Fig 1: Sec14l3/SEC14L2 interact with VEGFR2 and RAB4A/5 A via complementary domains. a SEC14L2 interacts with VEGFR2 endogenously in HUVECs. HUVEC cells were starved and stimulated with 100 ng ml−1 VEGFa for 5 min before harvest for IP and immunoblotting. TCL, total cell lysate. IgG serves as a negative control. Quantification of the interaction from three independent experiments is shown on the right (n = 3). *p < 0.05. b, c Sec14l3 shows a much stronger association with WT-VEGFR2 than its kinase domain mutation form (KM-VEGFR2). Different forms of VEGFR2 were co-expressed with Flag-tagged Sec14l3 in HEK293T cells. WB, western blot. Quantification of the interaction is shown on the right (n = 3). t-tests were used for statistical analyses in a–c. d Schematic diagrams of Sec14l3 and its truncated mutants. Numbers above the diagram indicate the corresponding amino acid positions. ΔN indicates N-terminal CARL-TRIO domain deletion; ΔS, Sec14 domain deletion; ΔG, GOLD domain deletion. e Sec14l3 interacts with VEGFR2 via its Sec14 domain. HEK293T cells were transfected with different Sec14l3 domain deletion forms with VEGFR2 plasmid respectively and harvested for IP. f Sec14l3 specifically interacts with RAB4A and RAB5A. Different RAB plasmids were co-transfected with VEGFR2 into HEK293T cells respectively for co-IP assay. g, h SEC14L2 interacts with RAB4 (g) and RAB5 (h) endogenously in HUVECs. i Sec14l3 interacts with RAB4A via its CRAL-TRIO and GOLD2 domains. Source data are provided as a Source Data file
Fig 2: Sec14l3/SEC14L2 promote RAB5A/4A activation. a Sec14l3 interacts preferentially with GDP-bound state of RAB4A/5 A. HEK293T cells were transfected with Sec14l3-GFP and lysed for in vitro incubation with GTPγS/GDP loaded-RAB4A/5 A respectively. Quantification data of the relative protein levels of bound Sec14l3 are shown on the right (n = 3). *p < 0.05. b Recombinant Sec14l3 protein purified from E. coli binds preferentially to GDP-bound RAB4A/5 A in vitro. Recombinant GST-Sec14l3 protein purified from E. coli was loaded with GTPγS and immobilized for incubation with the same amount of GTPγS/GDP loaded-RAB4A/5A. Quantification data of the relative protein levels of bound RAB4A/5A are shown on the right (n = 3). *p < 0.05. c Knockdown of SEC14L2 alleviates RAB5A-GTP levels in HEK293T cells. NC or SEC14L2 shRNA transfected cell lysates were immunoprecipitated by an anti-RAB5A-GTP antibody or subjected to GST-R5BD pull down. Quantification of the relative protein level of RAB5A-GTP is shown on the right (n = 3). **p < 0.01, *p < 0.05. d Knockdown of SEC14L2 alleviates RAB4A-GTP level in HEK293T cells. NC- or SEC14L2 shRNA transfected HEK293T cell lysates were immunoprecipitated by anti-RAB4A-GTP antibody and quantification of the relative protein level of RAB4A-GTP is shown on the right (n = 3). *p < 0.05. t-tests were used for statistical analyses in a–d. e Proposed working model of Sec14l3/SEC14L2 for RAB5A/4A-mediated VEGFR2 trafficking. Upon ligand stimulation, VEGFR2 could be internalized and recycled through RAB5A and RAB4A mediated pathways. Sec14l3/SEC14L2 are beneficial for increasing RAB5A/4A in their GTP-bound forms to accelerate the movement of VEGFR2-containing vesicles to early endosomes and subsequently recycling endosomes, consequently preventing VEGFR2 from PTP1B-mediated dephosphorylation
Fig 3: Pathogenic LRRK2 or knockdown of RAB8A causes accumulation of EGF in a RAB4-positive endocytic compartment. A, HeLa cells were transfected with either empty pCMV vector or pathogenic LRRK2 or cotransfected with GFP-tagged RAB4, and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. B, cells were transfected as indicated followed by quantification of internalized fluorescent EGF at 10 (left) and 30 min (right). n = 3 independent experiments. *, p < 0.05; **, p < 0.01. C, example of HeLa cells cotransfected with GFP-RAB4 and either empty pCMV vector or pathogenic LRRK2. Live pictures were taken 20 min upon fluorescent EGF internalization, and arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. Scale bar, 10 µm. D, quantification of colocalization of Alexa647-EGF with GFP-RAB4 (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 6 independent experiments. ***, p < 0.005. E, example of HeLa cells cotransfected with GFP-RAB4 and either ctrl-siRNA or RAB8A-siRNA. Live pictures were taken as described above. Arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. Scale bar, 10 µm. F, quantification of colocalization of Alexa647-EGF with GFP-RAB4 (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 3 independent experiments. **, p < 0.01. All error bars represent S.E.M.
Fig 4: Accumulation of EGF in a RAB4-positive endocytic compartment and deficits in EGFR recycling due to G2019S LRRK2 expression are rescued by active RAB7A expression. A, example of HeLa cells cotransfected with GFP-RAB4 and either empty pCMV vector or pathogenic LRRK2 with or without RAB7A-Q67L expression as indicated. Live pictures were taken 20 min upon fluorescent EGF internalization, and arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. An independent picture (543 HeNe laser line) was acquired to confirm coexpression of the distinct mRFP-tagged RAB7A constructs in all cases. Scale bar, 10 µm. B, quantification of colocalization of Alexa647-EGF with GFP-RAB4 in cells coexpressing empty pCMV vector or G2019S LRRK2 in the presence or absence of mRFP-tagged RAB7A constructs as indicated (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 3 independent experiments. **, p < 0.01. C, HeLa cells were transfected with the indicated constructs, and cell extracts (30 µg) were analyzed by Western blotting for FLAG-tagged G2019S-LRRK2, mRFP-RAB7A protein levels (anti-RAB7 antibody), and GAPDH as a loading control. D, HeLa cells were transfected with either empty pCMV vector or pathogenic G2019S LRRK2 in the presence or absence of mRFP-tagged RAB7A constructs as indicated, and EGFR surface levels and EGFR recycling were determined at the indicated time points. n = 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001. A.U., arbitrary units. All error bars represent S.E.M.
Fig 5: Expression of dominant-negative RAB7A causes defects in EGFR trafficking, accumulation of EGF in a RAB4-positive endocytic compartment, and deficits in EGFR recycling, which are reversed upon active RAB8A expression. A, HeLa cells were transfected with either empty pCMV vector (ctrl) or with dominant-negative RAB7A (RAB7A-T22N) in the presence or absence of active RAB8A (RAB8A-Q67L), and surface-bound fluorescent EGF was quantified. n = 3 independent experiments. *, p < 0.05. B, cells were transfected with the indicated constructs followed by quantification of internalized fluorescent EGF at 10 (left) and 30 min (right). n = 3 independent experiments. *, p < 0.05; **, p < 0.01. C, example of HeLa cells cotransfected with GFP-RAB4 and either mRFP-RAB7A-T22N or mRFP-RAB7A-T22N and FLAG-tagged RAB8A-Q67L as indicated. Live pictures were taken 20 min upon fluorescent EGF internalization, and arrows point to GFP-RAB4–positive vesicles containing Alexa647-EGF. An independent picture (543 HeNe laser line) was acquired to confirm coexpression of the mRFP-tagged RAB7A constructs in all cases. Scale bar, 10 µm. D, quantification of colocalization of Alexa647-EGF with GFP-RAB4 in the presence or absence of the distinct RAB7A constructs as indicated (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 3 independent experiments. **, p < 0.01. E, quantification of colocalization of Alexa647-EGF with GFP-RAB4 in the presence or absence of RAB7A-T22N and RAB8A-Q67L constructs as indicated (Manders' coefficient 1 × 100) from 15–20 cells per experiment. n = 3 independent experiments. *, p < 0.05. F, HeLa cells were transfected with the indicated constructs, and cell extracts (30 µg) were analyzed by Western blotting for mRFP-tagged RAB7A-T22N, FLAG-tagged RAB8A-Q67L, and GAPDH as a loading control. G, HeLa cells were transfected with either empty pCMV vector or dominant-negative RAB7A-T22N in the absence or presence of RAB8A-Q67L, and EGFR surface levels and EGFR recycling were determined at the indicated time points. n = 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005. A.U., arbitrary units. All error bars represent S.E.M.
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