A biosensor approach to probe the structure and function of the p85 alpha subunit of the phosphatidylinositol 3-kinase complex.
Phosphatidylinositol 3-kinase, which generates putative novel second messenger phospholipids, is a heterodimer composed of regulatory adaptor 85-kDa and catalytic 110-kDa subunits. The p85 alpha subunit contains a NH2-terminal src homology (SH) 3 domain, a region with homology to the product of the breakpoint cluster region (bcr) gene, and a COOH-terminal portion of the molecule which contains two SH2 domains, separated by a spacer region. In this study a panel of monoclonal antibodies (mAb) was raised against recombinant bovine p85 alpha to probe its multidomain structure in relation to function. These mAbs were characterized using a BIAcore biosensor instrument. Epitopes for nine mAbs were mapped in relation to the domain structure of p85 alpha using recombinant protein fragments expressed in bacteria. These mAbs were then used to map the sites on p85 alpha which are involved in growth factor receptor binding. Two interesting classes of functional mAbs were identified. First, mAb U14, whose epitope lies within the NH2-terminal SH2 domain of p85 alpha, blocked the interaction of p85 alpha with activated protein-tyrosine kinase receptors. Second, real-time binding experiments using phospholipid-containing vesicles showed that p85 alpha by itself could specifically bind certain phospholipids. Two mAbs (U9 and U15) with epitopes located in the inter-SH2 spacer region blocked the binding of lipids to this site. The relevance of these observations to understanding the relationship of structure to function of p85 and the phosphatidylinositol 3-kinase are discussed.
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Ludwig Institute for Cancer Research, London, United Kingdom.
Gout
I
I
Fry
M J
MJ
Panayotou
G
G
Dhand
R
R
Yonezawa
K
K
Kasuga
M
M
Waterfield
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015374:Biosensing Techniques; D001921:Brain / Q000201:enzymology; D002417:Cattle; D004229:Dithiothreitol / Q000494:pharmacology; D000939:Epitopes / Q000378:metabolism; D066246:ErbB Receptors / Q000276:immunology; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009857:Oncogenes; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010748:Phosphopeptides / Q000138:chemical synthesis; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051562:Proto-Oncogene Proteins c-bcr; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D010716:Phosphatidylinositols; D010743:Phospholipids; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D051562:Proto-Oncogene Proteins c-bcr; D004229:Dithiothreitol