The SH3 and BH domains of the p85alpha adapter subunit play a critical role in regulating class Ia phosphoinositide 3-kinase function.
We have investigated the role of the SH3 and BH domains in the function of the p85alpha adapter/regulatory subunit of PI 3-kinase. In these studies epitope-tagged adapter subunit constructs containing wild-type p85alpha, p85alpha lacking the SH3 domain (deltaSH3-p85alpha), or p85alpha lacking the Rac-GAP/BCR homology (BH) domain (deltaBH-p85alpha) were coexpressed with either the p110alpha or p110beta PI 3-kinase catalytic subunit in HEK293 cells. The deletion of either BH or SH3 domains had no effect on the intrinsic activity of the PI 3-kinase heterodimers. However, the ability of activated Rac to stimulate PI 3-kinase activity was only observed in heterodimers containing the p85alpha and deltaSH3-p85alpha, indicating that rac binding to the BH domain is responsible for rac-induced stimulation of class Ia PI 3-kinase. We also investigated the effect of SH3 and BH domain deletion on the ability of insulin to induce recruitment of these constructs into phosphotyrosine-containing signaling complexes. We find that p85alpha expressed alone is poorly recruited into such signaling complexes. However, when coexpressed with catalytic subunit, the p85alpha adapter subunit is recruited to an extent similar to that of endogenous p85alpha. Maximal insulin stimulation caused a similar level of recruitment of p85alpha, deltaSH3-p85alpha, and deltaBH-p85alpha to signaling complexes when these adapter subunits were coexpressed with catalytic subunit. However, there was a higher level of basal association of the deltaSH3-p85alpha and deltaBH-p85alpha with tyrosine-phosphorylated proteins, meaning that the insulin-induced fold increase in recruitment was lower for these forms of the adapter. These results indicate that the N-terminal domains of p85alpha play a critical role in the way the adapter subunit responds to growth factor stimulation.
Beeton
C A
CA
Department of Biochemistry and Molecular Biology, University College London, United Kingdom.
Das
P
P
Waterfield
M D
MD
Shepherd
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D007328:Insulin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D017384:Sequence Deletion; D018909:src Homology Domains
D007328:Insulin; D019869:Phosphatidylinositol 3-Kinases