Proteolytic cleavage of cyclin E leads to inactivation of associated kinase activity and amplification of apoptosis in hematopoietic cells

Suparna Mazumder, Bendi Gong, Quan Chen, Judith A. Drazba, Jeffrey C. Buchsbaum, Alexandru Almasan

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G1 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 E276-395 (where cyclin E276-395 is 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 E1-275, cyclin E276-395 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 E276-395, 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.

Original languageEnglish (US)
Pages (from-to)2398-2409
Number of pages12
JournalMolecular and Cellular Biology
Volume22
Issue number7
DOIs
StatePublished - 2002
Externally publishedYes

Fingerprint

Cyclin E
Phosphotransferases
Apoptosis
Cyclins
Caspases
Ketones
DNA Damage
Phosphatidylserines
Ionizing Radiation
Tumor Cell Line
Immunoprecipitation
Mutagenesis
Caspase 3
Proteolysis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Proteolytic cleavage of cyclin E leads to inactivation of associated kinase activity and amplification of apoptosis in hematopoietic cells. / Mazumder, Suparna; Gong, Bendi; Chen, Quan; Drazba, Judith A.; Buchsbaum, Jeffrey C.; Almasan, Alexandru.

In: Molecular and Cellular Biology, Vol. 22, No. 7, 2002, p. 2398-2409.

Research output: Contribution to journalArticle

Mazumder, Suparna ; Gong, Bendi ; Chen, Quan ; Drazba, Judith A. ; Buchsbaum, Jeffrey C. ; Almasan, Alexandru. / Proteolytic cleavage of cyclin E leads to inactivation of associated kinase activity and amplification of apoptosis in hematopoietic cells. In: Molecular and Cellular Biology. 2002 ; Vol. 22, No. 7. pp. 2398-2409.
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abstract = "Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G1 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 E276-395 (where cyclin E276-395 is 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 E1-275, cyclin E276-395 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 E276-395, 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.",
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AU - Mazumder, Suparna

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AU - Buchsbaum, Jeffrey C.

AU - Almasan, Alexandru

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