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. 2010 Dec 30;5(12):e15769. doi: 10.1371/journal.pone.0015769

Figure 2. Sequence determinants of calpain cleavage of SMN.

Figure 2

(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.