Structure of 85 kDa subunit of human phosphatidylinositol 3-kinase analyzed by using monoclonal antibodies.
An 85 kDa subunit (p85 alpha) of phosphatidylinositol 3-kinase (PI-3K) has one SH3 and two SH2 regions [SH2(N) and SH2(C)], which direct protein-protein interaction. We have established eighteen hybridomas producing monoclonal antibodies against p85 alpha to study the structure-function relationship of this protein. Epitope mapping using a series of deletion mutants expressed in E. coli showed that the monoclonal antibodies bound to at least 5 distinct epitope regions, which were well dispersed on p85 alpha except for its carboxyl-terminus. Monoclonal antibodies against amino-terminal regions and polyclonal antibodies against carboxyl-terminal regions immunoprecipitated p85 alpha expressed in human cells and in E. coli. On the other hand, monoclonal antibodies against the central part of p85 alpha failed to immunoprecipitate p85 alpha efficiently; however, they could immunoprecipitate p85 alpha mutants with deletion of either the amino- or the carboxyl-terminal region. Similar results were obtained by immunocytochemistry using confocal microscopy. These results suggested that steric hindrance prevents binding of monoclonal antibodies to the central part of p85 alpha where SH2(N) is located. The SH2(N) may have a distinct function from SH2(C), which is located at the carboxyl-terminal region and has been shown to mediate the binding of PI-3K to activated growth factor receptors.
Tanaka
S
S
Department of Pathology, National Institute of Health, Tokyo.
Matsuda
M
M
Nagata
S
S
Kurata
T
T
Nagashima
K
K
Shizawa
Y
Y
Fukui
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003429:Cross Reactions; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D012150:Polymorphism, Restriction Fragment Length; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D013329:Structure-Activity Relationship; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases; D011994:Recombinant Proteins