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. Author manuscript; available in PMC: 2016 Sep 1.
Published in final edited form as: Nat Cell Biol. 2016 Feb 8;18(3):319–327. doi: 10.1038/ncb3311

Figure 4.

Figure 4

IGFBP5 is a major mediator of mTORC1-dependent feedback inhibition of IGF-1 signaling. (A) IGFBP5 and Grb10 together account for a major faction of the IGF-1-inhibitory activity of mTORC1. We generated TSC2−/− MEFs with single IGFBP5 or Grb10 knockdown, as well as IGFBP5+Grb10 double knockdown. Where indicated, cells were also treated with rapamycin (20 nM for 24 hrs). Cells were stimulated with IGF-1 (at indicated concentrations). For site-specific phosphorylation, pIGF-1R(Y1135/1136), pAkt(S473), pS6K(T389) and pERK(T202/Y204) levels were analyzed. (B) Grb10 and IGFBP5 double knock down cells show increased proliferative responses in IGF-1-supplemented media. P <0.001 (two-way ANOVA test). n=9 independent biological replicate experiments. Error bars represent s.d. (C) Cancer-associated frameshift and nonsense mutations that have been reported for IGFBP5 (data from COSMIC). A complete list of the somatic mutations is shown in Supplementary Table 8. (D) and (E) IGFBP5 expression constructs harboring cancer-associated mutations were transfected into HEK293T cells. Cells were starved, and the corresponding CM was collected, mixed with IGF-1 and was incubated with wild-type MEFs (recipient cells). WCL was analyzed by immunoblotting experiments using the indicated antibodies. Cancer-associated mutations of IGFBP5 are shown that either disrupt (D) or maintain (E) its IGF-1-inhibitory activity. For site-specific phosphorylation, pIGF-1R(Y1135/1136) levels were analyzed. (F) NCI-H1435 cells harbor heterozygous IGFBP5 mutation (E202*). Genomic fragment in the 3rd exon of IGFBP5 from NCI-H1435 and HCC15 cell lines were amplified with PCR and were sequenced. The red arrows indicate the mutation (G to T) in the NCI-H1435 cell line. HCC15 cells contain wt IGFBP5. (G) NCI-H1435 cell line is sensitive to IGF-1R inhibitor, BMS-536924. NCI-H1435 and HCC15 NSCLC cell lines were seeded overnight in 6 well plates at the same density. After 48 hours treatment with BMS-536924 or Sunitinib, 6 well plates were fixed with methanol and then were stained with crystal violet.