Phosphorylation of Opaque2 changes diurnally and impacts its DNA binding activity.

Abstract

P i e t r o Ciceri,a.l E l i s a b e t t a Gianazza,b B a r b a r a Lazzari,a G u i d o Lippoli,a A n n a m a r i a Genga,a Gisela Hoschek,c R o b e r t J. Schmidt,c a n d A n g e l o Viottiag2 a lstituto Biosintesi Vegetali, Consiglio Nazionale delle Ricerche, Via Bassini 15, 1-201 33 Milan, ltaly lstituto di Scienze Farmacologiche, Universita degli Studi di Milano, Via Balzaretti 9, 1-201 33 Milan, ltalyDepartment of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-01 16In the maize endosperm, the Opaque2 (02) basic leucine zipper transcriptional activator regulates the expression of a subset of the zein seed storage protein gene family. lmmunodetection of wild-type or mutant 0 2 polypeptides fractionated by SDS-PAGE resolved a closely spaced doublet migrating in the 68-t o 72-kD range, whereas by using isoelectric focusing, seven t o nine isoforms were detected for each allele. Phosphatase treatment simplified the protein patterns t o a single band corresponding t o the nonphosphorylated component. In vivo and in vitro labeling confirmed that 0 2 can be phosphorylated. In protein gel blots probed with DNA, only the nonphosphorylated and hypophosphorylated 0 2 polypeptides were able t o bind an oligonucleotide containing the 0 2 binding sequence. Upon in situ dephosphorylation of the focused isoforms by phosphatase treatment of the isoelectric focusing filter, the hyperphosphorylated forms acquired DNA binding activity. The ratio among the various isoforms remained constant throughout the developmental stages of endosperm growth but changed from daytime t o nighttime, with a significant increase of the hyperphosphorylated forms during the night period. These results indicate that 0 2 exists in vivo as a pool of differently phosphorylated polypeptides and demonstrate that 0 2 DNA binding activity is modulated by a phosphorylation/dephosphorylation mechanism that appears t o be influenced by environmental conditions. INTRODUCTIONProtein phosphorylation and dephosphorylation, catalyzed by protein kinases and phosphatases, respectively, are important control mechanisms for severa1 biological processes in yeast, plant, and animal cells. These processes include cell division, perception and transduction of externa1 stimuli, hormone sensitivity, and control of metabolic pathways (Ranjeva and Boudet, 1987;Ma, 1993). The control of gene expression at the transcriptional leve1 is determined by cisacting elements and trans-acting factors that modulate the expressivity of a gene in response to the above-mentioned biological processes. In particular, transcription factors themselves are often regulated or modulated in their capacity to transactivate a gene by post-translational modifications involving phosphorylation (reviewed in Hunter and Karin, 1992;Karin and Hunter, 1995).During cereal seed maturation, starch, proteins, and oil, which are the main storage products, accumulate during de-' Current address: Department of Biology and Center for Molecular Genetics,...

Multifarious Roles of Intrinsic Disorder in Proteins Illustrate Its Broad Impact on Plant Biology

“…Another plant bZIP, Opaque-2 from maize, is likely phosphorylated by casein kinase II. Diurnal oscillation between hyper-and hypophosphorylated isoforms of Opaque-2 results in accumulation of the phosphorylated isoform during the night, and the DNA binding potential of the phosphorylated form is diminished (Ciceri et al, 1997). With regard to GRAS proteins, phosphorylation and dephosphorylation states of the DELLA subfamily are correlated with their stability, plant growth repressive activity, and gibberellic acid (GA)-induced degradation (Hussain et al, 2005;Itoh et al, 2005); reversible phosphorylation is required for the plant stress-induced response of tobacco (Nicotiana tabacum) GRAS1 (Czikkel and Maxwell, 2007); rice (Oryza sativa) CIGR1 and CIGR2 (for Chitininducible gibberellin-responsive protein) are induced by GA signals, depending on both phosphorylation and dephosphorylation events (Day et al, 2004).…”

Reconstruction of protein networks from an atlas of maize seed proteotypes

“…For example, an increase in bZIP family abundance corresponded to increases in bZIP phosphorylation, whereas the opposite pattern was observed for the ARF family. Considering that the DNA binding activity of OPAQUE2 (a maize bZIP) and ARF2 (in Arabidopsis) is abolished by hyperphosphorylation (41,42), our TF phosphorylation data may be used to infer TF activities during development.…”

Characterization of SBZ1, a soybean bZIP protein that binds to the chalcone synthase gene promoter

“…In particular, reversible phosphorylation is a common mechanism for the selective regulation of transcription factors. In higher plants, the phosphorylation of transcription factors might be an important mechanism for the selective control of gene expression in response to environmental signals (Wellmer et al 1999;Klimczak et al 1995;Ciceri et al 1997;Kircher et al 1998;Zhou et al 1997). In this study, SBZ1 was phosphorylated by a soybean cell extract in vitro ( Figure 5A).…”

“…Several reports have suggested that the activities of plant transcription factors can also be modulated by phosphorylation. Phosphorylation affects the affinities of some transcription factors for their DNA targets in vitro (Klimczak et al 1995;Ciceri et al 1997;Hardtke et al 2000). We examined, therefore, whether phosphorylation has any influence on the affinity of SBZ1 for the binding sequence ( Figure 6).…”