Interaction of p85 subunit of PI 3-kinase with insulin and IGF-1 receptors analysed by using the two-hybrid system.
Interaction of the p85 subunit of PI 3-kinase with the insulin receptor (IR) and the IGF-1 receptor (IGF-1R) was investigated using the two-hybrid system by assessing for his3 and lacZ activation in S. cerevisiae. The experiments were performed with the cytoplasmic beta domain (wild type or mutated) of IR and IGF-1R and p85 or its subdomains (N + C-SH2, N-SH2, C-SH2, SH3 + N-SH2). The results of his3 activation indicated that p85, N + C-SH2 and C-SH2 interact with both IR beta and IGF-1R beta, whereas N-SH2 and SH3 + N-SH2 interact only with IR beta. Interaction of p85 and N+C-SH2 with IR beta (delta C-43) or IGF-1R beta(delta C-43) in which the C-terminal 43 amino acids (including the YXXM motif) were deleted, persisted. The internal binding site thus revealed was not altered by further mutating Y960/F for IR or Y950/F for IGF-1R. Activation of lacZ upon interaction of p85 with IR beta(delta C-43) was 4-fold less as compared to IR beta. This activation with p85 and IGF-1R beta was 4-fold less as compared to IR beta and was somewhat increased (2-fold) for IGF-1R beta (delta C-43). Thus, the C-terminal domain in IGF-1R appears to exert a negative control on binding of p85 thereby providing a possible regulatory mechanism for direct activation of the PI 3-kinase pathway.
Lamothe
B
B
Institut Cochin de Génétique Moléculaire, INSERM U257, Paris, France.
Bucchini
D
D
Jami
J
J
Joshi
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004926:Escherichia coli; D007328:Insulin / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012697:Serine Endopeptidases; D001616:beta-Galactosidase / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D017931:DNA Primers; D007328:Insulin; D046911:Macromolecular Substances; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D017526:Receptor, IGF Type 1; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D001616:beta-Galactosidase; D000099160:LexA Repressor Protein