Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2

Abstract

Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. We have found that the protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3'-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1 compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. Our results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner.

Figures

FIG. 1.

EGF-induced association of p85 with Gab1 is enhanced in Shp2−/− cells. (A and B) WT and Shp2−/− cells were serum starved for 24 h and subsequently stimulated with EGF (50 ng/ml) for 5 min or left unstimulated, as indicated. Cell lysates were subjected to immunoprecipitation with anti-Gab1 antibodies followed by SDS-PAGE and immunoblotting with anti-Shc, anti-Grb2, or anti-p85 antibodies. The same membrane was stripped and reprobed with anti-Gab1 antibodies to determine the amount of Gab1 protein precipitated from each sample. (C) Unstimulated or EGF-stimulated cell lysates were subjected to immunoprecipitation with anti-EGFR or anti-Gab1 antibodies followed by SDS-PAGE and immunoblotting with antiphosphotyrosine (pY) antibodies. IP, immunoprecipitation.

FIG. 2.

Increased activity of the PI3K pathway in response to EGF stimulation of Shp2−/− cells. (A) Serum-starved cells were treated for 5 min with EGF (50 ng/ml), and cell lysates were subjected to immunoprecipitations with anti-Gab1 antibodies, followed by immune-complex PI3K activity assays. (B) WT and Shp2−/− cells were left unstimulated or stimulated with EGF for the indicated times. Total cellular proteins (50 μg) were resolved by SDS-PAGE and immunoblotted with anti-phospho-Erk or anti-phospho-Akt antibodies that recognize pSer473 or pThr308, as indicated. The same blot was stripped and reprobed with anti-total Akt and anti-total Erk1/2. (C) Lysates from control or EGF-stimulated cells were immunoprecipitated with anti-Akt antibodies, and kinase activities were determined with recombinant GST-GSK-3β as a substrate. The amount of Akt in the immune complexes was assessed by immunoblotting with anti-Akt. (D) The indicated cells were stimulated with EGF as described for panel C. Total cellular protein (50 μg) was subjected to immunoblotting with anti-phospho-GSK-3β antibodies. The blot was then stripped and reprobed with anti-total GSK-3β. IP, immunoprecipitation; MAPK, mitogen-activated protein kinase.

FIG. 3.

Increased Gab1-p85 association and elevated Akt activity in NIH 3T3 cells inducibly overexpressing catalytically inactive (C/S) Shp2. (A) Inducible Shp2 C/S cells were serum starved overnight in the presence (+Tet) or absence (−Tet) of tetracycline before stimulation with EGF (50 ng/ml) for 5 min. Total cellular proteins (50 μg) were resolved by SDS-PAGE and immunoblotted with anti-Shp2. Note that, following induction, expression of HA-Shp2 (C/S) was about twice that of endogenous Shp2. (B) Inducible Shp2 C/S cells were serum starved and stimulated as described for panel A. Gab1 immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with anti-p85 antibodies. The blot was then stripped and reprobed with anti-Gab1. Note that Gab1 associates with both p85α and p85β in these cells. (C) Inducible Shp2 C/S cells were serum starved and stimulated as for panel A. Akt phosphorylation was examined by immunoblotting with anti-phospho-Akt (Ser-473) antibodies, after which the membrane was stripped and reprobed with anti-total Akt. (D) 293 cells were transiently transfected with HA-WT Gab1 or HA-Gab1ΔShp2 constructs. After 24 h, cells were serum starved overnight and then stimulated with EGF (50 ng/ml) for 5 min. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies followed by SDS-PAGE and immunoblotting with anti-p85 antibodies. The blot was then stripped and reprobed with anti-HA antibodies to ensure comparable loading. IP, immunoprecipitation.

FIG. 4.

Far-Western analysis of Gab1-p85 interaction in WT and Shp2−/− cells. Gab1 immunoprecipitates, prepared from WT and Shp2−/− cells stimulated with EGF or left unstimulated as indicated, were resolved by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with GST-p85-N-SH2 (A) or GST-Shp2-N+C-SH2 (B), followed by anti-GST and secondary antibodies. Total Gab1 levels in the Gab1 immune complexes are shown at the bottom. IP, immunoprecipitation.

FIG. 5.

Reexpression of WT Shp2 in Shp2−/− cells. (A) WT Shp2 expression was restored to Shp2−/− cells by retroviral gene transduction (see Materials and Methods). Pooled puromycin-resistant clones were assessed for Shp2 expression by immunoblotting. The level of Shp2 expression in WT cells also is shown. Note the N-terminally truncated Shp2 protein in Shp2−/− cells and the near-WT levels of WT Shp2 in the reconstituted cells. (B) The indicated cell lines were serum starved and then stimulated with EGF (50 ng/ml) for 5 min or left unstimulated. Gab1 immunoprecipitates were subjected to SDS-PAGE and anti-p85 immunoblotting. The levels of Gab1 in the immunoprecipitates were detected by stripping the membrane and reprobing with anti-Gab1 antibodies. (C) The indicated cell lines were serum starved and stimulated with EGF for the indicated times or left unstimulated. Phospho-Akt and total Akt were detected by immunoblotting of total cell lysates. IP, immunoprecipitation.

FIG. 6.

PDGF- and IGF-1-evoked Akt activity is not enhanced in Shp2−/− cells. (A and B) Starved WT and Shp2−/− cells were stimulated with 50 ng of PDGF/ml (A) or 40 ng of IGF-1/ml (B) for the indicated times. Cell lysates were resolved by SDS-PAGE and subjected to immunoblotting with pSer473-specific Akt or pErk antibodies, as indicated. The membranes were then stripped and reprobed with anti-total Akt or anti-total Erk1/2 antibodies. (C) WT and Shp2−/− cells were serum starved for 24 h and subsequently stimulated with PDGF (50 ng/ml) for 5 min or left unstimulated, as indicated. Cell lysates were subjected to immunoprecipitation with anti-Gab1 antibodies, followed by SDS-PAGE and immunoblotting with anti-p85 antibodies. Total Gab1 levels in the Gab1 immune complexes are shown at the bottom. IP, immunoprecipitation; MAPK, mitogen-activated protein kinase.

FIG. 7.

Sustained EGF-induced relocalization of GFP-Gab1 to the plasma membrane in Shp2−/− cells. WT and Shp2−/− cells were transfected with a GFP-Gab1 expression vector. Forty-eight hours posttransfection, cells were stimulated with EGF (50 ng/ml) for the indicated times, fixed, and analyzed by fluorescence microscopy.

FIG. 8.

Model for regulation of kinetics of EGF-induced PI3K activation by Gab1-Shp2 interaction. Following EGF stimulation, the docking protein Gab1 is recruited to the plasma membrane through binding of its Met binding domain to the EGFR directly and through indirect recruitment via the Grb2 adapter protein. Phosphorylation of Gab1 by the EGFR and possibly other tyrosine kinases leads to recruitment and activation of multiple signal relay molecules, including PI3K. Activated PI3K catalyzes the production of PIP3. This initiates a positive feedback loop by recruiting more Gab1 molecules to the plasma membrane through binding of the PH domain of Gab1 to PIP3. By dephosphorylating the p85 binding sites on Gab1, Shp2 downregulates the Gab1-PI3K positive feedback loop, thereby controlling the extent, kinetics, and location of PI3K activation in response to EGF.