Cyclin G2 is a target of calpain-mediated proteolysis.

<p>A) OV2008 cells were transiently transfected with full-length cyclin G2 (CCNG2-V5), PEST-24, or ΔPEST and lysed in a buffer containing either calcium, purified calpain-1 (CAPN-1), or the combination of calcium and CAPN-1, with or without the calpain inhibitor, calpeptin. Calpain-1 induced the degradation of full length cyclin G2 and PEST-24 and this effect was attenuated by calpeptin. However, the level of ΔPEST was not affected by either calpain or calpeptin. B) Calpain promoted the degradation of cyclin G2 in ovarian cancer cells. ES2 and SKOV3.ip1 cells stably transfected with FLAG-CCNG2 were lysed in a buffer containing calcium, CAPN-1, and/or calpeptin, as indicated. The combination of calcium and calpain dramatically decreased cyclin G2 levels, whereas calpeptin reversed this effect. C) Inhibition of calpain activity enhanced cyclin G2 levels. OV2008 cells were transiently transfected with the FLAG-CCNG2 plasmid and treated with or without 50μM calpeptin for 2h or 6h. Western blot analysis demonstrated a protective effect of calpeptin on cyclin G2 stability. D) Silencing of calpains increased cyclin G2 levels. Top panel, OV2008 cells were transfected with siRNAs for calpain-1 (si-CAPN-1) or calpain-2 (si-CAPN-2) and casein zymography was performed to confirm the down-regulation and specificity of each siRNA for its respective calpain. Calpain-2 is identified by higher mobility on the gel. Bottom panel, OV2008 cells were transfected with si-CAPN-1 or si-CAPN-2 for 6 hours prior to overnight (16 hour) transfection of FLAG-CCNG2. Cells were recovered for 6 hours in the presence of CHX. Both calpain siRNAs increased cyclin G2 stability. NC, non-targeting control.