ABSTRACT
We studied a phosphate acceptor for casein kinase II (CK-II) in chloroplasts, and found a 56 kDa protein (p56) as an acceptor, which was partially purified from the stroma of spinach chloroplasts. The N-terminal amino acid sequence of p56 was identical with that of the beta subunit of chloroplast ATP synthase (CF0CF1-ATPase). In addition, the recombinant beta subunit of CF1 was phosphorylated when the subunit was incubated with CK-II. These results suggest that the beta subunit of CF1 is a substrate protein of CK-II in the chloroplast.
Subject(s)
Chloroplasts/enzymology , Protein Serine-Threonine Kinases/metabolism , Proton-Translocating ATPases/metabolism , Amino Acid Sequence , Casein Kinase II , Molecular Sequence Data , Phosphorylation , Proton-Translocating ATPases/chemistry , Recombinant Proteins/metabolism , Spinacia oleraceaABSTRACT
Chloroplast ribonucleoproteins (RNPs) encoded by the nuclear genome may play an important role in the post-transcriptional steps of light-induced chloroplast gene expression. To study the relationship between the synthesis of a 34 kDa ribonucleoprotein (p34) and light in spinach chloroplasts, a polyclonal antibody was raised against p34. Immunoblotting revealed that p34 synthesis was induced in a light dependent manner. In addition, levels of the large subunit of ribulose-1,5-bis phosphate carboxylase/oxygenase (Rubisco) encoded by the chloroplast genome fluctuated in relation to those of p34. These results suggest that the synthesis of p34 in response to light induces of chloroplast gene expression.
Subject(s)
Chloroplasts/metabolism , Light , Plant Proteins/biosynthesis , Ribonucleoproteins/biosynthesis , Spinacia oleracea/metabolism , Chloroplasts/radiation effects , Immunoblotting , Plant Proteins/immunology , Ribonucleoproteins/immunology , Spinacia oleracea/radiation effectsABSTRACT
Using ssDNA-cellulose column chromatography, a 34 kDa ribonucleoprotein (p34) has been purified from a 0.4 M KCl crude extract of spinach chloroplasts as an effective phosphate acceptor for casein kinase II (CK-II) in vitro. Monomeric and oligomeric CK-IIs were copurified with p34 by the column chromatography and the kinases were separated from p34 by means of Mono Q column chromatography. It was found that (i) the purified p34 (pI 4.9) was phosphorylated specifically by CK-II in vitro; and (ii) similar polypeptides, such as p35 (pI 4.7) and p39 (pI 4.9) in maize and p33 (pI 4.7) in liverwort, were detected as ssDNA-binding chloroplast proteins phosphorylated by CK-II in vitro. The findings suggest that (i) RNPs that function as phosphate acceptors for CK-II exist commonly in chloroplasts among plant cells; and (ii) the physiological activity of RNPs is regulated by their specific phosphorylation by CK-II in chloroplasts.
Subject(s)
Phosphates/metabolism , Plant Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Ribonucleoproteins/metabolism , Casein Kinase II , Chloroplasts/chemistry , Chromatography, Agarose , DNA, Single-Stranded/chemistry , Peptides/metabolism , Phosphorylation , Plant Proteins/isolation & purification , Plants/chemistry , Ribonucleoproteins/isolation & purification , Spinacia oleracea/chemistry , Substrate Specificity , Zea mays/chemistryABSTRACT
By means of successive GL-affinity and Mono S column chromatographies (HPLC), a 100 kDa GL-binding protein (gp100) was purified from the partially purified CK-II fraction of EAT cells as an effective phosphate acceptor for CK-II. It was found that (i) gp100 (pI 9.0) is copurified with CK-II, Hsp-90 and p34 or p70; (ii) gp100 cross-reacts with anti-human GR; (iii) phosphorylation of gp100 by CK-II is significantly stimulated by 1 microM GL or 0.3 microM oGA; and (iv) GL as well as DEX inhibit it at doses above 3 microM. Data are provided to suggest that the GL-induced selective inhibition of the CK-II catalyzed phosphorylation of gp100 may be involved in the anti-inflammatory effects of GL.
Subject(s)
Carrier Proteins/metabolism , Glycyrrhetinic Acid/analogs & derivatives , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Carcinoma, Ehrlich Tumor/enzymology , Carrier Proteins/isolation & purification , Casein Kinase II , Chickens , Chromatography, Affinity , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Dexamethasone/pharmacology , Electrophoresis, Polyacrylamide Gel , Glycyrrhetinic Acid/metabolism , Glycyrrhetinic Acid/pharmacology , Glycyrrhizic Acid , HSP90 Heat-Shock Proteins , Heat-Shock Proteins/isolation & purification , Heat-Shock Proteins/metabolism , Humans , Kinetics , Mice , Molecular Sequence Data , Molecular Weight , Phosphorylation , Protein Serine-Threonine Kinases/isolation & purification , Sequence Homology, Amino AcidABSTRACT
A 34 kDa ribonucleoprotein (p34) was purified to homogeneity from a 1.0 M KCl extract of spinach chloroplasts and characterized as an effective phosphate acceptor for casein kinase II (CK-II). The N-terminal 21 residues (W-V-A-Q-T-S-E-E-E-Q-E-G-S-T-N-A-V-L-E-G-E) of p34 were 95% identical with the sequence reported for 28RNP (plastid mRNA 3' end processing factor in chloroplast). Moreover, the findings that DNAs as well as RNAs significantly stimulate the CK-II catalyzed phosphorylation of p34 in vitro and induce its conformational change, suggest that the physiological activity of p34-bound RNA or DNA in chloroplast post-transcriptional regulation is controlled by specific p34 phosphorylation by CK-II.
