Dennd3 functions as a guanine nucleotide exchange factor for small GTPase Rab12 in mouse embryonic fibroblasts

Abstract

Small GTPase Rab12 regulates mTORC1 (mammalian target of rapamycin complex 1) activity and autophagy through controlling PAT4 (proton/amino acid transporter 4) trafficking from recycling endosomes to lysosomes, where PAT4 is degraded. However, the precise regulatory mechanism of the Rab12-mediated membrane trafficking pathway remained to be determined because a physiological Rab12-GEF (guanine nucleotide exchange factor) had yet to be identified. In this study we performed functional analyses of Dennd3, which has recently been shown to possess a GEF activity toward Rab12 in vitro. The results showed that knockdown of Dennd3 in mouse embryonic fibroblast cells caused an increase in the amount of PAT4 protein, the same as Rab12 knockdown did, and knockdown of Dennd3 and overexpression of Dennd3 were found to result in an increase and a decrease, respectively, in the intracellular amino acid concentration. Dennd3 overexpression was also found to reduce mTORC1 activity and promoted autophagy in a Rab12-dependent manner. Unexpectedly, however, Dennd3 knockdown had no effect on mTORC1 activity or autophagy despite increasing the intracellular amino acid concentration. Further study showed that Dennd3 knockdown reduced Akt activity, and the reduction in Akt activity is likely to have canceled out amino acid-induced mTORC1 activation through PAT4. These findings indicated that Dennd3 not only functions as a Rab12-GEF but also modulates Akt signaling in mouse embryonic fibroblast cells.

Keywords: Amino Acids Transport; Autophagy; Endosomes; G Proteins; Guanine Nucleotide Exchange Factor (GEF); Low Molecular Weight G Proteins; Membrane Trafficking; Rab Proteins; mTOR Complex (mTORC).

FIGURE 1.

Dennd3 regulates PAT4 degradation as an upstream activator of Rab12 in MEF cells. A, the efficiency of stealth RNAs against Dennd3 is shown. MEF cells stably expressing T7-Dennd3 were transfected with control or Dennd3 stealth RNAs. Cell lysates were analyzed by immunoblotting with the antibodies indicated. B, RNAi-mediated knockdown of Dennd3 in wild type MEF cells as demonstrated by RT-PCR. Gapdh was used as an internal control. The size of the molecular weight markers (bp) is shown on the left. C, Dennd3 knockdown resulted in an increase in the amount of PAT4 protein. MEF cells stably expressing HA-PAT4 were transfected with control or Dennd3 stealth RNAs. Cell lysates were analyzed by immunoblotting with the antibodies indicated. D, quantification of the results shown in C. E, the effect of Dennd3 knockdown on the degradation of PAT4 protein was canceled out by simultaneous expression of a constitutive active mutant of Rab12 (Rab12-QL). MEF cells stably expressing HA-PAT4 were transfected with control or Dennd3 stealth RNAs. One day after stealth RNA transfection, the cells were transfected with pEF-BOS control vector or pEF-FLAG-Rab12-QL, and 2 days after plasmid transfection cell lysates were analyzed by immunoblotting with the antibodies indicated. The positions of the molecular mass markers (in kDa) are shown on the left in A, C, and E. F, quantification of the results shown in E. *, p < 0.05; ***, p < 0.005. a.u., arbitrary units.

FIGURE 2.

Effect of Dennd3 knockdown on the level of active Rab12. A, cell lysates from control or Dennd3 knockdown MEF cells were incubated with beads coupled with T7-RILP-L1, a trapper of active Rab12 (15). Input and immunoprecipitates (IP) were analyzed by immunoblotting with the antibodies indicated. The positions of the molecular mass markers (in kDa) are shown on the left. B, quantification of the results shown in A. Note that Dennd3 knockdown caused a significant reduction in the amount of endogenous active Rab12. **, p < 0.01. a.u., arbitrary units.

FIGURE 3.

Effect of Dennd3 knockdown on the intracellular l-amino acid concentration. Control and Dennd3 knockdown cells were analyzed with an l-Amino Acid Quantitation kit (see “Experimental Procedures” for details). Note that knockdown of Dennd3 resulted in an increase in the amount of intracellular l-amino acid concentration. *, p < 0.05. a.u., arbitrary units.

FIGURE 4.

Effect of Dennd3 knockdown on autophagy. A, Dennd3 knockdown had no clear effect on autophagic flux. Control and Dennd3 knockdown MEF cells were cultured for 1 h under nutrient-rich (N) or starved (S) conditions in the absence or presence of 100 nm bafilomycin A1 (BafA1). Cell lysates were analyzed by immunoblotting with the antibodies indicated. B, Dennd3 knockdown had no clear effect on p62 degradation. Control and Dennd3 knockdown MEF cells were cultured under nutrient-rich or starved conditions for 2 h. Cell lysates were analyzed by immunoblotting with the antibodies indicated. The positions of the molecular mass markers (in kDa) are shown on the left in A and B. C, Dennd3 knockdown resulted in a slight decrease in the number of LC3-positive dots. Control and Dennd3 knockdown MEF cells were cultured for 1 h under nutrient-rich or starved conditions, fixed, and then immunostained with anti-LC3 antibody. Scale bar, 20 μm. D, quantification of the results shown in C. *, p < 0.05; **, p < 0.01; ***, p < 0.005. a.u., arbitrary units.

FIGURE 5.

Effect of Dennd3 knockdown on nutrient signals. A, Dennd3 knockdown resulted in a decrease in Akt activity. Control and Dennd3 knockdown MEF cells were starved for 1 h with HBSS and then restimulated for 15 min with culture medium before lysis. Cell lysates were analyzed by immunoblotting with the antibodies indicated. Note that Dennd3 knockdown had no effect on the phosphorylation level of S6K (p70S6K; second panel) but that a dramatic decrease in Akt activity (i.e. p-Atk) was observed (fourth panel). B, quantification of the results shown in A. *, p < 0.05; **, p < 0.01. a.u., arbitrary unit. C and D, insulin stimulation (C) or EGF stimulation (D) increased mTORC1 activity in Dennd3 knockdown cells in comparison with the control cells. Control and Dennd3 knockdown MEF cells were starved for 12 h with DMEM without FBS and then stimulated for 1 h with 250 nm insulin or for 30 min with 100 ng/ml EGF. Cell lysates were analyzed by immunoblotting with the antibodies indicated. It should be noted that insulin stimulation or EGF stimulation restored Akt activity in Dennd3 knockdown cells to the same level as in the control cells (middle panel in C and fourth panel in D, lanes 4–6) and increased mTORC1 activity (i.e. p-70S6K) (top panels in C and D, lanes 4–6). Increased phosphorylation of S6K was also observed in Dennd3 knockdown cells cultured in DMEM without FBS in the absence of insulin (top panel in C, lanes 1–3) or EGF (second panel in D, lanes 1–3). The positions of the molecular mass markers (in kDa) are shown on the left in A, C, and D.

FIGURE 6.

Effect of Dennd3 overexpression on mTORC1 activity and autophagy. A, Dennd3 overexpression resulted in a reduction in the intracellular l-amino acid concentration. Lysates of control and T7-Dennd3-expressing cells were analyzed with an l-Amino Acid Quantitation kit. *, p < 0.05. a.u., arbitrary unit. B, Dennd3 overexpression resulted in a reduction in the amount of TfR protein and mTORC1 activity independent of Akt activity. Control and T7-Dennd3-expressing MEF cells were starved for 1 h with HBSS and then restimulated for 15 min with culture medium. Cell lysates were analyzed by immunoblotting with the antibodies indicated. C, quantification of the results shown in B. *, p < 0.05; **, p < 0.01. D, addition of l-amino acids partially restored mTORC1 activity. Control and T7-Dennd3-expressing MEF cells were treated for 30 min with the l-amino acids indicated, and lysates of the cells were analyzed by immunoblotting with the antibodies indicated. E, quantification of the results shown in D. *, p < 0.05. F, Dennd3 expression resulted in an increase in the number of LC3-positive dots under nutrient-rich conditions. Control and T7-Dennd3-expressing MEF cells were cultured under nutrient-rich (N) or starved (S) conditions, fixed, and then immunostained with anti-LC3 antibody. Scale bar, 20 μm. G, quantification of the results shown in F. ***, p < 0.005. H, Dennd3 expression had virtually no effect on autophagic flux. Control and T7-Dennd3-expressing MEF cells were cultured for 1 h under nutrient-rich or starved conditions in the absence or presence of 100 nm bafilomycin A1 (BafA1). Cell lysates were analyzed by immunoblotting with the antibodies indicated. Note that the amount of LC3-II in T7-Dennd3-expressing cells increased only under nutrient-rich conditions, in comparison with the control cells (middle panel, compare lanes 1 and 4). The positions of the molecular mass markers (in kDa) are shown on the left in B, D, and H .

FIGURE 7.

Overexpression of Dennd3 modulates mTORC1 activity and autophagy in a Rab12-dependent manner. A, the effect of Dennd3 expression on mTORC1 activity was canceled out by knockdown of Rab12. Control and T7-Dennd3-expressing MEF cells were transfected with control siRNA or Rab12 siRNA, and cell lysates were analyzed by immunoblotting with the antibodies indicated. The positions of the molecular mass markers (in kDa) are shown on the left. B, quantification of the results shown in A. *, p < 0.05; **, p < 0.01. a.u., arbitrary unit. C, overexpression of Dennd3 resulted in an increase in the number of LC3-dots in a Rab12-dependent manner. Control and T7-Dennd3-expressing MEF cells were cultured as in A, fixed, and then immunostained with anti-LC3 antibody.