Characterization of the Nucleotide-Binding Capacity and the ATPase Activity of the PIP3-Binding Protein JFC1

In this work, we demonstrate that the phosphatidylinositol 3,4,5-trisphosphate-binding protein JFC1 is an ATP-binding protein with magnesium-dependent ATPase activity. We show that JFC1 specifically binds to the ATP analog 8-azido-[α-32P]ATP. The affinity of JFC1 for [α-32P]ATP was 10× greater than its affinity for [α-32P]ADP; the protein did not appear to bind to [α-32P]GTP. JFC1 hydrolyzed [α-32P]ATP in a Mg2+-dependent manner. JFC1, which also hydrolyzed dATP, has a relatively high affinity for ATP, with a KMvalue of 58 µM, and a kcatvalue of 2.27 per min. The predicted amino acid sequence of JFC1 denotes a putative nucleotide-binding site similar to those in the GHKL ATPase/kinase superfamily. However, a truncation of JFC1 that contains boxes G2 and G3 but not boxes N and G1 of the Bergerat-binding site showed residual ATPase activity. Secondly, the antitumor ATP-mimetic agent geldanamycin, which inhibits the ATPase activity of Hsp-90, did not affect JFC1 ATPase. Therefore, the characteristics of the ATP-binding site of JFC1 are unique. Phosphatidylinositol 3,4,5-trisphosphate, a highaffinity ligand of JFC1 did not affect its ATPase kinetics parameters, suggesting that the phosphoinositide have a different role in JFC1 function.