Multisite phosphorylation of Arabidopsis HFR1 by casein kinase II and a plausible role in regulating its degradation rate.
Arabidopsis Long Hypocotyl in Far-Red Light 1 (HFR1), a bHLH transcription factor, plays a critical role in promoting seedling photomorphogenesis and in balancing the shade-avoidance response under canopy shade conditions. Previous studies have established that HFR1 protein is degraded in darkness and is stabilized under light conditions to promote light signaling. How light regulates HFR1 stability is not well understood. In this study, we show that Arabidopsis HFR1 can be phosphorylated by recombinant casein kinase II (CKII) and plant extract in vitro and that phosphorylation of HFR1 can be effectively reduced by treatments with two CKII-specific inhibitors, 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (DRB) and heparin. We demonstrate that HFR1 physically interacts with the CKB1 and CKB2 regulatory subunits of CKII. Mutagenesis studies indicate that HFR1 is phosphorylated at multiple serine (Ser) residues in the N-terminal regulatory domain of HFR1. We also show that phosphorylation of HFR1 is promoted by light and that a predicted CKII site, Ser(122), represents a major phosphorylation site of HFR1 under both dark and light conditions. Comparison of wild-type, phosphorylation-deficient, and phosphorylation-mimic mutant proteins suggests that phosphorylation acts to reduce the degradation rate of HFR1. Together, our results suggest that CKII-mediated phosphorylation represents an important post-translational modification influencing the stability and signaling activity of Arabidopsis HFR1.
Park
Hee-Jin
HJ
Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853, USA.
Ding
Lei
L
Dai
Mingqiu
M
Lin
Rongcheng
R
Wang
Haiyang
H
D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D047390:Casein Kinase II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008027:Light; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D019697:Onions; D010766:Phosphorylation; D017434:Protein Structure, Tertiary
D029681:Arabidopsis Proteins; D047390:Casein Kinase II; D004268:DNA-Binding Proteins; D004004:Dichlororibofuranosylbenzimidazole; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; C414438:At1g02340 protein, Arabidopsis