DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli.
To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBF1), we have obtained large amounts of this protein by expression in Escherichia coli. Bacterial GBF1 was shown to be phosphorylated very efficiently by nuclear extracts from broccoli. The phosphorylation activity was partially purified by chromatography on heparin-Sepharose and DEAE-cellulose and was characterized. It showed the essential features of casein kinase II activity: utilization of GTP in addition to ATP as a phosphate donor, strong inhibition by heparin, preference for acidic protein substrates, salt-induced binding to phosphocellulose, and salt-dependent deaggregation. The very low Km value for GBF1 (220 nM compared to approximately 10 microM for casein) was in the range observed for identified physiological substrates of casein kinase II. Phosphorylation of GBF1 resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex. The conditions of this stimulatory reaction fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors. The DNA binding activity of the endogenous plant GBF was shown to be reduced by treatment with calf alkaline phosphatase; this reduction was diminished by addition of fluoride and phosphate or incubation in the presence of casein kinase II and ATP.
Klimczak
L J
LJ
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
Schindler
U
U
Cashmore
A R
AR
D001937:Brassica / Q000201:enzymology*; D047390:Casein Kinase II; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D010944:Plants / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid
D004268:DNA-Binding Proteins; D009711:Nucleotides; D010750:Phosphoproteins; D008345:Manganese; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D008274:Magnesium