A plant 126-kDa phosphatidylinositol 4-kinase with a novel repeat structure - Cloning and functional expression in baculovirus-infected insect cells

Phosphatidylinositol metabolism plays a central role in signaling pathways in animals and is also believed to be of importance in signal transduction in higher plants, We report here the molecular cloning of a cDNA encoding a previously unidentified 126-kDa phosphatidylinositol (PI) 4-kinase (AtPI4K beta) from the higher plant Arabidopsis thaliana. The novel protein posses ses the conserved domains present in animal and yeast PI 4-kinases, namely a lipid kinase unique domain and a catalytic domain. An additional domain, approximately 300 amino acids long, containing a high percentage (46%) of c harged amino acids is specific to this plant enzyme. Recombinant AtPI4K bet a expressed in baculovirus-infected insect (Spodoptera frugiperda) cells ph osphorylated phosphatidylinositol exclusively at the D4 position of the ino sitol ring. Recombinant protein was maximally activated by 0.6% Triton X-10 0 but was inhibited by adenosine with an IC50 of similar to 200 mu M. Wortm annin at a concentration of 10 mu M inhibited AtPI4K beta activity by simil ar to 90%. AtPI4K beta transcript levels were similar in all tissues analyz ed. Light or treatment with hormones or salts did not change AtPI4K beta tr anscript levels to a great extent, indicating constitutive expression of th e AtPI4K beta gene.