Calpain 3 Is a Rapid-Action, Unidirectional Proteolytic Switch Central to Muscle Remodeling
Figure 2
Verification of putative CAPN3 targets.
A,B,D) Skeletal muscle homogenates of 5–7 Calpainopathy patients (P1-7), 1–2 healthy controls (H1-2) and 2–4 unrelated muscular dystrophies (OPMD, MH, FSHD) were analyzed on western blot with specific antibodies for Tropomyosin (A, at 35 kDa), BOC (B, at 110 and 45 kDa) and PIAS3 (D, at 60 kDa). The arrows denote the detected protein bands. C) Schematic model of PIAS3 E3 SUMO ligase activity, regulated by CAPN3. PIAS3 is involved in the covalent conjugation of Small Ubiquitin Like Modifier (SUMO) proteins to target proteins. PIAS3 has a CAPN3 cleavage motif at the C-terminus. E) FLAG tagged PIAS1, 2, 3, and 4 were transiently expressed with active or inactive CAPN3, and cleavage was analyzed on western blot with a FLAG specific antibody. Upon co-expression with active CAPN3 PIAS3, PIAS1 and PIAS2 are cleaved at the C-terminus as predicted. PIAS4 is not cleaved. The arrows denote uncleaved and cleaved FLAG-PIAS. Nt means not transfected, + and – refer to active and inactive CAPN3 respectively. F) PIAS3 was transiently expressed in an in vivo sumoylation assay with HIS6-SUMO1 or HIS6-SUMO2 and active or inactive CAPN3. Cells were lysed directly in sample buffer and sumoylation levels were estimated on western blot with a HIS antibody (upper panel). Correct expression and CAPN3 mediated cleavage was confirmed with a FLAG antibody (lower panel).