Fig 1.
Cyclin G2 is an unstable protein and can be degraded through a calcium-mediated mechanism.
A) OV2008, SKOV3.ip1, or ES2 cells stably transfected with a FLAG-tagged cyclin G2 (FLAG-CCNG2) were seeded at equal densities and treated with 10μg/ml cychoheximide (CHX) to block de novo protein synthesis. Cells were lysed before CHX treatment (time 0) or at 1 to 5 hour after CHX treatment. Cyclin G2 levels were determined by Western blotting using an anti-FLAG antibody. B) Prediction of calpain cleavage site using CALPCLEAV. Many potential sites were found; however, the one located in the PEST domain between position 319 and 320 had the highest score. C) Lystaes of OV2008 or ES2 cells stably transfected with FLAG-CCNG2 were incubated in a buffer containing different concentrations of CaCl2, for 1–2 hours (OV2008) or 1 hour (ES2). Cyclin G2 levels were analyzed by Western blotting. Increased calcium concentration resulted in decreased amounts of cyclin G2. D) Upper panel: Schematic representation of three cyclin G2 constructs, full-length-CCNG2 (CCNG2), PEST-24 (containing the first 24 amino acid of the PEST domain), and ΔPEST (complete removal of the PEST domain). Lower panel: Cyclin G2 wild type and deletion constructs were used to transfect OV2008 cells. Following transfection, cells were treated with either DMSO as a control or 1μM of the calcium ionophore, A23187, with or without pre-incubation with 20μM ALLN for 30 minutes. Cells were lysed and Western blot analyses were performed. Treatment with A23187 decreased the levels of full length and PEST-24 cyclin G2 while the protease inhibitor, ALLN, protected cyclin G2 from degradation. A23187 and ALLN had no effect on the level of ΔPEST.
Fig 2.
Cyclin G2 is a target of calpain-mediated proteolysis.
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.
Fig 3.
Phosphorylation is involved in calpain-mediated cyclin G2 degradation.
A) OV2008 cells stably transfected with FLAG-CCNG2 were pretreated with CIP to dephosphorylate proteins and then further incubated with purified calpain-1 (CAPN-1) or calpain-2 (CAPN-2) and calcium. CIP treatment abolished the effect of calpains on cyclin G2 degradation. B) OV2008 cells were transfected with CCNG2-V5 and recovered in the presence of various concentrations of either DMAT, a CKII inhibitor, GF109203X, a PKC inhibitor, or PNU 112455A, a Cdk2/5 inhibitor. Cyclin G2 levels were analyzed by Western blot. CKII, PKC, and CDK5 inhibitors did not affect cyclin G2 stability. C) Effects of various kinase inhibitors on cyclin G2 degradation. MG-132 was used as a positive control. Western blot analysis showed that treatment with 10μM Tyrphostin AG1478, which inhibits EGFR, and PP2, which inhibits Src and EGFR, protected cyclin G2 from degradation. D) Aligned conserved residues of cyclin G2 from amino acid 262 to 311 across various species. The predicted EGFR and Src phosphorylation sites, at position 284 and 285, are well conserved in vertebrates. E) OV2008 stable cells that express cyclin G2 were treated with either DMSO or AG1478, for 30 minutes prior to lysing the cells. Whole cell lysates were further incubated with 5 mM calcium chloride and purified calpain-2 for 1 hour. Inhibition of EGFR blocked the effect of calpain on cyclin G2 degradation.
Fig 4.
EGF enhances cyclin G2 degradation.
A) OV2008 cells stably expressing cyclin G2 were treated with 10 μM MG-132, to block protein degradation, or 20ng/mL EGF, and their vehicle control for 1 hour, and chased with CHX to block translation for 2 hours. Treatment with MG-132 protected cyclin G2 from degradation, while treatment with EGF decreased cyclin G2 stability. B) ES2 cells were transiently transfected with a FLAG-CCNG2 construct or its vector control for 16h. The FLAG-CCNG2-transfected cells were then treated with CHX in the presence of various concentrations of EGF for 1h. Protein lysates were probed for anti-phospho-tyrosine (p-Tyr) and a band corresponding to the molecular size of EGFR was induced by EGF. A dose-dependent decrease of cyclin G2 stability was also observed. C) OV2008 cells were transiently transfected with FLAG-CCNG2 overnight and then treated with calpeptin (50 μM) for 30min, followed by EGF (20ng/ml) and CHX treatment for 2h. Inhibition of calpain blocked the effect of EGF on cyclin G2 degradation. D) SKOV3.ip1 transiently transfected with FLAG-CCNG2 were treated with or without EGF, in the presence or absence of MG-132. MG-132 attenuated the effect of EGF on cyclin G2 degradation. E) OV2008 cells were transfected with either full-length cyclin G2-V5 or PEST deletion mutants (PEST-24 or ΔPEST) and treated with EGF for 30 min before addition of CHX for another 2 hours. EGF increased degradation of the wild-type (WT) and PEST-24 cyclin G2, whereas removal of PEST domain abolished EGF-induced cyclin G2 degradation. F) OV2008 cells were treated with EGF (20 ng) or PBS as the control for 15 min prior to lysis. Casein zymography was performed to detect calpain 1 and calpain 2 activity. Clearing bands on the zamogram reveraled that EGF increased calpain 2, but not calpain 1, activity.
Fig 5.
Proposed role of EGF in cyclin G2 degradation.
EGF binds to its receptor, EGFR, to induce cyclin G2 phosphorylation, either directly or indirectly via downstream kinases. EGF may also activate calpain-2 to enhance its activity. Phosphorylated cyclin G2 is recognized and degraded by calpains.