Figure 1.
Western analysis of calpain assays detects two SMN cleavage products.
(A) 1 mM CaCl2 and the indicated units of Calpain1 were incubated with U2-OS cell lysates. 30 µg total protein was used in each reaction. Both N-terminal and C-terminal cleavage products were observed with the indicated SMN antibodies (left). (B) Cells were mock transfected or transfected with either EGFP empty vector or EGFP-Calpastatin (CAST). Lysates were incubated in the absence (-) or presence (+) of 1 mM CaCl2 to activate SMN cleavage by endogenous calpains. Overexpression of calpastatin blocked calpain cleavage of SMN. (C) Cells were transfected with either EGFP-SMN or EGFP-SMN(D252A) and 1 mM CaCl2 (+, E, I) was added to the lysates. Where indicated, calpain cleavage was inhibited by addition of EGTA (E) or ALLN (I). Full-length GFP-SMN and cleavage products were detected by Western analysis using either N- or C-terminal SMN antibodies. As expected, the mock-transfected sample (M) did not contain GFP-tagged proteins. GAPDH was used as a loading control. *In the absence of calpain activation and protease inhibitors EGFP-SMN was subject to unknown protease(s), unrelated to calpains. Calpain cleavage products of EGFP-SMN that correlated to those observed upon calpain cleavage of endogenous SMN were studied.
Figure 2.
Sequence determinants of calpain cleavage of SMN.
(A) Schematic of SMN protein, showing relevant domains and amino acids. The Tudor domain, proline-rich (P-rich) region, and YG box are labeled. Solid and dotted lines indicate the strong and weak PEST motifs, respectively. The calpain cleavage region (CCR) and mapped calpain cleavage site (CCS) are labeled. (B-F) Internal deletions were created in EGFP-SMN and transiently expressed in U2-OS cells. Endogenous calpain cleavage assays and subsequent Western analysis was performed to determine calpain cleavage susceptibility. Full-length GFP-SMN and cleavage products were detected using N- or C-terminal SMN antibodies. (B) The PEST motif and CCR are necessary for calpain cleavage, whereas the Tudor domain is dispensable. (C) Smaller deletions within the PEST domain allow for calpain cleavage. The entire PEST motif is not necessary for calpain cleavage. (D–E) Sequence determinants of calpain within the CCR. The CCR was progressively refined within residues 183–189 and 192–194.
Figure 3.
Mapping the calpain cleavage site.
(A) Coomassie stained gel of HIS6-SMN/GST-Gemin2 heterodimers cleaved in vitro with indicated units of Calpain1 for 1 h. at 30°C. Full-length SMN (FL-SMN) as well as the N-terminal (N-SMN) and C-terminal (C-SMN) cleavage products are indicated with arrows. The C-terminal cleavage fragments were subjected to peptide fingerprint analysis. Asterisks (*) indicate full-length and truncated GST-Gemin2 proteins (see Fig. S2). (B) Western blot analysis of in vitro calpain assays. Antibodies recognizing the N- or C-terminus of SMN detected FL-SMN and SMN calpain cleavage products. The fraction of SMN cleavage was directly proportional to the amount of exogenous Calpain1 added. (C) Endogenous calpain cleavage assays were performed with EGFP-SMN containing small deletions within the calpain cleavage site. Double deletions blocked calpain cleavage, whereas single deletions did not.
Table 1.
Peptide fingerprint analysis of C-terminal SMN cleavage product.
Figure 4.
Deletion of the SMN C-terminus affects calpain cleavage.
(A) A representative Western blot (for illustrative purposes only) demonstrating the calpain cleavage susceptibility of several C-terminal mutants. (B) Quantification of GFP-SMN cleavage was determined from Western blots probed with N-terminal SMN antibodies followed by Cy3 conjugated secondary antibodies (see Methods). The average fraction (expressed as %) of calpain cleavage was calculated from six independent cell-free cleavage assays. Removal of amino acids 268-294 (ΔYG+) in SMN renders it more susceptible to calpain cleavage, whereas mutations in the YG box or exon 7 (ΔEx7) did not. Error bars represent the SEM. Asterisks (**) indicate p value <0.001, determined by two-tailed Student T-test.
Figure 5.
SMA mutations affect calpain cleavage.
(A, C, E) Illustrative Western blot demonstrating the calpain cleavage susceptibility of several N-terminal mutants. (B, D, F) Quantification of GFP-SMN cleavage was determined from Western blots probed with N-terminal SMN antibodies followed by Cy3 conjugated secondary antibodies (see Methods). The average % of calpain cleavage was calculated from six independent cell-free cleavage assays. Error bars represent the SEM. Asterisks indicate p value, where p<0.005 (*) or p<0.001 (**), determined by two-tailed Student T-test. (A, B) Calpain cleavage of SMN(D30N) was similar to WT, whereas SMN(D44V) was drastically reduced, below the limits of quantification. Therefore, GFP antibodies were used only for detection of the EGFP-SMN(D44V) protein. (C, D) Three mutations within the Tudor domain (I116F, E134K, and Q146Q) showed slightly reduced susceptibility to calpain cleavage, whereas A111G behaved similar to WT. (E, F) Calpain cleavage of A188S was modestly reduced, but its deletion (ΔA188) greatly reduced calpain cleavage, below the limits of quantification.
Figure 6.
Endogenous calpain cleaves SMN in the cytoplasm.
Cytoplasmic and nuclear extracts were prepared from U2-OS cells and used for cell-free calpain assays. Western blotting was performed to detect calpain cleavage of SMN, calpain activation, and fractionation efficiency. (A) Endogenous SMN was cleaved by endogenous calpain in the cytoplasm, but not in the nucleus. Extracts (30 µg) were left untreated (-), treated with 1 mM CaCl2 (+), or treated with calpain inhibitior (I) ALLN prior to CaCl2 addition. (B) Nuclear SMN was resistant to calpain cleavage even with the addition of exogenous Calpain1. Extracts (30 µg) were left untreated (-), treated with 1 mM CaCl2 and 1U of exogenous Calpain1 (++), or treated with calpain inhibitior (I) ALLN prior to addition of CaCl2 and Calpain1.