Reconstitution of Arabidopsis casein kinase II from recombinant subunits and phosphorylation of transcription factor GBF1.

Abstract

In contrast to the well-defined tetrameric structure of animal and yeast casein kinase II (CKII), plant CKII is found in two forms: a monomeric form and an oligomeric form whose subunit composition is not well defined. The Arabidopsis homologs of the catalytic subunit alpha (CKA1) and the regulatory subunit beta (CKB1) of CKII were expressed in Escherichia coli to examine their ability to form complexes, the effect of CKB1 on the catalytic activity, and the relationship of the recombinant enzymes to those isolated from plant material. Both subunits were found mainly in the inclusion body fraction in the bacterial expression strains, and they were solubilized and renatured with the recovery of catalytic (CKA1) and stimulatory (CKB1) activities. The combination of purified CKA1 and CKB1 proteins resulted in up to 100-fold stimulation of casein kinase activity compared with the CKA1 activity alone, showing that CKB1 has biochemical properties similar to those of the beta subunit from animals. CKA1 and CKB1 spontaneously assembled into a tetrameric complex, CKA1(2)CKB12, which had properties very similar to those of the oligomeric CKII form isolated from broccoli. However, the properties of the catalytic subunit CKA1 alone differed from those of the broccoli monomeric form of CKII-like activity. Phosphorylation of transcription factor GBF1 with the reconstituted CKA1(2)CKB1(2) enzyme resulted in stimulation of its DNA binding activity and retardation of the protein-DNA complex; these results are identical to those obtained previously with isolated nuclear CKII from broccoli.

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).…”

“…Some transcription factors are regulated by CKII, a ubiquitous tetrameric serine/threonine kinase composed of two catalytic (a/aЈ) and two regulatory subunits. Interaction studies of CKII have shown that the basic domain is the main interaction site between bZIP proteins and CKII (Klimczak et al 1995;Yamaguchi et al 1998). In vitro kinase assays using the catalytic a subunit of recombinant CKII demonstrated that CKII also phosphorylates SBZ1 ( Figure 5D).…”

“…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).…”

Plant Genes for Abiotic Stress

“…The phosphorylation of TFs is a common modification that can influence their ability to bind to promoters. For example, the level of G-Box Binding Factor 1 (GBF1) is constant, but its affinity for the G-box is modulated by its phosphorylation status: its phosphorylation by nuclear Casein Kinase II (CKII) enables Gbox binding (Klimczak et al, 1995). In the dark, some TFs that positively regulate gene expression in response to light, such as Long After Farred Light 1 (LAF1), are ubiquitylated by Constitutive Photomorphogenic 1 (COP1), a ring-finger-type ubiquitin E3 ligase.…”

Multiple Isoforms of Arabidopsis Casein Kinase I Combine Conserved Catalytic Domains with Variable Carboxyl-Terminal Extensions

“…It appears that the CKI enzymes may strongly interfere with cellular functions, possibly with the translational apparatus, which would prevent higher levels of accumulation. Low protein expression is generally common for protein kinases present in the soluble fraction of bacterial cells (due to their toxic effects), and higher amounts are usually observed only when inactive proteins are sequestered in inclusion bodies (Russo et al, 1992;Klimczak et al, 1995).…”