Direct Association of Grb2 with the p85 Subunit of Phosphatidylinositol 3-Kinase

Abstract

Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to play a key role in growth factor signaling pathways, although its signaling mechanism has not been fully elucidated. Using the yeast interaction trap system, we have identified Grb2 as a PI 3-kinase interacting protein. Our experiments demonstrate that p85, the regulatory subunit of PI 3-kinase, interacts with Grb2 in vivo, and this interaction is independent of growth factor stimulation. The direct association between Grb2 and p85 was reconstituted in vitro with glutathione S-transferase fusion proteins. Domain analyses and peptide competition indicate that the association is mediated by the SH3 domains of Grb2 and the proline-rich motifs of p85 and that only one SH3 domain is required for minimal binding. The interaction does not displace the catalytic subunit of PI 3-kinase but is exclusive of Sos. Signaling through PI 3-kinase, therefore, may involve the ubiquitous adapter Grb2, which serves as a convergence point for multiple pathways.

Nerve growth factor induced stimulation of Ras requires Trk interaction with Shc but does not involve phosphoinositide 3-OH kinase

“…It is unclear how the appearance of PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates correlates with the level of PI 3-kinase product lipids in whole cells: the maximal amount of PI 3-kinase activity found in antiphosphotyrosine immunoprecipitates is only 4% of that which can be detected in anti-p85a immunoprecipitates. Despite reports of an inducible association between p85 and Shc in Bcr-Abl transformed chronic myelogenous leukemia cells (Harrison-Findik et al, 1995) and constitutive association between p85 and Grb2 in ®broblasts (Wang et al, 1995), we were unable to detect PI 3-kinase activity in Shc immunoprecipitates (data not shown). It has been suggested that a 115 kDa tyrosine phosphoprotein of unknown identity can associate with p85 following NGF treatment of PC12 cells (Ohmichi et al, 1994): this may account for PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates.…”

An oncogenic fusion product of the phosphatidylinositol 3-kinase p85β subunit and HUMORF8, a putative deubiquitinating enzyme

“…Therefore, signaling of PI 3-kinase through its speci®c phosphoinositide lipid products may remain intact and activate various downstream targets such as protein kinase C isomers, ras and akt (Carpenter and Cantley, 1996;Hemmings, 1997;Toker and Cantley, 1997). As in wildtype p85b the BCR-homology region of the chimeric protein could interact with the GTPbound forms of rac and Cdc42 (Carpenter and Cantley, 1996), while its proline-rich region and SH3 domain could bind abl, lck, lyn, fyn, dynamin, a, b and g tubulin, p36/38, GRB2, actinin, SHC, src, cbl, phosphoinositides and p85 to itself (Kapeller et al, 1994;1995;Pleiman et al, 1994;Gout et al, 1993, Susa et al, 1996Fukazawa et al, 1995;Wang et al, 1995;Shibasaki et al, 1994;Harrison-Findik et al, 1995;Liu et al, 1993;Meisner et al, 1995). Membrane localization of PI 3-kinase, either via N-terminal myristoylation or via C-terminal farnesylation signals, is su cient to stimulate downstream targets (Klippel et al, 1996).…”

Grb2 downregulation leads to Akt inactivation in heregulin-stimulated and ErbB2-overexpressing breast cancer cells

“…This is because in addition to binding to Sos, Grb2 can use its SH3 domain to bind to other proline-rich containing proteins, such as PI3 kinase (Wang et al, 1995), and Grb2-associated binder protein-1 (Gab-1) (HolgadoMadruga et al, 1996). Both PI3 kinase and Gab-1 can stimulate Akt activity (Holgado-Madruga et al, 1997;Wang et al, 1995).…”

“…This is because in addition to binding to Sos, Grb2 can use its SH3 domain to bind to other proline-rich containing proteins, such as PI3 kinase (Wang et al, 1995), and Grb2-associated binder protein-1 (Gab-1) (HolgadoMadruga et al, 1996). Both PI3 kinase and Gab-1 can stimulate Akt activity (Holgado-Madruga et al, 1997;Wang et al, 1995). The activation of Akt by PI3 kinase or Gab-1 appears to be Ras-independent because the binding of Grb2 to PI3 kinase or Gab-1 can exclude Sos from binding to Grb2, and could potentially stimulate Akt activity without stimulating Erk1,2 activity (Holgado-Madruga et al, 1997; Wang et al, 1995).…”