The regulatory subunits of PI3K, p85alpha and p85beta, interact with XBP-1 and increase its nuclear translocation

Abstract

Despite the fact that X-box binding protein-1 (XBP-1) is one of the main regulators of the unfolded protein response (UPR), the modulators of XBP-1 are poorly understood. Here, we show that the regulatory subunits of phosphotidyl inositol 3-kinase (PI3K), p85alpha (encoded by Pik3r1) and p85beta (encoded by Pik3r2) form heterodimers that are disrupted by insulin treatment. This disruption of heterodimerization allows the resulting monomers of p85 to interact with, and increase the nuclear translocation of, the spliced form of XBP-1 (XBP-1s). The interaction between p85 and XBP-1s is lost in ob/ob mice, resulting in a severe defect in XBP-1s translocation to the nucleus and thus in the resolution of endoplasmic reticulum (ER) stress. These defects are ameliorated when p85alpha and p85beta are overexpressed in the liver of ob/ob mice. Our results define a previously unknown insulin receptor signaling pathway and provide new mechanistic insight into the development of ER stress during obesity.

Figure 1

p85α and p85β interact with XBP-1s. (a) Silver staining of immunoprecipitated proteins with XBP-1 after infecting MEFs with Ad-XBP-1s and cross-linking with DSP (see Online Methods for details). (b) Tandem mass spectroscopy analysis of the band indicated with an arrowhead in a. (c) Immunoblotting for SH2 of p85 and XBP-1s proteins after immunoprecipitation (IP) of XBP-1 from MEFs infected with the indicated adenoviruses. WCL, whole cell lysate. (d) Immunoblots of SH2 and XBP-1 after XBP-1s immunoprecipitation. Total lysates were immunoblotted for SH2 and tubulin. (e) Western blot analysis for XBP-1 and HA after immunoprecipitation with HA-specific antibody crosslinked to beads. (f) Expression of XBP-1 target genes, Dnajb9, Pdia3 and Herpud1, in MEFs infected with Ad-XBP-1s, Ad-p85α, or Ad-XBP-1s and Ad-p85α. (g) mRNA levels of Dnajb9, Pdia3 and Herpud1 in MEFs infected with Ad-XBP-1s, Ad-p85β or Ad-XBP-1s and Ad-p85β. Experiments in c–g were independently repeated three times. Error bars are means ± s.e.m.; P values were determined by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2

p85α and p85β increase nuclear translocation of XBP-1s. (a,b) Nuclear protein amounts of XBP-1s and p85 in MEFs infected with Ad-XBP-1s and with increasing doses of Ad-p85α (a) or Ad-p85β (b). Lamin A/C was used as a control for nuclear protein level. (c) Left, western blot for HA in MEFs infected with p85αN-HA. Right, nuclear protein amounts of XBP-1s, p85αN-HA and lamin A/C in MEFs expressing XBP-1s together with increasing amounts of p85αN-HA. (d) Left, western blot for SH2 in MEFs infected with p85αΔBH-HA. Right, immunoblotting for HA in XBP-1 immunoprecipitates from MEFs infected with Ad-XBP-1s and with either p85α-HA or p85αΔBH-HA. (e) Immunoblotting of MEFs infected with Ad-XBP-1s or with Ad-XBP-1s and Ad-p85αΔBH-HA together. Nuclear proteins were immunoblotted with the indicated antibodies. (f) Western blot for p110 in XBP-1 immunoprecipitates and for XBP-1s, SH2 and p110 in whole-cell lysates from cells infected with Ad-XBP-1s, Ad-p110, Ad-XBP-1s and Ad-p110, Ad-XBP-1s, Ad-p110 and Ad-p85α or Ad-XBP-1s, Ad-p110 and Ad-p85β. (g) XBP-1s and lamin A/C protein levels after infection of MEFs with a constant dose of Ad-XBP-1s and increasing doses of Ad-p110. (h,i) Nuclear protein amounts of XBP-1s and lamin A/C in cells infected with Ad-XBP-1s and Ad-p85α (h) or Ad-p85β (i) and with increasing doses of Ad-p110. (j) XBP-1s nuclear protein amounts in the presence or absence of wortmannin (100 μM). Experiments were independently repeated three times.

Figure 3

Insulin increases nuclear transport of XBP-1s. (a) Nuclear and cytoplasmic protein amounts of XBP-1s, p85α and p85β in MEFs infected with a constant dose of XBP-1s and p85α-Flag and increasing doses of p85β-HA. (b) Nuclear and cytoplasmic protein amounts of XBP-1s, p85α and p85β in MEFs infected with a constant dose of XBP-1s and p85β-HA and increasing doses of p85α-Flag. (c) HA and SH2 blotting in Flag immunoprecipitates of cells infected with Ad-p85α-Flag and Ad-p85β-HA. (d) HA and Flag immunoblotting in p85α-Flag immunoprecipitates after insulin (500 nM) stimulation. (e) Nuclear protein amounts of XBP-1s in insulin (500 nM)-stimulated XBP-1s–infected MEFs. (f) mRNA levels of p85α and p85β in the p85α and p85β DKD cells. (g) Protein amount of p85 in the DKD cells. (h) Nuclear and total protein levels of XBP-1s in DKD cells that were infected with Ad-XBP-1s and stimulated with insulin for 10 and 15 min. (i) XBP-1 splicing assay in PLKO and DKD cells that were stimulated with tunicamycin (0.75 μg ml–1) for the indicated time periods. (j) Nuclear and total XBP-1s protein amount in PLKO and DKD cells after tunicamycin (2 μg ml–1) stimulation. Each experiment was independently repeated three times.

Figure 4

XBP-1s import to the nucleus is impaired in the ob/ob mice. (a) XBP-1 mRNA splicing in the WT and ob/ob liver during refeeding. (b) XBP-1s protein abundance in total liver lysates. The graph next to the blots depicts the XBP-1s/tubulin ratio at fasting and after 1 h of refeeding. (c) Nuclear XBP-1s protein amounts in WT and ob/ob liver during refeeding (top). Lysates were subjected to immunoprecipitation with XBP-1s–specific antibody and immunoblotted with SH2-specific antibody (bottom). (d–g) Gene expression level of Herpud1 (d), Dnajb9 (e), Pdia3 (f) and Calr (g) at fasting and during refeeding. (h) PERK phosphorylation on Thr980 in the liver of WT and ob/ob mice during refeeding. Experiments were repeated in three independent cohorts. Error bars are means ± s.e.m.; P values were determined by Student’s t test (#P = 0.6, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5

Overexpression of p85α and p85β in the liver of ob/ob mice increases glucose tolerance and establishes euglycemia. (a–f) Eight-week-old, male ob/ob mice were injected with Ad-LacZ (2.5 · 109 plaque-forming units (PFU) per mouse) (n = 7) or Ad-p85α (3 · 109 PFU per mouse) (n = 7) or Ad-p85β (2.5 · 109 PFU per mouse) (n = 7) through the tail vein. (a) Liver p85 abundance on day 8 after the injections. (b) Blood glucose concentrations (mg dl–1) after 6-h fasting on day 4 of the injections. (c) GTT on day 6 after injection. (d) XBP-1s nuclear protein amounts and PERK phosphorylation on Thr980 at fasting, and 1, 3 and 5 h after refeeding. (e,f) mRNA levels of Dnajb9 and Herpud1 in the liver of Ad-LacZ–injected (e) or Ad-p85α–injected (f) ob/ob mice during refeeding. (g–j) Eight-week-old, male ob/ob mice were injected with Ad-LacZ (2.5 · 109 PFU per mouse) (n = 6), Ad-p85αΔBH (2.5 · 109 PFU per mouse) (n = 6) or Ad-p85βΔBH (2.5 · 109 PFU per mouse) (n = 6) through the tail vein. (g) Western blotting for SH2 at day 8 after injection. (h) Blood glucose concentration (mg dl–1) after 6-h fasting on day 4 of the injections. (i) GTT on day 6 after injection. (j) XBP-1s nuclear protein amounts and Lamin A/C control levels in the liver at fasting and 1, 3 and 5 h after refeeding. Each experiment was repeated independently three times. Error bars are means ± s.e.m.; P values were determined by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6

Nuclear translocation of XBP-1s is impaired in the liver-specific Pik3r1−/−;Pik3r2−/− mice. Seven-week-old, male Pik3r1f/f;Pik3r2−/− mice were injected with Ad-LacZ (n = 6) (7.5 · 109 PFU per mouse) and Ad-Cre (n = 6) (7.5 · 109 PFU per mouse) via tail vein. (a) Schematic characterization of Cre-mediated recombination of Pik3r1 allele (left) and PCR products after recombination (right). (b) p85 protein amounts at day 15 after injection. (c) Nuclear protein amounts of XBP-1s at fasting and 1 h after refeeding. (d–f) mRNA levels of Dnajb9 (d), Herpud1 (e) and Pdia3 (f) at fasting and after 1 h refeeding. Experiments were repeated in three independent cohorts. Error bars are means ± s.e.m.; P values were determined by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).