenberg, G. Roman, K. Feldmann, J. Ecker, Cell 72, 427 (1993)]. This cDNA was polymerase chain reaction-amplified with Bam HI adapters and cloned into pCMX-PL1. In vitro transcription/translation was performed with a Promega TnT kit. A PHYA-GST fusion protein was created by cloning the Arabidopsis PHYA cDNA with a Bam HI site at the 5′ end and a Not I site after the last amino acid into pDS472a [S. L. Forsburg and D. A. Sherman, Gene 191, 191 (1997)] and expressed in Schizosaccharomyces pombe as described [S. L. Forsburg, Nucleic Acids Res. 21, 2955 (1993)]. A PHYB-GST fusion protein was created by adding a Not I site after the last amino acid of PHYB in clone p41 and adding an in-frame GST cDNA with Not I and Xba I adapters. GST, PHYA-GST, or PHYB-GST (1 μg) were bound onto glutathione-agarose beads (G4510, Sigma). The beads were washed three times with extraction buffer (EB). [T. D. Elich and J. Chory, Plant Cell 9, 2271 (1997)]; in vitro transcribed and translated PKS1 (diluted 1:10 in EB) was added to the beads and incubated for 60 min at 4°C with gentle shaking. The beads were washed five times with EB; the bound proteins were eluted with EB containing 10 mM glutathione, then separated by SDS-polyacrylamide gel electrophoresis (PAGE) [U. K. Laemmli, Nature 227, 680 (1970)], stained, and exposed.
18. A full-length PKS1 clone was obtained from an Arabidopsis cDNA library [J. Kieber, M. Rothenberg, G. Roman, K. Feldmann, J. Ecker, Cell 72, 427 (1993)]. This cDNA was polymerase chain reaction-amplified with Bam HI adapters and cloned into pCMX-PL1. In vitro transcription/translation was performed with a Promega TnT kit. A PHYA-GST fusion protein was created by cloning the Arabidopsis PHYA cDNA with a Bam HI site at the 5′ end and a Not I site after the last amino acid into pDS472a [S. L. Forsburg and D. A. Sherman, Gene 191, 191 (1997)] and expressed in Schizosaccharomyces pombe as described [S. L. Forsburg, Nucleic Acids Res. 21, 2955 (1993)]. A PHYB-GST fusion protein was created by adding a Not I site after the last amino acid of PHYB in clone p41 and adding an in-frame GST cDNA with Not I and Xba I adapters. GST, PHYA-GST, or PHYB-GST (1 μg) were bound onto glutathione-agarose beads (G4510, Sigma). The beads were washed three times with extraction buffer (EB). [T. D. Elich and J. Chory, Plant Cell 9, 2271 (1997)]; in vitro transcribed and translated PKS1 (diluted 1:10 in EB) was added to the beads and incubated for 60 min at 4°C with gentle shaking. The beads were washed five times with EB; the bound proteins were eluted with EB containing 10 mM glutathione, then separated by SDS-polyacrylamide gel electrophoresis (PAGE) [U. K. Laemmli, Nature 227, 680 (1970)], stained, and exposed.
19. A full-length PKS1 clone was obtained from an Arabidopsis cDNA library [J. Kieber, M. Rothenberg, G. Roman, K. Feldmann, J. Ecker, Cell 72, 427 (1993)]. This cDNA was polymerase chain reaction-amplified with Bam HI adapters and cloned into pCMX-PL1. In vitro transcription/translation was performed with a Promega TnT kit. A PHYA-GST fusion protein was created by cloning the Arabidopsis PHYA cDNA with a Bam HI site at the 5′ end and a Not I site after the last amino acid into pDS472a [S. L. Forsburg and D. A. Sherman, Gene 191, 191 (1997)] and expressed in Schizosaccharomyces pombe as described [S. L. Forsburg, Nucleic Acids Res. 21, 2955 (1993)]. A PHYB-GST fusion protein was created by adding a Not I site after the last amino acid of PHYB in clone p41 and adding an in-frame GST cDNA with Not I and Xba I adapters. GST, PHYA-GST, or PHYB-GST (1 μg) were bound onto glutathione-agarose beads (G4510, Sigma). The beads were washed three times with extraction buffer (EB). [T. D. Elich and J. Chory, Plant Cell 9, 2271 (1997)]; in vitro transcribed and translated PKS1 (diluted 1:10 in EB) was added to the beads and incubated for 60 min at 4°C with gentle shaking. The beads were washed five times with EB; the bound proteins were eluted with EB containing 10 mM glutathione, then separated by SDS-polyacrylamide gel electrophoresis (PAGE) [U. K. Laemmli, Nature 227, 680 (1970)], stained, and exposed.
20. Amino acid abbreviations are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; S. Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
21. D. E. Somers and P. H. Quail, Plant J. 7, 413 (1995); L. Goosey, L. Palecanda, R. A. Sharrock, Plant Physiol. 115, 959 (1997).
22. D. E. Somers and P. H. Quail, Plant J. 7, 413 (1995); L. Goosey, L. Palecanda, R. A. Sharrock, Plant Physiol. 115, 959 (1997).
23. X. Yang, E. J. Hubbard, M. Carlson, Science 257, 680 (1992).
24. 2-terminal 215 amino acids of PKS1 were fused to GST in pGEX-4T1 (Pharmacia). The fusion protein was expressed and purified. Phospho-amino analysis was performed as described [K. Beemon and T. Hunter, J. Virol. 28, 551 (1978)].
25. V. N. Lapko, X.-Y. Jiang, D. L. Smith, P.-S. Song, Protein Sci. 8, 1 (1999); R. W. J. McMichael, thesis, University of California, Davis (1991). The S599K mutant oat PHYA was obtained by site-directed mutagenesis, expressed, and purified as described (7). Recombinant S599K had wild-type spectral properties (10).
26. V. N. Lapko, X.-Y. Jiang, D. L. Smith, P.-S. Song, Protein Sci. 8, 1 (1999); R. W. J. McMichael, thesis, University of California, Davis (1991). The S599K mutant oat PHYA was obtained by site-directed mutagenesis, expressed, and purified as described (7). Recombinant S599K had wild-type spectral properties (10).
27. Y.-S. Wong, R. W. J. McMichael, J. C. Lagarias, Plant Physiol. 91, 709 (1989); M. Ahmad, J. A. Jarillo, O. Smirnova, A. R. Cashmore, Mol. Cell 1, 939 (1998).
28. Y.-S. Wong, R. W. J. McMichael, J. C. Lagarias, Plant Physiol. 91, 709 (1989); M. Ahmad, J. A. Jarillo, O. Smirnova, A. R. Cashmore, Mol. Cell 1, 939 (1998).
29. After germination, seedlings were grown for 5 days at 20°C in constant light on 1X MS media (Gibco-BRL). Light treatments were as described [M. M. Neff and J. Chory, Plant Physiol. 118, 27 (1998)]. Red light-grown seedlings were extracted as described [D. M. Lagarias, M. W. Crepeau, M. D. Malnes, J. C. Lagarias, Plant Cell 9, 675 (1997)] except that 0.1% NP40 and 25 mM KF were added to the extraction buffer. An antibody to the GST-PKS1 protein (15) was produced and affinity-purified. PKS1 was immunoprecipitated with this antibody covalently attached to protein A-agarose (Pierce). The immunoprecipitates were treated with calf intestine phosphatase (CIP; Boehringer Mannheim) with or without phosphatase inhibitors [S. Lanker, M. H. Valdivieso, C. Wittenberg, Science 271, 1597 (1996)]. Samples were separated by 9% SDS-PAGE, subjected to protein immunoblotting, and probed with affinity-purified antibody to PKS1. Binding of a horseradish peroxidase-coupled secondary antibody was revealed with SuperSignal (Pierce).
30. After germination, seedlings were grown for 5 days at 20°C in constant light on 1X MS media (Gibco-BRL). Light treatments were as described [M. M. Neff and J. Chory, Plant Physiol. 118, 27 (1998)]. Red light-grown seedlings were extracted as described [D. M. Lagarias, M. W. Crepeau, M. D. Malnes, J. C. Lagarias, Plant Cell 9, 675 (1997)] except that 0.1% NP40 and 25 mM KF were added to the extraction buffer. An antibody to the GST-PKS1 protein (15) was produced and affinity-purified. PKS1 was immunoprecipitated with this antibody covalently attached to protein A-agarose (Pierce). The immunoprecipitates were treated with calf intestine phosphatase (CIP; Boehringer Mannheim) with or without phosphatase inhibitors [S. Lanker, M. H. Valdivieso, C. Wittenberg, Science 271, 1597 (1996)]. Samples were separated by 9% SDS-PAGE, subjected to protein immunoblotting, and probed with affinity-purified antibody to PKS1. Binding of a horseradish peroxidase-coupled secondary antibody was revealed with SuperSignal (Pierce).
31. After germination, seedlings were grown for 5 days at 20°C in constant light on 1X MS media (Gibco-BRL). Light treatments were as described [M. M. Neff and J. Chory, Plant Physiol. 118, 27 (1998)]. Red light-grown seedlings were extracted as described [D. M. Lagarias, M. W. Crepeau, M. D. Malnes, J. C. Lagarias, Plant Cell 9, 675 (1997)] except that 0.1% NP40 and 25 mM KF were added to the extraction buffer. An antibody to the GST-PKS1 protein (15) was produced and affinity-purified. PKS1 was immunoprecipitated with this antibody covalently attached to protein A-agarose (Pierce). The immunoprecipitates were treated with calf intestine phosphatase (CIP; Boehringer Mannheim) with or without phosphatase inhibitors [S. Lanker, M. H. Valdivieso, C. Wittenberg, Science 271, 1597 (1996)]. Samples were separated by 9% SDS-PAGE, subjected to protein immunoblotting, and probed with affinity-purified antibody to PKS1. Binding of a horseradish peroxidase-coupled secondary antibody was revealed with SuperSignal (Pierce).
32. Proteins from 5-day-old seedlings grown in the dark or in red light were extracted, run on SDS-PAGE, subjected to protein immunoblotting, and probed with PKS1 antibody (18). PhyB was detected with the mBA2 antibody [T. Shinomura et al., Proc. Natl. Acad. Sci. U.S.A. 93, 8129 (1996)].
33. Plants overexpressing PKS1 were obtained by in planta transformation of wild-type Col or phyA-205 with a pCGN18 vector [T. Jack, G. L. Fox, E. M. Meyerowitz, Cell 76, 703 (1994)] containing the PKS1 cDNA in the Bam HI site. Homozygous lines with a single insertion were selected for analysis. Proteins were extracted by grinding 20 1-week-old seedlings in 50 μl of final sample buffer. Protein immunoblots were performed as described (18).
34. J. M. Brown and R. A. Firtel, Curr. Biol. 8, R662 (1998); C. Chang and R. C. Stewart, Plant Physiol. 117, 723 (1998).
35. J. M. Brown and R. A. Firtel, Curr. Biol. 8, R662 (1998); C. Chang and R. C. Stewart, Plant Physiol. 117, 723 (1998).
36. W. Krek, Curr. Opin. Genet. Dev. 8, 36 (1998).
37. I. Stancovski and D. Baltimore, Cell 91, 299 (1997).
38. M. Ni, J. M. Tepperman, P. H. Quail, ibid. 95, 657 (1998).
39. We thank the Arabidopsis Biological Resource Center for EST clone 43C7T7, R. Evans for pCMX-PL1, L. Barden for help with artwork, M. Neff for advice on seedling measurements, L. Li for providing the oat PHYA S599K clone, J. Meisenhelder for help with phospho-amino analysis, and M. Gahrtz, T. Hunter, M. Neff, and D. Weigel for comments on the manuscript Supported by NIH grant RO1GM52413 (J.C.), NSF grant MCB96-04511 (J.C.L.), and HFSP and Swiss National Science Foundation fellowships (C.F.). J.C. is an investigator of the Howard Hughes Medical Institute.