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-[alpha-(32)P]ATP. The affinity of JFC1 for [alpha-(32)P]ATP was 10x greater than its affinity for [alpha-(32)P]ADP; the protein did not appear to bind to [alpha-(32)P]GTP. JFC1 hydrolyzed [alpha-(32)P]ATP in a Mg(2+)-dependent manner. JFC1, which also hydrolyzed dATP, has a relatively high affinity for ATP, with a K(M) value of 58 microM, and a k(cat) value 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 high-affinity ligand of JFC1 did not affect its ATPase kinetics parameters, suggesting that the phosphoinositide have a different role in JFC1 function.
Catz
S D
SD
Department of Molecular and Experimental Medicine, Division of Biochemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
Johnson
J L
JL
Babior
B M
BM
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism*; D016227:Benzoquinones; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D047029:Lactams, Macrocyclic; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
D000345:Affinity Labels; D001386:Azides; D016227:Benzoquinones; D003432:Cross-Linking Reagents; D003838:Deoxyadenine Nucleotides; D047029:Lactams, Macrocyclic; D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D011809:Quinones; D011993:Recombinant Fusion Proteins; C431146:SYTL1 protein, human; C060974:phosphatidylinositol 3,4,5-triphosphate; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; D008274:Magnesium; C026600:2'-deoxyadenosine triphosphate; C001277:geldanamycin