Proteolytic Cleavage of Cyclin E Leads to Inactivation of Associated Kinase Activity and Amplification of Apoptosis in Hematopoietic Cells

Abstract

Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G 1 to S in mammalian cells and has an established role in oncogenesis. Here we examined the role of deregulated cyclin E expression in apoptosis. The levels of p50-cyclin E initially increased, and this was followed by a decrease starting at 8 h after treatment with genotoxic stress agents, such as ionizing radiation. This pattern was mirrored by the cyclin E-Cdk2-associated kinase activity and a time-dependent expression of a novel p18-cyclin E. p18-cyclin E was induced during apoptosis triggered by multiple genotoxic stress agents in all hematopoietic tumor cell lines we have examined. The p18-cyclin E expression was prevented by Bcl-2 overexpression and by the general caspase and specific caspase 3 pharmacologic inhibitors zVAD-fluoromethyl ketone (zVAD-fmk) and N-acetyl-Asp-Glu-Val-Aspaldehyde (DEVD-CHO), indicating that it was linked to apoptosis. A p18-cyclin E 276-395 (where cyclin E 276-395is the cyclin E fragment containing residues 276 to 395) was reconstituted in vitro, with mutagenesis experiments, indicating that the caspase-dependent cleavage was at amino acid residues 272 to 275. Immunoprecipitation analyses of the ectopically expressed cyclin E 1-275 , cyclin E 276-395 deletion mutants, and native p50-cyclin E demonstrated that caspase-mediated cyclin E cleavage eliminated interaction with Cdk2 and therefore inactivated the associated kinase activity. Overexpression of cyclin E 276-395 , but not of several other cyclin E mutants, specifically induced phosphatidylserine exposure and caspase activation in a dose-dependent manner, which were inhibited in Bcl-2-overexpressing cells or in the presence of zVAD-fmk. Apoptosis and generation of p18-cyclin E were significantly inhibited by overexpressing the cleavage-resistant cyclin E mutant, indicating a functional role for caspase-dependent proteolysis of cyclin E for apoptosis of hematopoietic tumor cells.The cyclins and their catalytic subunits, the cyclin-dependent kinases (CDKs), control cell cycle progression by regulating events that drive the transitions between cell cycle phases (13,14). The activity of these CDKs is regulated positively by cyclins, their associated catalytic partners, and negatively by binding of CDK inhibitors (CKIs). Activation of cyclin/CDK complexes results in a cascade of protein phosphorylations that ultimately induces cell cycle progression. Cyclins were first identified in clam and sea urchin embryos, where they were observed to accumulate during interphase and to be degraded during mitosis (16). The human G 1 cyclins, the D-and E-type cyclins, were identified functionally by screening of human cDNA libraries for sequences that could complement G 1 cyclin mutations in Saccharomyces cerevisiae (31,35,65). The cyclin E mRNA levels show a periodic pattern of expression, being synthesized during the G 1 phase of the cell cycle, with levels increasing sharply in late G 1 , followed by accumulation of cyclin E protein and then down regulation in S phase (1...

Analysis of cyclin B1 and CDK activity during apoptosis induced by camptothecin treatment

“…The cyclin E1 complex was active at 16 h after treatment, which was earlier than cyclin A or cyclin B1 complexes. In contrast to previous reports, we did not detect small p18 forms of cyclin E that might induce activated CDK complexes (Porter et al, 2001;Mazumder et al, 2002) although the same antibody was used. A role for cyclin B1 in apoptosis A Borgne et al Several lines of evidence support the hypothesis that active CDK complexes participate in CPT-induced apoptosis.…”

Cell survival, cell death and cell cycle pathways are interconnected: Implications for cancer therapy

“…During apoptosis it is cleaved into a 18-kDa fragment (p18-cyclin E) by caspases and it can no longer bind to the CDKs. Overexpression of Bcl2 protein downregulated the apoptotic process and at the same time reduced the expression of p18-cyclin E (Mazumder et al, 2002). These results throw some light on the roles of cyclin E in apoptosis.…”

“…Recent investigations revealed that cyclin E is responsible for induction of apoptosis in hematopoietic cells (Mazumder et al, 2002). Cyclin E activity is increased substantially in the apoptotic process mediated by irradiation of RPMI 8226 cells.…”

The role of the ubiquitin/proteasome system in cellular responses to radiation

“…If the level of cyclin E is insufficient, cell cycle arrest at G1/S occurs, while too much cyclin E results in premature entry into S phase, mutations, and genomic instability (Spruck et al, 1999). Irradiation of hematopoietic cancer cells was found to increase cyclin E levels in a time-and dose-dependent manner (Mazumder et al, 2000;Mazumder et al, 2002), and this could influence cell cycle arrest. Importantly, mutations in the F box protein hCDC4 have been implicated in the pathogenesis of various forms of cancer and may be responsible for some cases of elevated cyclin E levels that are frequently associated with human cancers (Koepp et al, 2001;.…”

“…Levels and cleavage products of various cyclins have been implicated in apoptosis induction (Finkielstein et al, 2002;Mazumder et al, 2002). Also, accumulation of CDKI P27 Kip1 and cyclin E in mice deficient in its E3 ligase Skp2 results in increased spontaneous apoptosis (Nakayama et al, 2000), and the level of Skp2 in gastric carcinomas modulates the phenotype of the cancer, presumably by affecting P27 expression (Masuda et al, 2002).…”