Article Figures & Data
Figures
- FIG. 1.
SMN is ubiquitinated and degraded by the UPS. (A) Treatment of 3813 SMA patient-derived fibroblast with the proteasome inhibitor, MG132 (10 μM); the cell-permeable calpain inhibitor, Calpeptin; the lysosome inhibitor, ammonium chloride (NH4Cl); and an autophagy inhibitor, 3-MA. Quantification is shown in lower panel. (B) 3813 cells treated with 1, 5, or 10 μM concentrations of the proteasome inhibitor lactacystin. The data represent mean ± the standard error of the mean (SEM) of five independent experiments. *, P < 0.05. (C) Cell lysates were denatured with 1% SDS and then renatured with 4.5% Triton X-100 prior to immunoprecipitation with anti-SMN antibody. In addition, cell lysis buffer was supplemented with 2.5 μM ubiquitin aldehyde to inhibit deubiquitinating enzymes. Western bots were probed with antibodies to SMN, Gemin 2, Gemin 3, and Gemin 6. (D) Western blot probed with an antibody to ubiquitin after immunoprecipitation of SMN from cells in the presence or absence of 10 μM lactacystin.
- FIG. 2.
FL-SMN and SMNΔ7 are ubiquitinated and degraded by the proteasome. (A) Quantification of TCA-soluble SMN fragments after incubation of ubiquitinated or nonubiquitinated 125I-GST-FL-SMN with 5 nM 26S proteasome after 30 min. The data represent mean ± the SEM of three independent experiments. *, P < 0.05. (B) Rate of degradation of ubiquitinated 125I-GST-SMN and 125I-GST-SMNΔ7 after incubation with 5 nM purified 26S proteasome and precipitation, at the designated times, on ice with 10% TCA. The data represent mean ± the SEM of three independent experiments.
- FIG. 3.
SMNΔ7 is more rapidly degraded than FL-SMN. (A) Pulse-chase analysis of endogenous SMN, myc-SMN, and GFP-SMN in HEK293T cells. (B) Pulse-chase analysis of myc-SMN and SMNΔ7. (C) Pulse-chase analysis of myc-SMN and SMNΔ7 in the absence or presence of 10 μM MG132. (D) Pulse-chase analysis of SMNΔ7 in HEK293T cells after knockdown of endogenous SMN 72 h after transfection of shRNA targeting SMN. The data represent mean ± the SEM of three independent experiments. Inset shows Western blots of endogenous SMN, the shRNA-resistant myc-SMNΔ7 (designated by an asterisk) and actin at various time points after SMN shRNA transfection.
- FIG. 4.
Residues that are critical for oligomerization modulate SMN stabilization. (A) Pulse-chase analysis of SMN deletion mutants lacking exons 1 through 7. The data represent mean ± the SEM of three experiments. (B) Pulse-chase assay performed on FL-SMN, as well as SMN mutants Y272C and G279V, which show deficits in SMN complex formation, and E134K, which does not affect complex formation. The data represent mean ± the SEM of three experiments.
- FIG. 5.
SMN is stabilized by incorporation into the SMN complex. (A) Pulse-chase assay performed using Gemin 2 and Gemin 3 antibodies to identify SMN that may be in complex. The data represent the means ± the SEM of four experiments. (B) SMN from cells starved of cysteine-methionine for 2 and 12 h and pulse-labeled with radioactive cysteine-methionine, followed by 10 to 30% sucrose gradient separation. (C) Pulse-chase analysis of SMN from cells starved of cysteine-methionine for 2 and 12 h. The data represent mean ± the SEM of five experiments.
- FIG. 6.
SMN is stabilized by PKA. (A) Western blot of SMN from 3813 cells treated with forskolin (10 μM) for the indicated times. (B) Quantification of blot in panel A normalized to actin. (C) Western blot of SMN from cells overexpressing constitutively active PKA. (D) Autoradiograph of SMN and point mutants following in vitro PKA assay using purified catalytic subunit of PKA and purified GST-SMN as the substrate. (E) SMN turnover in the presence or absence of constitutively active PKA and the PKA inhibitor PKI.
- FIG. 7.
PKA increases the amount of SMN in complex. (A) Western blot of SMN immunoprecipitated from cells either expressing constitutively active PKA or the empty vector. Blots were probed with antibodies to Gemin 5, Gemin 3, Gemin 2, and SMN. (B) The relative association of gemins with SMN was obtained by normalizing the amount of coimmunoprecipitated Gemin to total SMN protein. Bar graph represents the relative amounts of coimmunoprecipitated Gemin in the presence of PKA to that in the absence of PKA. The data represent mean ± the SEM of three experiments. *, P < 0.05. (C) Quantification of SMN in sucrose gradient fractions from cells expressing constitutively active PKA or empty vector. The data represent mean ± the SEM of four experiments. *, P < 0.05.
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Fig. S1 (MG132 does not increase ROS).
Zipped TIF file, 15K. - Supplemental file 2
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Fig. S2 (Ubiquitination of GST-SMN and GST-SMNΔ7).
Zipped TIF file, 43K. - Supplemental file 3
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Fig. S3 (Cysteine/methionine starvation shifts Gemins sedimentation patterns).
Zipped TIF file, 158K. - Supplemental file 4
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Legends to Fig. S1 to S3.
Zipped MS Word document, 5K.
- Supplemental file 1
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Fig. S1 (MG132 does not increase ROS).
