Project 4 - Covalent modification of a bifunctional enzyme responsible for fructose 2,6-bisphosphate metabolism in plants

Fructose 2,6-bisphosphate (Fru-2,6-P2) is a key signal metabolite contributing to the regulation of partitioning of recently assimilated carbon between sucrose and starch during photosynthesis, and also influencing the interconversion of critical metabolite pools in non-photosynthetic tissues. In higher plants the steady-state level of Fru-2,6-P2 is maintained by the complementary activities of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase that are responsible for its synthesis and degradation, respectively. Both enzymatic activities are found on a single bifunctional protein. Previous studies have shown that this enzyme is sensitive to allosteric modulation by a range of metabolic intermediates (e.g. Markham, J.E. and Kruger, N.J. (2002) Kinetic properties of a recombinant spinach leaf 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Eur. J. Biochem. - in press), and these properties have provided the basis for a detailed explanation for the regulation of photosynthetic carbon partitioning in leaves. Recently, a short publication has suggested that the bifunctional enzyme from arabidopsis leaves (like its counterpart in mammals) is susceptible to reversible (de)phosphorylation in vivo (Furumoto et al. (2001) Plant Cell Physiol. 42, 1044-1048), with the implication (by analogy to other organisms) that such covalent modification alters the kinetic characteristics of the enzyme and is important in physiological regulation of the Fru-2,6-P2 level.

The aim of this project is to determine whether the bifunctional enzyme in a cell suspension culture of arabidopsis undergoes reversible (de)phosphorylation and to assess the extent to which the phosphorylation state of this protein changes under different physiological conditions. We have chosen to work with a heterotrophic cell suspension culture because it is possible to grow a relatively homogeneous population of cells under closely defined conditions, alter the metabolic state of the cells through defined perturbations, and conveniently apply metabolic inhibitors and radiolabelled substrates to the medium. The work is likely to centre on the use of SDS-PAGE and 2D-electrophoresis followed by immunodetection (western blotting) of the bifunctional protein using available antibodies to determine the phosphorylation state of the enzyme. These studies will probably be complemented by direct measurement of the extent of phosphorylation following incubation of the cells in 32Pi and immunopurification of the bifunctional protein, again using specific antibodies. The project will ask "is the bifunctional enzyme sensitive to phosphorylation?", "does the phosphorylation state vary with the metabolic state of the cells?" and "does the phosphorylation state alter (or correlate with) the level of Fru-2,6-P2 in vivo?". Studies of the effect of known inhibitors of protein kinases and phosphoprotein phosphatase may be used to establish to what extent, and under which conditions, changes in phosphorylation state contribute to changes the level of this important signal metabolite.

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