Fig 2: The crystal structure of AP2M1 bound to pS616-IRS1. a Ribbon diagram of the crystal structure of AP2M1 (residues 160–435) bound to pS-IRS1. pS-IRS1 is shown as sticks. b Surface drawing of AP2M1, with pS-IRS1 shown as sticks. c A close-up view of the surface drawing of AP2M1 colored by its electrostatic potential (blue, positive; red, negative; white, neutral). pS-IRS1 is shown as sticks

Fig 3: The SHP2-MAPK pathway promotes insulin-activated IR endocytosis. a HepG2 cells expressing IR-GFP WT were starved, treated with the indicated inhibitors for 2 h, treated without or with 100 nM insulin for 20 min, and stained with anti-GFP (IR; green) and DAPI (blue). Scale bar, 5 µm. b Quantification of the ratios of PM and IC IR-GFP signals of cells in a (DMSO, n = 47; DMSO + Ins, n = 56; IRi, n = 50; IRi + Ins, n = 65; U0126, n = 96; U0126 + Ins, n = 111; PD0325901, n = 26; PD0325901 + Ins, n = 54; SHP099, n = 43; SHP099 + Ins, n = 49; GDC0941, n = 57; GC0941 + Ins, n = 48; AKTi, n = 65; AKTi + Ins, n = 96; BI2536, n = 26; BI2536 + Ins, n = 24; Reversine, n = 37; Reversine + Ins, n = 27; mean ± s.d.; ****p < 0.0001; two-tailed unpaired t test). c Binding of IRS1 peptides to AP2M1 (residues 160–435). Input and proteins bound to IRS1 peptide beads were analyzed by SDS-PAGE and stained with Coomassie (CBB). The relative band intensities are shown below (mean ± s.d.; n = 5 independent experiments). d Isothermal titration calorimetry (ITC) analysis of binding between IRS1 peptides and AP2M1 (residue 160–435), with Kd indicated. e The IRS1 peptides were incubated with active SHP2 for the indicated durations, spotted onto membranes, and detected with the anti-pY612-IRS1 antibody. f Quantification of the relative SHP2 activity in e (mean ± s.d.; n = 4 independent experiments; ****p < 0.0001; two-tailed unpaired t test). g Model of the regulation of insulin-activated IR endocytosis by a phosphorylation switch on IRS1/2. Insulin-bound IR phosphorylates itself and IRS1/2, and activates the PI3K-AKT and MAPK pathways. SHP2 acts upstream of RAS-RAF and promotes the activation of MAPK pathway. p31comet binds to the IR-bound MAD2 and blocks IR-AP2 association to prevent premature IR endocytosis. In feedback regulation, activated ERK1/2 phosphorylate S616 and other sites on IRS1. SHP2 binds to the C-terminal phosphotyrosine site on IRS1 and dephosphorylates pY612 of the doubly phosphorylated IRS1 (pY612/pS616), thus promoting IRS1–AP2M1 association. p31comet is released from MAD2 by an unknown mechanism, allowing the assembly of an MCC-like complex on IR. MAD2- and IRS1/2-dependent AP2 recruitment and clustering trigger clathrin-mediated IR endocytosis. Source data are provided as a Source Data file

Fig 4: MYC-mediated IRS2 regulation of breast cancer stemness(A) ALDH activity of IRS1−/−, IRS2−/− SUM-159 cells expressing EV or IRS2 after treatment with the Myc inhibitor 10074-G5. The ALDH inhibitor DEAB was included as a negative control.(B) IRS1−/−, IRS2−/− SUM-159 cells expressing EV or IRS2 were assayed for mammospheres in the absence or presence of 10074-G5 in B27+ supplement.(C) Irs2−/− PyMT cells expressing EV, Myc-WT, and Myc-T58A were analyzed for mammospheres in B27+ supplement.(D) In vitro limiting-dilution assay of Irs2−/− PyMT cells expressing EV, Myc-WT, and Myc-T58A.(E) Model for IRS2/PI3K-dependent regulation of breast cancer stemness. IRS2/PI3K mediates IIS-induced inactivation of GSK3β through phosphorylation on serine 9. Inactive pS9-GSK3β lacks the ability to phosphorylate threonine 58 of MYC, thus blocking proteasome-mediated degradation of MYC and allowing active pS62-MYC to accumulate. Created with BioRender.com.The mammosphere data shown in all graphs represent the mean ± SD of a representative experiment performed three times independently. The ALDH data shown represent the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.See also Figure S4.

Fig 5: IRS2 supports self-renewal of CSC in a PI3K-dependent manner(A and B) IRS1−/−, IRS2−/− SUM-159 cells (A) and Irs1−/−, Irs2−/− PyMT cells (B) expressing empty vector (EV), IRS1, or IRS2 were assayed for mammospheres in B27+ supplement.(C) Irs1−/−, Irs2−/− PyMT cells expressing EV or Irs2 were assayed for mammospheres with B27+ or B27− supplement.(D and E) IRS2−/− SUM-159 cells (D) or Irs2−/− PyMT cells (E) were assayed for mammospheres in B27+ supplement.(F and G) ALDH activity was examined in SUM-159 cells (F) and PyMT cells (G). The ALDH inhibitor DEAB was included as a negative control.(H) Irs2fl/fl PyMT and Irs2−/− PyMT cells were injected into the mammary fat pads of NOD/SCID mice in 10-fold serial dilutions.(I and J) IRS1−/−, IRS2−/− SUM-159 cells (I) and Irs1−/−, Irs2−/− PyMT cells (J) expressing EV, Irs2-WT (WT), or Irs2-Y5F (Y5F) were assayed for mammospheres in B27+ supplement.(K and L) IRS1−/−, IRS2−/− SUM-159 cells (K) and Irs1−/−, Irs2−/− PyMT cells (L) expressing EV, Irs2-WT, or Irs2-Y5F were assayed for ALDH activity.(M) Irs1−/−, Irs2−/− PyMT cells expressing EV, Irs2-WT, or Irs2-Y5F were injected into the mammary fat pads of NOD/SCID mice in 10-fold serial dilutions. For (H) and (M), data are presented as a log-log plot, and the frequency of stem cells is calculated by extreme limiting-dilution analysis. The mammosphere data shown represent the mean ± SD of a representative experiment performed three times independently. The ALDH data shown represent the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.See also Figures S2 and S3.