Mutations in the inter-SH2 domain of the regulatory subunit of phosphoinositide 3-kinase: effects on catalytic subunit binding and holoenzyme function.
Class IA phosphoinositide 3-kinases (PI3Ks) represent a group of heterodimeric lipid kinases with important functions in cellular signal transduction. The regulatory p85 subunit constitutively binds to the catalytic p110 subunit and mediates the recruitment of the heterodimer to various membrane-localized proteins upon activation by a vast array of stimuli. The functional characterization of protein domains that mediate p85 function has been hampered by a lack of structural data. Therefore, we investigated a 35-aa region in the inter-SH2 domain of p85, reported to be necessary for binding of p110, by site-directed mutagenesis and evaluated the importance of individual amino acids for PI3K heterodimer formation. This approach led to the identification of an 11-aa region required for p110 binding in vitro and mesoderm induction during early Xenopus development in vivo. Further analyses revealed two pairs of hydrophobic amino acids within this region, which are particularly important for high-affinity intersubunit interaction. Thus, our data provide further insight into the molecular mechanisms of PI3K intersubunit interaction and led to the identification of new p85 mutant proteins with varying degrees of dominant-negative effects that will be helpful for future PI3K-related research.
Elis
Winfried
W
Department of Molecular Immunology, Albert Ludwigs University of Freiburg, Max Planck Institute for Immunobiology, D-79108 Freiburg, Germany.
Lessmann
Eva
E
Oelgeschlager
Michael
M
Huber
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D017931:DNA Primers; D019281:Dimerization; D008969:Molecular Sequence Data; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D018909:src Homology Domains / Q000235:genetics*
D017931:DNA Primers; D019869:Phosphatidylinositol 3-Kinases