A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2

Abstract

Using a series of immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

Figure 1

(A)The workflow of the cross-species PI3K immunoprecipitation (IP)-LC-MS/MS proteomics strategy from human vs. drosophila cells. The immunoprecipitation, tryptic digestion and high resolution shotgun LC-MS/MS analyses from both drosophila and human cells separately result in the Venn diagram showing the numbers of identified proteins and overlapping proteins. (B) The 49 possible PI3K interaction candidates of biological importance under various levels of insulin stimulation from drosophila S2R+ cells.

Figure 2

(A) The fold change of PI3K subunit IPs (Pi3k21b (p85) regulatory and Pi3k92e (p110) catalytic) in drosophila S2R+ cells. These 49 proteins represent the results of the human-drosophila reciprocal BLAST overlap analysis. Chico (IRS) is mainly associated with the catalytic subunit and Csw (SHP2) is mainly associated with the free regulatory subunit. (B) The p85 immunoprecipitation in 293T, NCI H929, BCR-ABL H929, MOLP8, and K562 cells and a mock IP control with no cell lysate immunoblotted for p110, GAB2, p85, SHP2 and IgG. (C) SHP2 immunoprecipitation in 293T, NCI H929, BCR-ABL H929, MOLP8, and K562 cells were immunoblotted for p110, GAB2, p85, SHP2 and IgG. All cell lines show a significant interaction of p85 with SHP2 even in the absence of p110. (D) Blots for p85, SHP2, GAB2 and actin control in H929 whole cell lysate under serum starved, full serum and insulin stimulation to test for protein expression levels of the core p85-SHP2-GAB2 ternary complex components.

Figure 3

(A) The p85 IP in H929 cells was immunoblotted for p110, GAB2, p85 and SHP2 under treatment with various tyrosine kinase inhibitors (imatinib, BKM120, NVP-AEW154, and GSK11220212). The intensity of each band was calculated by measuring the band intensity with ImageJ and averaging the band intensity of duplicates. (B) The SHP2 IP in H929 cells with immunoblots for p110, GAB2, p85 and SHP2 under treatment with various kinase inhibitors (GSK11220212, NVP-AEW154, BKM120, imatinib).The blot showed the complex with GAB2 and p85 was only disrupted by imatinib and p110 was unable to bind the p85-SHP2 complex. (C) A P85 IP after stimulation with growth factors (FCS, Starvation, IGF1, EGF, PDGF, and Insulin) followed by immunoblots for p110, GAB2, p85 and SHP2 showed the disruption of the ternary complex only with insulin and IGF1. (D) A SHP2 IP after stimulation with growth factors (FCS, Starvation, PDGF, EGF, IGF1, and Insulin) followed by immunoblots for p110, p85, GAB2 and SHP2 showed the disruption of the ternary complex only with insulin and IGF1.

Figure 4

(A) The immunoblots of p85 (free p85 + p110 heterodimer) and p110 (only p110 heterodimer) IPs in H929 cells for quantification of SHP2 with equal p85 levels. The bar blots were generated by measuring the band intensity with ImageJ. (B) Immunoblots of GAB2 and actin control from stable GAB2 shRNA KD H929 cells. (C) IPs of p85 and SHP2 was performed in GAB2 KD and control cells the under starvation and serum (FCS) stimulation followed by blots for p85 and SHP2. (D) Immunoblots for SHP2, GAB2 and actin control from stable SHP2 shRNA KD of H929 cells. A p85 IP was performed in SHP2 KD cells and control H929 cells followed by blots for GAB2, SHP2, p85 and IgG control. (E) p85α, GAB2 and actin control immunoblots in stable p85α shRNA KD and control cells SHP2 IP was performed in p85 KD cells and control cells with blots for GAB2, SHP2, p85 and IgG control. (F) In H929 cells, FLAG tagged -p85 wt, FLAG tagged p85 without iSH2, FLAG tagged iSH2, FLAG tagged p85 without cSH2, FLAG tagged p85 without nSH2 and a negative control (mock) were transient transfected, followed by a FLAG IP. The eluent of the IP was blotted for FLAG peptide and SHP2. (G) Far-Western blots were performed from SHP2 IPs of serum stimulated or starved cells incubated with either GST-tagged p85, GST- tagged SH2 domain, SHP2 antibody or GST control followed by secondary antibody. The tagged p85 and the GST- tagged SH2 domain but not the control binds positively to SHP2 under both conditions. (H) Far-Western blots were performed by from p85 IPs of serum stimulated or starved cells incubated with a GST-tagged SHP2, p85 antibody or GST control followed by secondary antibody against the GST tag. The tagged SHP2 but not the control binds positively to p85 under both conditions.

Figure 5. Model for BCR-ABL positive H929 signaling and the SHP2-p85 direct complex.

Under normal growth conditions, SHP2 is bound to the majority of p85 on its SH2 domains initiating a complex with GAB2. A minor portion of p85 forms the PI3-kinase with p110 and GAB2 to produce low levels of AKT activity. When H929 cells is stimulated with insulin, p85 dissociates from SHP2 resulting in a disruption of the SHP2-p85-GAB2 complex. This causes p85 to mainly form a heterodimer complex with p110, resulting in AKT activation.