Phosphorylation by CK2 Enhances the Rapid Light-induced Degradation of Phytochrome Interacting Factor 1 in Arabidopsis
2011; Elsevier BV; Volume: 286; Issue: 14 Linguagem: Inglês
10.1074/jbc.m110.186882
ISSN1083-351X
AutoresQingyun Bu, Ling Zhu, Michael D. Dennis, Lu Yu, Sheen X. Lu, Maria D. Person, Elaine M. Tobin, Karen Browning, Enamul Huq,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoThe phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four β subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis. The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four β subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis. IntroductionBecause light is an essential environmental signal for modulating plant growth and development throughout the life cycle, plants have several different classes of photoreceptors sensing and responding to major bandwidths of the light spectrum (1Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (789) Google Scholar, 2Bae G. Choi G. Annu. Rev. Plant Biol. 2008; 59: 281-311Crossref PubMed Scopus (339) Google Scholar). Of these photoreceptors, the phytochrome (phy) 3The abbreviations used are: phy, phytochrome; R, red; FR, far-red; PIF, phytochrome interacting factor; MOPS, 4-morpholinepropanesulfonic acid; LUC, luciferase; APA, active phyA binding domain; APB, active phyB binding domain. family (which consists of five members, phyA to phyE, in Arabidopsis) senses and responds to a broad spectrum (red, far-red, and blue) of light signals and pleiotropically regulates plant growth from seed germination to flowering time (3Franklin K.A. Quail P.H. J. Exp. Bot. 2010; 61: 11-24Crossref PubMed Scopus (565) Google Scholar, 4Castillon A. Shen H. Huq E. Genetics. 2009; 182: 161-171Crossref PubMed Scopus (36) Google Scholar, 5Schaefer E. Nagy F. Photomorphogenesis in Plants and Bacteria. 3rd Ed. Springer, Dordrecht, The Netherlands2006Crossref Scopus (6) Google Scholar). Within a cell, phys are synthesized as an inactive Pr conformer that resides in the cytosol. Exposure to red (R) light triggers a conformation shift to a biologically active Pfr form, which migrates into the nucleus and initiates phy signaling (6Fankhauser C. Chen M. Trends Plant Sci. 2008; 13: 596-601Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The active Pfr form can be reverted back to the inactive Pr form by exposure to far-red (FR) light. The active Pfr form interacts with multiple signaling partners within the nucleus (7Huq E. Quail P.H. Briggs W.R. Spudich J.L. Handbook of Photosensory Receptors. Wiley-VCH, Weinheim, Germany2005: 151-170Google Scholar) and controls the expression of a large number (10–30% of the genome) of genes to regulate photomorphogenesis (8Ma L. Li J. Qu L. Hager J. Chen Z. Zhao H. Deng X.W. Plant Cell. 2001; 13: 2589-2607Crossref PubMed Scopus (458) Google Scholar, 9Quail P.H. J. Integr. Plant Biol. 2007; 49: 11-20Crossref Scopus (42) Google Scholar, 10Jiao Y. Lau O.S. Deng X.W. Nat. Rev. Genet. 2007; 8: 217-230Crossref PubMed Scopus (747) Google Scholar).One of the ways phys exert this strong effect on gene expression is by differentially regulating the stability of positively and negatively acting transcription factors functioning in light signaling pathways (11Huq E. Trends Plant Sci. 2006; 11: 4-7Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Henriques R. Jang I.C. Chua N.H. Curr. Opin. Plant Biol. 2009; 12: 49-56Crossref PubMed Scopus (64) Google Scholar). For example, the positively acting transcription factors (e.g. HY5, HFR1, LAF1, and possibly others) are degraded in the dark and stabilized under red, far-red, and blue light conditions. By contrast, the negatively acting transcription factors (e.g. the phytochrome interacting factors (PIFs)) are stable in the dark and degrade in response to light. The net effect of this stabilization and destabilization of transcription factors shapes the transcriptome that regulates photomorphogenesis.PIFs have been shown to play central roles in phy signaling pathways (13Castillon A. Shen H. Huq E. Trends Plant Sci. 2007; 12: 514-521Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 14Duek P.D. Fankhauser C. Trends Plant Sci. 2005; 10: 51-54Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 15Leivar P. Quail P.H. Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar). They belong to the bHLH class of transcription factors and are encoded by six family members in Arabidopsis (PIF1 and PIF3–7) (13Castillon A. Shen H. Huq E. 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Moreover, quadruple (pifQ) mutant seedlings of pif1, pif3, pif4, and pif5 are constitutively photomorphogenic, suggesting that PIFs repress photomorphogenesis in the dark (24Leivar P. Monte E. Oka Y. Liu T. Carle C. Castillon A. Huq E. Quail P.H. Curr. Biol. 2008; 18: 1815-1823Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar, 27Shin J. Kim K. Kang H. Zulfugarov I.S. Bae G. Lee C.H. Lee D. Choi G. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7660-7665Crossref PubMed Scopus (326) Google Scholar). This morphological phenotype is reflected at the gene expression level, where a majority of the light-regulated genes are constitutively expressed in the pifQ mutant in the dark (27Shin J. Kim K. Kang H. Zulfugarov I.S. Bae G. Lee C.H. Lee D. Choi G. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7660-7665Crossref PubMed Scopus (326) Google Scholar, 28Leivar P. Tepperman J.M. Monte E. Calderon R.H. Liu T.L. Quail P.H. Plant Cell. 2009; 21: 3535-3553Crossref PubMed Scopus (205) Google Scholar). In wild-type seedlings, light signals perceived by phys promote degradation of PIFs through the ubiquitin/26 S proteasomal pathway to derepress gene expression and promote photomorphogenesis (13Castillon A. Shen H. Huq E. Trends Plant Sci. 2007; 12: 514-521Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 15Leivar P. Quail P.H. Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 29Chen M. Galvão R.M. Li M. Burger B. Bugea J. Bolado J. Chory J. Cell. 2010; 141: 1230-1240Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar).Although the mechanisms of dark-induced degradation of positively acting transcription factors are well understood (12Henriques R. Jang I.C. Chua N.H. Curr. Opin. Plant Biol. 2009; 12: 49-56Crossref PubMed Scopus (64) Google Scholar), the light-induced degradation of PIFs is only beginning to be understood. All PIFs except PIF7 are rapidly phosphorylated and ubiquitinated in response to light in vivo prior to their degradation (26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar, 30Al-Sady B. Ni W. Kircher S. Schäfer E. Quail P.H. Mol. Cell. 2006; 23: 439-446Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 31Shen Y. Khanna R. Carle C.M. Quail P.H. Plant Physiol. 2007; 145: 1043-1051Crossref PubMed Scopus (195) Google Scholar). The mutant PIF1 and PIF3 that fail to interact with the Pfr forms of phyA and phyB do not undergo light-dependent phosphorylation, suggesting that phy interaction is necessary for the light-dependent phosphorylation and subsequent degradation of PIFs. However, the putative kinase(s) and the putative E3 ligase(s) responsible for phosphorylation of PIFs and recognition of the phosphorylated forms for subsequent ubiquitination and degradation are still unknown.CK2 (formerly known as casein kinase II), a ubiquitous Ser/Thr kinase, has been implicated in regulating light signaling and circadian rhythms in Arabidopsis (32Lee Y. Lloyd A.M. Roux S.J. Plant Physiol. 1999; 119: 989-1000Crossref PubMed Scopus (50) Google Scholar, 33Sugano S. Andronis C. Ong M.S. Green R.M. Tobin E.M. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 12362-12366Crossref PubMed Scopus (192) Google Scholar, 34Hardtke C.S. Gohda K. Osterlund M.T. Oyama T. Okada K. Deng X.W. EMBO J. 2000; 19: 4997-5006Crossref PubMed Scopus (284) Google Scholar, 35Park H.J. Ding L. Dai M. Lin R. Wang H. J. Biol. Chem. 2008; 283: 23264-23273Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 36Daniel X. Sugano S. Tobin E.M. Proc. Natl. Acad. Sci. 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A CK2-like activity has been shown to phosphorylate HY5, a positively acting component in a phy signaling pathway (34Hardtke C.S. Gohda K. Osterlund M.T. Oyama T. Okada K. Deng X.W. EMBO J. 2000; 19: 4997-5006Crossref PubMed Scopus (284) Google Scholar). In addition, CK2 phosphorylates HFR1, a positively acting HLH transcription factor functioning under FR and blue light pathways (35Park H.J. Ding L. Dai M. Lin R. Wang H. J. Biol. Chem. 2008; 283: 23264-23273Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Phosphorylation of both HY5 and HFR1 has been shown to stabilize these transcription factors (34Hardtke C.S. Gohda K. Osterlund M.T. Oyama T. Okada K. Deng X.W. EMBO J. 2000; 19: 4997-5006Crossref PubMed Scopus (284) Google Scholar, 35Park H.J. Ding L. Dai M. Lin R. Wang H. J. Biol. Chem. 2008; 283: 23264-23273Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Because CK2-mediated phosphorylation stabilizes the positively acting transcription factors involved in light signaling in Arabidopsis, we reasoned that CK2 might also phosphorylate the negatively acting transcription factors (e.g. PIFs) and regulate their stability/function in Arabidopsis. Here we show that CK2 phosphorylates PIF1 at multiple sites in vitro. In addition, CK2-mediated phosphorylation promotes rapid light-induced degradation of PIF1 to fine-tune photomorphogenesis.DISCUSSIONPreviously, it was shown that PIF1 and PIF3–6 were phosphorylated in response to light before being degraded through the 26 S proteasomal pathway (13Castillon A. Shen H. Huq E. Trends Plant Sci. 2007; 12: 514-521Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 15Leivar P. Quail P.H. Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar). Interactions with both phyA and phyB are necessary for the light-induced phosphorylation and degradation under all three light conditions (4Castillon A. Shen H. Huq E. Genetics. 2009; 182: 161-171Crossref PubMed Scopus (36) Google Scholar, 13Castillon A. Shen H. Huq E. Trends Plant Sci. 2007; 12: 514-521Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 15Leivar P. Quail P.H. Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar). This study provides biochemical evidence that PIF1 is a substrate for Arabidopsis CK2, a ubiquitous Ser/Thr kinase present in all organisms. Phosphorylation by CK2 appears to be necessary for the rapid light-induced degradation of PIF1.Several lines of evidence suggest that PIF1 is a bona fide substrate for Arabidopsis CK2. First, in silico studies predicted that PIF1 has multiple CK2 phosphorylation sites (supplemental Table S2). Second, in vitro kinase assays demonstrated that PIF1 is strongly phosphorylated by purified Arabidopsis CK2 α and β subunit holoenzyme combinations (Fig. 1A). Third, CK2-mediated phosphorylation of PIF1 is inhibited by a specific inhibitor, heparin, in a concentration-dependent manner (Fig. 1B). Fourth, mapping of the phosphorylation sites identified seven phosphorylation sites scattered throughout the PIF1 polypeptide with a cluster of sites at the C-terminal end (Fig. 2, A–C, and supplemental Fig. S1). Fifth, CK2-mediated phosphorylation enhances the rapid light-induced degradation of PIF1 (FIGURE 3, FIGURE 4). Sixth, overexpression of CK2 β subunits accelerates the light-induced degradation of native PIF1 in vivo (Fig. 5A). These data provide strong evidence that PIF1 is a substrate for Arabidopsis CK2 and that CK2 phosphorylation specifically plays a role in the light-induced degradation of PIF1 to promote photomorphogenesis. However, the above data do not show that PIF1 is a substrate for CK2 in vivo. Further experiments are necessary to demonstrate whether CK2 phosphorylates PIF1 in vivo.Previously, CK2 substrates have been identified in plants. These include translation initiation factors (e.g. eIF2α, eIF2β, eIF3c, eIF4B, and eIF5) (40Dennis M.D. Browning K.S. J. Biol. Chem. 2009; 284: 20602-20614Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 41Dennis M.D. Person M.D. Browning K.S. J. Biol. 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Biochem. 2003; 42: 3503-3508Crossref PubMed Scopus (31) Google Scholar), abscisic acid responsive protein Rab17 in maize (48Riera M. Figueras M. López C. Goday A. Pagès M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9879-9884Crossref PubMed Scopus (114) Google Scholar), and positively acting transcription factors (e.g. HY5 and HFR1) involved in light signaling pathways (34Hardtke C.S. Gohda K. Osterlund M.T. Oyama T. Okada K. Deng X.W. EMBO J. 2000; 19: 4997-5006Crossref PubMed Scopus (284) Google Scholar, 35Park H.J. Ding L. Dai M. Lin R. Wang H. J. Biol. Chem. 2008; 283: 23264-23273Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). However, phosphorylation by CK2 has been shown to either stabilize or modulate the activity of these factors (34Hardtke C.S. Gohda K. Osterlund M.T. Oyama T. Okada K. Deng X.W. EMBO J. 2000; 19: 4997-5006Crossref PubMed Scopus (284) Google Scholar, 35Park H.J. Ding L. Dai M. Lin R. Wang H. J. Biol. Chem. 2008; 283: 23264-23273Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 40Dennis M.D. Browning K.S. J. Biol. Chem. 2009; 284: 20602-20614Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In contrast, our data show that phosphorylation by CK2 promotes the light-induced degradation of PIF1 through the ubiquitin/26 S proteasomal pathway (Fig. 3A). This is similar to the posttranslational regulation of mammalian IκBα and promyelocytic leukemia proteins, where CK2-mediated phosphorylation enhanced their polyubiquitination and proteasomal degradation (49Scaglioni P.P. Yung T.M. Cai L.F. Erdjument-Bromage H. Kaufman A.J. Singh B. Teruya-Feldstein J. Tempst P. Pandolfi P.P. Cell. 2006; 126: 269-283Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 50Kato Jr., T. Delhase M. Hoffmann A. Karin M. Mol. Cell. 2003; 12: 829-839Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Similar to PIF1, CK2 phosphorylates a cluster of sites at the C terminus of IκBα, and this phosphorylation is UV-light inducible (50Kato Jr., T. Delhase M. Hoffmann A. Karin M. Mol. Cell. 2003; 12: 829-839Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Therefore, CK2-mediated stabilization and destabilization of proteins might represent an evolutionarily conserved mechanism.Although our data provide strong evidence that CK2 promotes the light-induced degradation of PIF1 in vivo, PIF1-6M (which lacks the majority of the CK2 phosphorylation sites) and the PIF1(S464–466A) mutants are still robustly phosphorylated in response to light in vivo as observed previously for wild-type PIF1 (Figs. 3A and 4A and supplemental Fig. S3) (26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar). These data suggest that either CK2 phosphorylates PIF1 in response to light at different sites than the ones identified and mutated, or a separate light-specific kinase may phosphorylate PIF1 at different Ser/Thr residues under light. Because phy interaction is necessary for the light-induced phosphorylation and degradation of PIFs (12Henriques R. Jang I.C. Chua N.H. Curr. Opin. Plant Biol. 2009; 12: 49-56Crossref PubMed Scopus (64) Google Scholar, 13Castillon A. Shen H. Huq E. Trends Plant Sci. 2007; 12: 514-521Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar) and because phyA has been shown to have Ser/Thr kinase activity (51Yeh K.C. Lagarias J.C. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13976-13981Crossref PubMed Scopus (351) Google Scholar), it is possible that phys might directly phosphorylate PIFs in response to light. However, convincing in vivo evidence for the phyA kinase hypothesis is still lacking. Therefore, these data suggest that phosphorylation of PIF1 by CK2 and phosphorylation by either phys directly or a phy-associated kinase is necessary for the rapid light-induced degradation of PIF1. Further work is needed to identify the additional phosphorylation sites by either phytochromes or a phytochrome-associated kinase.In addition, the mechanism of CK2-mediated enhanced degradation of PIF1 is still unknown. It is possible that phosphorylation of PIF1 by CK2 enhances the affinity of PIF1 for phys, as phy interaction has been shown to be necessary for rapid degradation of PIF1 (26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar). However, this is unlikely, as the isolated APA and APB domains, which are located within the first 150 amino acids, are both necessary and sufficient for physical interaction with phyA and phyB, respectively (Fig. 2A) (26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar, 52Khanna R. Huq E. Kikis E.A. Al-Sady B. Lanzatella C. Quail P.H. Plant Cell. 2004; 16: 3033-3044Crossref PubMed Scopus (282) Google Scholar). Although PIF1 has one CK2 phosphorylation site near the APB sequence (Thr-10) (Fig. 2A), the results show that the CK2 sites at the C-terminal end (Ser-464, Ser-465 and Ser-466) play the major role in PIF1 degradation compared with other CK2 sites (Figs. 2, 3A, and 4A). An alternative hypothesis is that CK2-mediated phosphorylation of PIF1 at the C terminus enhances the interaction of PIF1 with substrate recognition factors responsible for polyubiquitination and subsequent degradation (e.g. F-box proteins in Skp-Cullin-F-box (SCF) complex). This is more likely because enhancement of proteasomal degradation of multiple factors by signal-induced phosphorylation has been demonstrated (50Kato Jr., T. Delhase M. Hoffmann A. Karin M. Mol. Cell. 2003; 12: 829-839Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 53Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4043) Google Scholar). This possibility is also consistent with our previous data that isolated N- (1–150 amino acids) and C-terminal (151–478 amino acids) regions are not phosphorylated and degraded in response to light (26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar). Conversely, both N- and C-terminal regions are necessary for the light-induced degradation of PIF1 in vivo (26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar). Identification of factors responsible for recognition and polyubiquitination of PIF1 will help distinguish these possibilities.In summary, our data and those of others show that light stabilizes positively acting transcription factors (e.g. HY5, LAF1, and HFR1) (34Hardtke C.S. Gohda K. Osterlund M.T. Oyama T. Okada K. Deng X.W. EMBO J. 2000; 19: 4997-5006Crossref PubMed Scopus (284) Google Scholar, 35Park H.J. Ding L. Dai M. Lin R. Wang H. J. Biol. Chem. 2008; 283: 23264-23273Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 54Seo H.S. Yang J.Y. Ishikawa M. Bolle C. Ballesteros M.L. Chua N.H. Nature. 2003; 423: 995-999Crossref PubMed Scopus (394) Google Scholar) and induces proteasomal degradation of negatively acting transcription factors (e.g. PIFs) to promote photomorphogenesis (Fig. 6). CK2 phosphorylates both positively and negatively acting transcription factors involved in light signaling. However, the significance of CK2 phosphorylation is opposite for these two classes of transcription factors. CK2-mediated phosphorylation enhances the stability of the positively acting factors, whereas it decreases the stability of the negatively acting factors (e.g. PIF1 and possibly other PIFs). This contrasting but apparently synergistic effect is expected to promote robust photomorphogenesis. However, our phenotypic analyses showed that the CK2 β subunit overexpression lines have modest seedling deetiolation and circadian phenotypes (Figs. 5, B and C, and supplemental Fig. S7). Moreover, a stable PIF1 is expected to promote hypocotyl growth as observed (FIGURE 3, FIGURE 4) (26Shen H. Zhu L. Castillon A. Majee M. Downie B. Huq E. Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (206) Google Scholar), whereas a CK2 dominant-negative mutant displayed a short hypocotyl phenotype in the dark (45Moreno-Romero J. Espunya M.C. Platara M. Ariño J. Martínez M.C. Plant J. 2008; 55: 118-130Crossref PubMed Scopus (53) Google Scholar). These apparent contradictions might be explained in part by the fact that CK2-mediated phosphorylation of HY5 reduces the DNA binding ability of HY5 (34Hardtke C.S. Gohda K. Osterlund M.T. Oyama T. Okada K. Deng X.W. EMBO J. 2000; 19: 4997-5006Crossref PubMed Scopus (284) Google Scholar). In addition, all organisms have multiple CK2 substrates in vivo that may function in multiple pathways. More than 300 substrates have been identified for CK2 in an animal system (55Meggio F. Pinna L.A. FASEB J. 2003; 17: 349-368Crossref PubMed Scopus (1098) Google Scholar). Therefore, the phenotypes of the CK2 mutants and overexpression lines will reflect the net effect of CK2 on these diverse pathways that regulate plant growth and development. Identification and characterization of these substrates will shed light on how CK2 optimizes photomorphogenesis. IntroductionBecause light is an essential environmental signal for modulating plant growth and development throughout the life cycle, plants have several different classes of photoreceptors sensing and responding to major bandwidths of the light spectrum (1Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (789) Google Scholar, 2Bae G. Choi G. Annu. Rev. Plant Biol. 2008; 59: 281-311Crossref PubMed Scopus (339) Google Scholar). 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Genet. 2007; 8: 217-230Crossref PubMed Scopus (747) Google Scholar).One of the ways phys exert this strong effect on gene expression is by differentially regulating the stability of positively and negatively acting transcription factors functioning in light signaling pathways (11Huq E. Trends Plant Sci. 2006; 11: 4-7Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 12Henriques R. Jang I.C. Chua N.H. Curr. Opin. Plant Biol. 2009; 12: 49-56Crossref PubMed Scopus (64) Google Scholar). For example, the positively acting transcription factors (e.g. HY5, HFR1, LAF1, and possibly others) are degraded in the dark and stabilized under red, far-red, and blue light conditions. By contrast, the negatively acting transcription factors (e.g. the phytochrome interacting factors (PIFs)) are stable in the dark and degrade in response to light. The net effect of this stabilization and destabilization of transcription factors shapes the transcriptome that regulates photomorphogenesis.PIFs have been shown to play central roles in phy signaling pathways (13Castillon A. Shen H. Huq E. Trends Plant Sci. 2007; 12: 514-521Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 14Duek P.D. Fankhauser C. Trends Plant Sci. 2005; 10: 51-54Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 15Leivar P. Quail P.H. Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar). They belong to the bHLH class of transcription factors and are encoded by six family members in Arabidopsis (PIF1 and PIF3–7) (13Castillon A. Shen H. Huq E. Trends Plant Sci. 2007; 12: 514-521Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 14Duek P.D. Fankhauser C. 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A CK2-like activity has been shown to phosphorylate HY5, a positively acting component in a phy signaling pathway (34Hardtke C.S. Gohda K. Osterlund M.T. Oyama T. Okada K. Deng X.W. EMBO J. 2000; 19: 4997-5006Crossref PubMed Scopus (284) Google Scholar). In addition, CK2 phosphorylates HFR1, a positively acting HLH transcription factor functioning under FR and blue light pathways (35Park H.J. Ding L. Dai M. Lin R. Wang H. J. Biol. Chem. 2008; 283: 23264-23273Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Phosphorylation of both HY5 and HFR1 has been shown to stabilize these transcription factors (34Hardtke C.S. Gohda K. Osterlund M.T. Oyama T. Okada K. Deng X.W. EMBO J. 2000; 19: 4997-5006Crossref PubMed Scopus (284) Google Scholar, 35Park H.J. Ding L. Dai M. Lin R. Wang H. J. Biol. Chem. 2008; 283: 23264-23273Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Because CK2-mediated phosphorylation stabilizes the positively acting transcription factors involved in light signaling in Arabidopsis, we reasoned that CK2 might also phosphorylate the negatively acting transcription factors (e.g. PIFs) and regulate their stability/function in Arabidopsis. Here we show that CK2 phosphorylates PIF1 at multiple sites in vitro. In addition, CK2-mediated phosphorylation promotes rapid light-induced degradation of PIF1 to fine-tune photomorphogenesis.
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