Volume 13, Issue 3, Pages 341-355 (February 2004) Shp2 Regulates Src Family Kinase Activity and Ras/Erk Activation by Controlling Csk Recruitment  Si Qing Zhang, Wentian Yang, Maria I. Kontaridis, Trever G. Bivona, Gengyun Wen, Toshiyuki Araki, Jincai Luo, Julie A. Thompson, Burkhart L. Schraven, Mark R. Philips, Benjamin G. Neel  Molecular Cell  Volume 13, Issue 3, Pages 341-355 (February 2004) DOI: 10.1016/S1097-2765(04)00050-4 Copyright © 2004 Cell Press Terms and Conditions

Figure 1 Decreased SFK Activation in Shp2−/− Cells (A and B) SFK activation site tyrosines are hypophosphorylated and C-terminal inhibitory tyrosines are hyperphosphorylated in Shp2−/− cells. Serum-starved WT, Shp2−/−, and Rescue cells were treated with PDGF (50 ng/ml), EGF (50 ng/ml), or FGF (40 ng/ml). (A) Lysates resolved by SDS-PAGE were immunoblotted with phospho-specific Src antibodies. (B) Immunoprecipitates with rabbit anti-Src Ab (left) or MAb 327 (right) were immunoblotted with the indicated p-Src Ab. Blots were stripped and reprobed with rabbit anti-Src Ab. (C) Src activity is decreased in Shp2−/− cells. Starved or EGF-stimulated cell lysates were subjected to immune complex kinase assays. (D) Defective β1 Integrin-evoked SFK activation in Shp2−/− cells. Shp2−/− and Rescue cells were plated onto FN-coated plates in serum-free medium, and Src phosphorylation was analyzed by immunoblotting. To control for loading, blots were reprobed with rabbit anti-total Src Ab. Results shown for each panel are representative of two to five experiments. Numbers below each panel represent intensities of pSrc416 or pSrc529 adjusted to total Src levels, and normalized to a value of 1.0 for unstimulated Rescue cells. Molecular Cell 2004 13, 341-355DOI: (10.1016/S1097-2765(04)00050-4) Copyright © 2004 Cell Press Terms and Conditions

Figure 2 Multiple SFK Targets Are Hypophosphorylated in Shp2−/− Cells (A) Shp2−/− and Rescue cells were starved, then stimulated with EGF (50 ng/ml). Vav2, p62Dok, and Plcγ1 immunoprecipitates were subjected to SDS-PAGE and anti-pY or anti-pPlcγ1 immunoblotting, as indicated, then stripped and reprobed with anti-total p62Dok, Vav2, or Plcγ1 Ab, respectively. (B) The indicated cells were plated onto FN-coated plates in serum-free medium. Anti-Fak and anti-p190RhoGAP immunoprecipitates were subjected to anti-pY immunoblotting, then blots were reprobed for Fak and p190RhoGAP. (C) Hypophosphorylated proteins in Shp2−/− cells are SFK targets. The indicated immunoprecipitates from SYF−/−+Src and SYF−/− cells, respectively, were subjected to anti-pY immunoblotting. Blots were stripped and reprobed with anti-total p62Dok, Vav2, Plcγ1, or p190RhoGAP Ab. Molecular Cell 2004 13, 341-355DOI: (10.1016/S1097-2765(04)00050-4) Copyright © 2004 Cell Press Terms and Conditions

Figure 3 Acute Deletion of Shp2 Also Leads to Defective SFK Activation flShp2/flShp2 fibroblasts were infected with AdGFP or AdCre/GFP, followed by sorting for GFP+ cells. After stimulation with EGF (50 ng/ml), FGF (40 ng/ml), or PDGF (50 ng/ml) for 5 min, WT (AdGFP-infected) and KO (AdCre/GFP) lysates were immunoblotted with the indicated Ab. Under these conditions, about 80% of cells were deleted for Shp2. Numbers beneath the panel represent normalized intensities as in Figure 1, corrected for deletion efficiency (by applying a weighted average for the respective contribution of deleted and nondeleted cells). Molecular Cell 2004 13, 341-355DOI: (10.1016/S1097-2765(04)00050-4) Copyright © 2004 Cell Press Terms and Conditions

Figure 4 Shp2 Regulates Tyrosyl Phosphorylation of PAG (A) Left: 293 cells were transfected with Flag-PAG (2 μg), WT Shp2 (0.5–4 μg), and/or the Shp C459S (C/S) mutant (0.5–4 μg). After 24 hr, lysates from randomly growing cells were subjected to anti-Flag immunoprecipitations and anti-pY immunoblotting, and then reprobed with Flag Ab. Right: 293 cells transfected with Flag-PAG (2 μg) and either Shp2 WT or C/S (4 μg), as indicated, were lysed, immunoprecipitated with Flag Ab, and immunoblotted with Shp2 Ab. WT and mutant Shp2 expression in the total cell lysate also is shown. Note that Shp2 associates with PAG and this association is enhanced in cells expressing the substrate-trapping mutant Shp2 C/S. (B) 293 cells were transfected with the indicated combinations of Flag-PAG (2 μg), WT Shp2 (4 μg), and/or Shp2C/S (4 μg). After 24 hr, cells were starved overnight and stimulated with EGF (50 ng/ml) for 10 min or left unstimulated. PAG phosphorylation was analyzed as in (A). (C) 293 cells transfected as in (B) were plated onto FN-coated plates, and PAG phosphorylation was measured as in (A). Numbers beneath each panel represent Flag-PAG tyrosyl phosphorylation normalized to Flag-PAG levels and adjusted so that basal Flag-PAG tyrosyl phosphorylation equals 1.0. Molecular Cell 2004 13, 341-355DOI: (10.1016/S1097-2765(04)00050-4) Copyright © 2004 Cell Press Terms and Conditions

Figure 5 Shp2 Regulates Csk Localization and SFK Activation via PAG (A) Shp2 regulates endogenous PAG tyrosyl phosphorylation. Lysates from Rescue and Shp2−/− cells were subjected to immunoprecipitation with nonimmune (NI) or Csk Ab (left), or PAG Ab (right), followed by anti-pY immunoblotting. Blots were reprobed with Csk MAb or PAG Ab, respectively. The Csk-associated 90 kDa phosphotyrosyl protein (pp90) comigrates with tyrosyl phosphorylated PAG (data not shown). The blots shown represent one of three experiments. (B) Shp2 regulates Csk localization. Shp2−/− and Rescue cells on coverslips were transfected with Csk-GFP (5 μg), starved, and stimulated with EGF (50 ng/ml) for 10 min or left untreated. GFP images were obtained with a confocal microscope. (C) Stable overexpression of dominant-negative PAG in Shp2−/− cells. Shp2−/− cells were infected with pBabePAGY314F, pBabeShp2 (Rescue), or pBabe alone. PAG expression was detected by immunoblotting. (D) Dominant-negative PAG enhances RTK-evoked SFK activation. Shp2−/− cells expressing Shp2 (rescue), empty vector (pBabe), or PAGY314F were stimulated with EGF (50 ng/ml) or PDGF (50 ng/ml), and lysates were immunoblotted with anti-pSrc Ab. (E) Csk binding site in Paxillin is hyperphosphorylated in the absence of functional Shp2. Rescue and Shp2−/− cells were stimulated with EGF (50 ng/ml) for 5 min. Lysates were subjected to immunoblotting with the indicated Ab. Molecular Cell 2004 13, 341-355DOI: (10.1016/S1097-2765(04)00050-4) Copyright © 2004 Cell Press Terms and Conditions

Figure 6 Regulation of EM Ras and Erk Activation by Shp2 (A) WT, Shp2−/−, or SYF−/− fibroblasts were cotransfected with GFP-RBD and H-Ras. After 24, cells were starved, stimulated with EGF (40 ng/ml), and imaged by confocal microscopy. Ras activation (detected by GFP-RBD) occurs on PM (arrows) and EM (arrowheads) in WT cells, but only on the PM in cells lacking Shp2 or SFKs. (B) WT, Shp2−/−, or SYF−/− fibroblasts were transfected with GFP-H-Ras (top row) or GFP-RBD and untagged H-Ras alone (middle row) or with Shp2 or Src, respectively (bottom row). Transfected cells were grown in serum overnight and imaged as in (A). Reconstituting Shp2 expression to Shp2−/− cells or Src expression to SYF−/− cells restores EM Ras activation. (C) Growth factor-induced Erk activity is decreased in SYF−/− cells. SYF−/−+Src and SYF−/− cells were starved and stimulated with EGF (50 ng/ml) or PDGF (50 ng/ml). Lysates were immunoblotted with p-Erk Ab and then reprobed with Erk Ab. Note that Erk1 activation (p44) is defective in cells lacking SFKs. (D) Increasing Src activity in Shp2−/− cells enhances Erk activation. Left: Shp2−/− cells were infected with pBabe WT Src or parental pBabe. Stably infected pools were starved and then stimulated with EGF or left unstimulated. Lysates were analyzed by immunoblotting with the indicated Ab and compared to analogous lysates from Rescue cells. Right: Shp2−/− and Rescue cells were transfected with 20 nM Csk siRNA or mock-transfected, starved, and then stimulated with EGF (50 ng/ml) for 10 min. Lysates were immunoblotted with the indicated Ab. Csk siRNA significantly increases Erk activation even though Csk levels are reduced by only ∼40%. (E) PAGY314F increases growth factor-evoked Erk activation. Lysates from starved or EGF-stimulated Rescue cells, vector, or PAGY314F-infected Shp2−/− cells were immunoblotted with p-Erk Ab. The membrane was then reprobed with anti-p42 Erk Ab. Molecular Cell 2004 13, 341-355DOI: (10.1016/S1097-2765(04)00050-4) Copyright © 2004 Cell Press Terms and Conditions

Figure 7 Model for Regulation of Growth Factor and Integrin Signaling by Shp2 (A) Upon RTK stimulation, Shp2 is recruited to the PM, most likely by scaffolding adapters such as Gab1, and promotes SFK activation by dephosphorylating PAG, thereby regulating Csk recruitment. A similar mechanism, perhaps involving proteins such as Shps1/Sirpα1, may explain Shp2 activation of SFK during integrin signaling. (B) Activated SFKs are required for phosphorylation of several key targets, leading to the activation of distinct downstream signaling pathways. One important downstream pathway leads to EM Ras activation and late stage Erk activation via the Plcγ1/RasGRP pathway. Defective phosphorylation/activation of other SFK targets may help explain altered cell spreading, focal adhesions, and/or stress fibers in Shp2-deficient cells. See text for details. Molecular Cell 2004 13, 341-355DOI: (10.1016/S1097-2765(04)00050-4) Copyright © 2004 Cell Press Terms and Conditions