Rab7 mutants associated with Charcot-Marie-Tooth disease exhibit enhanced NGF-stimulated signaling - PubMed (original) (raw)

Rab7 mutants associated with Charcot-Marie-Tooth disease exhibit enhanced NGF-stimulated signaling

Soumik BasuRay et al. PLoS One. 2010.

Abstract

Missense mutants in the late endosomal Rab7 GTPase cause the autosomal dominant peripheral neuropathy Charcot-Marie-Tooth disease type 2B (CMT2B). As yet, the pathological mechanisms connecting mutant Rab7 protein expression to altered neuronal function are undefined. Here, we analyze the effects Rab7 CMT2B mutants on nerve growth factor (NGF) dependent intracellular signaling in PC12 cells. The nerve growth factor receptor TrkA interacted similarly with Rab7 wild-type and CMT2B mutant proteins, but the mutant proteins significantly enhanced TrkA phosphorylation in response to brief NGF stimulation. Two downstream signaling pathways (Erk1/2 and Akt) that are directly activated in response to phospho-TrkA were differentially affected. Akt signaling, arising in response to activated TrkA at the plasma membrane was unaffected. However Erk1/2 phosphorylation, triggered on signaling endosomes, was increased. Cytoplasmic phospho-Erk1/2 persisted at elevated levels relative to control samples for up to 24 h following NGF stimulation. Nuclear shuttling of phospho Erk1/2, which is required to induce MAPK phosphatase expression and down regulate signaling, was greatly reduced by the Rab7 CMT2B mutants and explains the previously reported inhibition in PC12 neurite outgrowth. In conclusion, the data demonstrate a mechanistic link between Rab7 CMT2B mutants and altered TrkA and Erk1/2 signaling from endosomes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Rab7 CMT2B Mutants are Membrane Bound and Interact with TrkA.

(A) BHK cells were transfected with GFP-tagged Rab7 wild-type and CMT2B disease mutants. After 16 h of transfection, cells were fixed and permeabilized by saponin treatment. Enlarged perinuclear vesicular staining was observed for the CMT2B disease mutants. (B) PC12 cells were stimulated with NGF (200 ng/ml) for 15–60 min as indicated. Cells were lysed and samples of cell lysates were immunoblotted for TrkA and Rab7. Lysate samples containing equal amounts of protein were immunoprecipitated (IP) with a pAb directed against Rab7 and immunoblotted for TrkA. Subsequently, the membrane was stripped and reprobed for Rab7. (C) PC12 cells were transfected with wild-type GFP-Rab7 and stimulated with NGF for 60 min. Samples containing equal amounts of protein were immunoprecipitated with antibodies against GFP to isolate the GFP-Rab7 and subsequently immunoblotted for TrkA. The membrane was stripped and reprobed with antibodies against GFP. (D) PC12 cells were transfected with GFP-tagged Rab7 CMT2B disease mutants and stimulated with NGF for 60 min. Equal amounts of lysate samples were immunoprecipitated with antibodies against GFP to precipitate the GFP-Rab7 and subsequently immunoblotted for TrkA. The membrane was stripped and reprobed with antibodies against GFP. (B–D) Representative blots from one of three independent experiments are shown in each case.

Figure 2

Figure 2. Cells Expressing Rab7 CMT2B Mutants Exhibit Higher Levels of Phospho-TrkA Following NGF Stimulation.

(A) Transiently transfected PC12 cells expressing GFP, GFP-Rab7wt, GFP-Rab7L129F, GFP-Rab7K157N, GFP-Rab7N161T, GFP-Rab7 V162M were briefly stimulated with NGF (200 ng/ml) followed by surface stripping of ligand and reincubation in 0.1% BSA-DMEM for 2 h, 6 h and 24 h respectively. Subsequently, cells were lysed and equal amounts of protein were immunoblotted and probed for total and phosphorylated TrkA. Significantly, higher levels of phosphorylated TrkA were found at 2 h and 6 h. Equal amounts of protein from cell lysates were probed with antibodies against GFP. GFP-Rab7wt and GFP-Rab7 CMT2B mutant proteins are uniformly expressed in PC12 cells under these conditions as shown in the adjacent representative blot. (B) Films from four independent experiments were quantified by Image J analysis. Bar graphs show pTrkA in GFP-Rab7wt and CMT2B mutant expressing cells. In each case the amount of pTrkA was normalized to the amount of GFP protein expressed. The bar graph shows the fold-change in pTrkA as a function of pTrkA in the GFP only control at each respective time point. Error bars indicating mean±S.E.M., n = 4, **p<0.01, ***p<0.001. For detailed numerical values see Table 1.

Figure 3

Figure 3. Cells Expressing Rab7 CMT2B Mutants Exhibit Enhanced Erk Signaling Following NGF Stimulation.

(A) Transiently transfected PC12 cells expressing GFP, GFP-Rab7wt, GFP-Rab7L129F, GFP-Rab7K157N, GFP-Rab7N161T, GFP-Rab7 V162M were briefly stimulated with NGF (200 ng/ml) followed by surface stripping of ligand and reincubation in 0.1% BSA-DMEM for 2–24 h. Subsequently, cells were lysed and equal amounts of protein were immunoblotted and probed for total and phosphorylated Erk1/2. (B) Films from three independent experiments were quantified by Image J analysis. Bar graph shows levels of pErk1/2 in GFP-Rab7wt and CMT2B mutant expressing cells. In each case the amount of pErk1/2 was normalized to the amount of GFP protein expressed. The bar graph shows the fold-change in pErk1/2 as a function of pErk1/2 in the GFP only control at each respective time point. Error bars indicating mean±S.E.M., n = 3,*p<0.05; **p<0.01, ***p<0.001. For detailed numerical values see Table 1.

Figure 4

Figure 4. Cells Expressing Rab7 CMT2B Mutants Have no Effect on Akt Signaling.

(A) Transiently transfected PC12 cells expressing GFP, GFP-Rab7wt, GFP-Rab7L129F, GFP-Rab7K157N, GFP-Rab7N161T, GFP-Rab7 V162M were briefly stimulated with NGF (200 ng/ml) followed by surface stripping of ligand and reincubation in 0.1% BSA-DMEM for 2–24 h. Subsequently, cells were lysed and equal amounts of protein were immunoblotted and probed for total and phosphorylated Akt. (B) Films from three independent experiments were quantified by Image J analysis. Bar graph shows levels of pAkt in GFP-Rab7wt and CMT2B mutant expressing cells. In each case the amount of pAkt was normalized to the amount of GFP protein expressed. The bar graph shows the fold-change in pAkt as a function of pAkt in the GFP only control at each respective time point. Error bars indicating mean±S.E.M. n = 3, p values were not significant, indicating no differences between samples.

Figure 5

Figure 5. Cells Expressing Rab7 CMT2B Mutants Exhibit Altered Cytoplasmic and Nuclear Partitioning of Activated Erk1/2.

Transiently transfected PC12 cells expressing GFP, GFP-Rab7wt, GFP-Rab7L129F, GFP-Rab7K157N, GFP-Rab7N161T, GFP-Rab7 V162M were briefly stimulated with NGF (200 ng/ml) followed by surface stripping of ligand and reincubation in 0.1% BSA-DMEM for (A) 2 h and (B) 24 h. Subsequently, cells were lysed and subjected to subcellular fractionation and immunoblotted with antibodies directed against phosphorylated Erk1/2. The purity of fractions was confirmed by probing for the nuclear marker lamin B.

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