Phosphorylation of a bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase, is regulated physiologically and developmentally in rosette leaves of Arabidopsis thaliana.
The phosphorylation status of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase (EC 2.7.1.105/ EC 3.1.3.46) in rosette leaves of Arabidopsis was examined. Immunoblotting with specific antisera detected 96-kDa and 92-kDa bands in the crude protein extracts from rosette leaves of Arabidopsis. Incubation of protein samples with alkaline phosphatase before SDS-PAGE reduced the 96-kDa band with concomitant increase of the 92-kDa band, suggesting that the former is a phosphorylated form of the latter. In accordance with this result, 96-kDa and 92-kDa bands were immuno-precipitated from the crude protein extracts from [(32)P]orthophosphate-labeled rosettes of Arabidopsis; and, the former was heavily labeled, the latter faintly labeled. Analysis of phospho-amino acid residues derived from the [(32)P]-labeled 96-kDa band revealed that the phosphorylation occurred on serine and threonine residues, excluding the possibility that the phosphorylated band represent a phospho-histidine intermediate that is known to form in the phosphatase reaction. The relative level of the 96-kDa band over the 92-kDa band in whole rosette extracts changed diurnally and was highest at the beginning of nighttime. Furthermore, the 96-kDa band was highly enriched in the extracts of very young rosette leaves, suggesting that the phosphorylation status of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase is regulated physiologically and developmentally in Arabidopsis.
Furumoto
T
T
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan. furumoto@lif.kyoto-u.ac.jp
Teramoto
M
M
Inada
N
N
Ito
M
M
Nishida
I
I
Watanabe
A
A
D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D025481:Phosphofructokinase-2 / Q000378:metabolism*; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology*
D017931:DNA Primers; D025481:Phosphofructokinase-2