The second messenger binding site of inositol 1,4,5-trisphosphate 3-kinase is centered in the catalytic domain and related to the inositol trisphosphate receptor site.
A segment of inositol 1,4,5-trisphosphate 3-kinase responsible for inositol 1,4,5-trisphosphate (InsP(3)) binding was characterized and confirmed by three different approaches employing the fully active expressed catalytic domain of the enzyme. Part of this moiety was protected from limited tryptic proteolysis by InsP(3). Sequencing of two fragments of 16 and 21 kDa, generated in the absence or presence of InsP(3), respectively, identified segment Glu-271 to Arg-305 as being protected. 15 monoclonal antibodies, all binding to epitopes within this region, inhibited enzyme activity and interfered with inositol phosphate binding. Detailed enzyme kinetic parameters of 32 site-directed mutants revealed residues Arg-276 and Lys-303 in this segment and Arg-322, located nearby, as directly involved in and five other closely neighbored residues, all located within a segment of 73 amino acids, as also influencing InsP(3) binding. Part of this region is similar in sequence to an InsP(3) binding segment in InsP(3) receptors. Combined with the finding that mutants influencing only ATP binding all lie outside this region, these data indicate that an InsP(3) binding core domain is inserted between two segments acting together in ATP binding and phosphate transfer.
Bertsch
U
U
Institut für Medizinische Biochemie und Molekularbiologie, Universitäts-Krankenhaus Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany.
Deschermeier
C
C
Fanick
W
W
Girkontaite
I
I
Hillemeier
K
K
Johnen
H
H
Weglöhner
W
W
Emmrich
F
F
Mayr
G W
GW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D000939:Epitopes / Q000737:chemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015290:Second Messenger Systems; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D000939:Epitopes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D014357:Trypsin