Fig. 5: RAPL inhibits the phosphorylation of LC3 at Thr50 by Mst1. | Nature Communications

Fig. 5: RAPL inhibits the phosphorylation of LC3 at Thr50 by Mst1.

From: The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling

Fig. 5

a Direct binding between RAPL and ATG8 family proteins. Pull-down assays were performed using purified GST-fused RAPL and 293T lysates expressing CLIP-LC3 or flag-GABARAP. Three independent experiments were performed. b Interaction of RAPL with purified LC3. Immunoprecipitation of myc-tagged RAPL in 293T cell lysate was followed by the addition of recombinant LC3b. Myc-RAPL and LC3 were detected with anti-Myc and anti-LC3 antibodies, respectively. Two independent experiments were performed. c FRET assay between RAPL and LC3. FRET between mCherry-RAPL and YPet-LC3 in adhered lymphocytes was measured (number of cells measured: n = 20). Lymphocytes expressing YPet-FL-mCherry24 (positive control, PC, n = 17), or mCherry-RAPL with YPet alone (n = 20) were used as positive and negative controls, respectively. d Role of LC3 phosphorylation in the canonical autophagy pathway. In the presence of nutrient starvation, Mst1 phosphorylates LC3b at Thr50 to inhibit binding of LC3b to FYCO1, an adaptor protein for kinesin, thereby promoting dynein-dependent transport of autophagosomes to the perinuclear region42. e Specific inhibition of Mst1-mediated LC3 phosphorylation at Thr50 by RAPL in 293T cells. Indicated tagged proteins were overexpressed in 293T cells and detected by western blotting with lysates and tag-specific antibodies. For Nore1A, α-tubulin and phosphorylated LC3 (p-LC3), antigen specific antibodies were used. N = 7. f Increased inhibition of LC3 phosphorylation caused by Rap1 activation in 293T cells. N = 7. g Effect of RAPL knockdown on LFA1 accumulation at the contact surface. BAF/LFA1 cells infected with virus harboring control shRNA (shCont): n = 40 or shRNA against RAPL (shRAPL): n = 44. h Effect of RAPL knockdown on LFA1(β2) and LC3 clustering. shCont: n = 38; shRAPL: n = 39. The error bars represent the SEM. Statistical analyses were performed using unpaired (g, h) nonparametric two-sided Student’s t tests, or one-way ANOVA with Dunnett correction (e), or Turkey correction (c, f) for multiple comparisons. The error bars represent the SEM. Source data are provided as a Source Data file.

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