A Plant 126-kDa Phosphatidylinositol 4-Kinase with a Novel Repeat Structure

Abstract

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 encod- ing a previously unidentified 126-kDa phosphatidyli- nositol (PI) 4-kinase (AtPI4K beta ) from the higher plant Arabidopsis thaliana. The novel protein possesses the conserved domains present in animal and yeast PI 4-kinases, namely a lipid kinase unique domain and a cata- lytic domain. An additional domain, approximately 300 amino acids long, containing a high percentage (46%) of charged amino acids is specific to this plant enzyme. Recombinant AtPI4K beta expressed in baculovirus-in- fected insect (Spodoptera frugiperda) cells phosphoryl- ated phosphatidylinositol exclusively at the D4 position of the inositol ring. Recombinant protein was maximally activated by 0.6% Triton X-100 but was inhibited by adenosine with an IC50 of ~200 µM. Wortmannin at a concentration of 10 µM inhibited AtPI4K beta activity by ~90%. AtPI4K beta transcript levels were similar in all tis- sues analyzed. Light or treatment with hormones or salts did not change AtPI4K beta transcript levels to a great extent, indicating constitutive expression of the AtPI4K beta gene.