Fig. 8: AAMDC interacts with the GTPase activating protein RabGAP1L. | Nature Communications

Fig. 8: AAMDC interacts with the GTPase activating protein RabGAP1L.

From: The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer

Fig. 8

a Colocalization of endogenous AAMDC and RabGAP1L proteins in luminal breast cancer cells assessed by immunofluorescence (IF). Hoechst 33258-stained nuclei (blue), α-AAMDC antibody (green), and α-RabGAP1L antibody (red). Arrows indicate regions of strong signal overlap. b Localization of AAMDC and RabGAP1L in adjacent representative sections of normal breast, and in selected estrogen receptor negative (ER−) or invasive ductal carcinoma (IDC) breast cancer specimens assessed by immunohistochemistry (IHC). c Superposition of the crystal structure of bacterial (1IHN, cyan, https://doi.org/10.2210/pdb1IHN/pdb)11,54 and human (2AB1, orange, https://doi.org/10.2210/pdb2AB1/pdb)55,74 AAMDC proteins (left) and the Phyre2 (v2.0) homology model of human RabGAP1L. The phosphotyrosine-binding (PTB) domain is shown in red and the C-terminal Tre-2/Bub2/CdC16 (TBC) Rab-binding domain in violet (right). d–f Interaction between AAMDC, RabGAP1L, and Rab7a by immunoprecipitation (IP) in HEK293T cells transiently transfected with full-length (d) or deletion mutants (e–f) of the tagged cDNAs: HA-RabGAP1L, FLAG-AAMDC, and Myc-Rab7a. The IPs were immunoblotted with an α-HA antibody to detect HA-RabGAP1L (98 kDa) or α-FLAG to detect FLAG-AAMDC (17 kDa). Deletion of the PTB domain is indicated by HA-RabGAP1LΔPTB(471) (48 kDa) (e), and deletion of the Rab-binding TBC domain by HA-RabGAP1LΔTBC(585) (64 kDa) (f); α-IgG-conjugated beads and beads only are used as a control. g Bioluminescence resonance energy transfer (BRET) assays in HEK293FT cells transiently overexpressing Venus-HA-Rab7a or Venus-Rab22a, RLuc8-AAMDC, full-length RabGAP1L, RabGAP1LΔPTB, or RabGAP1LΔTBC. Control cells (Ctrl) are transfected with Venus-Rab and RLuc8-AAMDC only. The BRET ratio values are normalized to the respective controls. The individual values for n = 4 biological replicates (Rab7a) and for n = 3 biological replicates (Rab22a) are shown as mean values ± SEM, and statistical significance is determined using Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparison test (Rab7a: **p = 0.0096 for Ctrl vs. RabGAP1L WT, **p = 0.0069 for RabGAP1L WT vs. RabGAP1LΔPTB, and not significant (ns) for RabGAP1L WT vs. RabGAP1LΔTBC and for RabGAP1LΔPTB vs. RabGAP1LΔTBC; Rab22a: **p = 0.0024 for Ctrl vs. RabGAP1L WT, **p = 0.0045 for RabGAP1L WT vs. RabGAP1LΔPTB, **p = 0.0020 for RabGAP1L WT vs. RabGAP1LΔTBC, and ns for RabGAP1LΔPTB vs. RabGAP1LΔTBC).

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