Regulation of Cyclin G2 Degradation in Human Ovarian Cancer Cells

Epithelial ovarian cancer has the highest mortality rate among gynecological malignancies. Uncontrolled cell growth lies at the core of EOC. Normal cell cycle progression is governed by temporal expression of canonical cyclin molecules, which then bind to and activate CDKs. Cyclin G2, an unconventional cyclin, negatively regulates cell cycle and its levels are lowered in various types of cancer. Our lab has previously reported that cyclin G2 is an unstable protein, which is degraded by the UPP. Through a series of in vitro assays, we showed that cyclin G2 is a target of calpains proteolytic activity in a number of human ovarian cancer cells. Furthermore, we found that inhibition of EGFR in those cells led to protection of cyclin G2 from degradation. Finally, we demonstrated that EGF stimulation also resulted in degradation of cyclin G2. Considered together, these findings suggest that activation of EGFR and calpain promote cyclin G2 degradation

Epidermal growth factor promotes cyclin G2 degradation via calpain-mediated proteolysis in gynaecological cancer cells

<div><p>Cyclin G2 (CCNG2) is an atypical cyclin that functions to inhibit cell cycle progression and is often dysregulated in human cancers. We have previously shown that cyclin G2 is highly unstable and can be degraded through the ubiquitin/proteasome pathway. Furthermore, cyclin G2 contains a PEST domain, which has been suggested to act as a signal for degradation by multiple proteases. In this study, we determined if calpains, a family of calcium-dependent proteases, are also involved in cyclin G2 degradation. The addition of calpain inhibitors or silencing of calpain expression by siRNAs strongly enhanced cyclin G2 levels. On the other hand, incubation of cell lysates with purified calpains or increasing the intracellular calcium concentration resulted in a decrease in cyclin G2 levels. Interestingly, the effect of calpain was found to be dependent on the phosphorylation of cyclin G2. Using a kinase inhibitor library, we found that Epidermal Growth Factor (EGF) Receptor is involved in cyclin G2 degradation and treatment with its ligand, EGF, induced cyclin G2 degradation. In addition, the presence of the PEST domain is necessary for calpain and EGF action. When the PEST domain was completely removed, calpain or EGF treatment failed to trigger degradation of cyclin G2. Taken together, these novel findings demonstrate that EGF-induced, calpain-mediated proteolysis contributes to the rapid destruction of cyclin G2 and that the PEST domain is critical for EGF/calpain actions.</p></div

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.</p

Proposed role of EGF in cyclin G2 degradation.

<p>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.</p

Cyclin G2 is an unstable protein and can be degraded through a calcium-mediated mechanism.

<p>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 <i>de novo</i> 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 CaCl_2, 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.</p

The Solution Structures of Two Prophage Homologues of the Bacteriophage λ Ea8.5 Protein Reveal a Newly Discovered Hybrid Homeodomain/Zinc-Finger Fold

A cluster of genes in the <i>exoxis</i> region of bacteriophage λ are capable of inhibiting the initiation of DNA synthesis in <i>Escherichia coli</i>. The most indispensible gene in this region is <i>ea8.5</i>. Here, we report the nuclear magnetic resonance structures of two <i>ea8.5</i> orthologs from enteropathogenic <i>E. coli</i> and <i>Pseudomonas putida</i> prophages. Both proteins are characterized by a fused homeodomain/zinc-finger fold that escaped detection by primary sequence search methods. While these folds are both associated with a nucleic acid binding function, the amino acid composition suggests otherwise, leading to the possibility that Ea8.5 associates with other viral and host proteins