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Altered subcellular localization of tumor-specific cyclin E isoforms affects cyclin-dependent kinase 2 complex formation and proteasomal regulation.

ABSTRACT: In tumors, alternative translation and posttranslational proteolytic cleavage of full-length cyclin E (EL) produces tumorigenic low molecular weight cyclin E (LMW-E) isoforms that lack a portion of the EL amino-terminus containing a nuclear localization sequence. Therefore, we hypothesized that LMW-E isoforms have altered subcellular localization. To explore our hypothesis, we compared EL versus LMW-E localization in cell lysates and in vivo using fractionation and protein complementation assays. Our results reveal that LMW-E isoforms preferentially accumulate in the cytoplasm where they bind the cyclin E kinase partner, cyclin-dependent kinase 2 (Cdk2), and have associated kinase activity. The nuclear ubiquitin ligase Fbw7 targets Cdk2-bound cyclin E for degradation; thus, we examined if altered subcellular localization affected LMW-E degradation. We found that cytoplasmic LMW-E/Cdk2 was less susceptible to Fbw7-mediated degradation. One implication of our findings is that altered LMW-E and LMW-E/Cdk2 subcellular localization may lead to aberrant LMW-E protein interactions, regulation, and activity, ultimately contributing to LMW-E tumorigenicity.

SUBMITTER: Delk NA

PROVIDER: S-EPMC2669888 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Altered subcellular localization of tumor-specific cyclin E isoforms affects cyclin-dependent kinase 2 complex formation and proteasomal regulation.

Delk Nikki A NA Hunt Kelly K KK Keyomarsi Khandan K

Cancer research 20090324 7

In tumors, alternative translation and posttranslational proteolytic cleavage of full-length cyclin E (EL) produces tumorigenic low molecular weight cyclin E (LMW-E) isoforms that lack a portion of the EL amino-terminus containing a nuclear localization sequence. Therefore, we hypothesized that LMW-E isoforms have altered subcellular localization. To explore our hypothesis, we compared EL versus LMW-E localization in cell lysates and in vivo using fractionation and protein complementation assays ...[more]

PMID: 19318554

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Project description:In higher eukaryotes many genes encode protein isoforms whose properties and biological roles are often poorly characterized. Here we describe systematic approaches for detection of either distinct isoforms, or separate pools of the same isoform, with differential biological properties. Using information from ion intensities we have estimated protein abundance levels and using rates of change in stable isotope labeling with amino acids in cell culture isotope ratios we measured turnover rates and subcellular distribution for the HeLa cell proteome. Protein isoforms were detected using three data analysis strategies that evaluate differences between stable isotope labeling with amino acids in cell culture isotope ratios for specific groups of peptides within the total set of peptides assigned to a protein. The candidate approach compares stable isotope labeling with amino acids in cell culture isotope ratios for predicted isoform-specific peptides, with ratio values for peptides shared by all the isoforms. The rule of thirds approach compares the mean isotope ratio values for all peptides in each of three equal segments along the linear length of the protein, assessing differences between segment values. The three in a row approach compares mean isotope ratio values for each sequential group of three adjacent peptides, assessing differences with the mean value for all peptides assigned to the protein. Protein isoforms were also detected and their properties evaluated by fractionating cell extracts on one-dimensional SDS-PAGE prior to trypsin digestion and MS analysis and independently evaluating isotope ratio values for the same peptides isolated from different gel slices. The effect of protein phosphorylation on turnover rates was analyzed by comparing mean turnover values calculated for all peptides assigned to a protein, either including, or excluding, values for cognate phosphopeptides. Collectively, these experimental and analytical approaches provide a framework for expanding the functional annotation of the genome.

Dataset Information

Altered subcellular localization of tumor-specific cyclin E isoforms affects cyclin-dependent kinase 2 complex formation and proteasomal regulation.

Publications

Altered subcellular localization of tumor-specific cyclin E isoforms affects cyclin-dependent kinase 2 complex formation and proteasomal regulation.

Similar Datasets

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