Novel splice variants of cyclin E with altered substrate specificity.
Cyclin E, a G(1) cyclin, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In this study we have examined the possibility that the shortened mRNA splice variants could give rise to tumor-specific cyclin E LMW proteins. We used the Splice Capture method to identify, enumerate and isolate known spliced mRNAs and to look for previously undetected mRNA forms of cyclin E that might be translated into the LMW proteins. We show that a new splice variant of cyclin E found in tumor cells isolated by the Splice Capture strategy, named Delta48, activates CDK2 more robustly than full-length cyclin E when assayed from transiently transfected cells with the natural substrate GST-Rb. We also found the Splice Capture method to be superior to the conventional RNase protection assay in analyzing the cyclin E mRNA present in normal and tumor cells. Splice Capture enumerated the relative abundance of known forms of cyclin E mRNA and easily discovered new splice variants in both normal and tumor cells. We conclude that the abundance of cyclin E splice variants in cells may represent a novel form of regulation of cyclin E, and if translated they show altered substrate specificity compared to the full length form of cyclin E.
Porter
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Division of Molecular Medicine, Wadsworth Center, Albany, NY 12201-0509, USA.
Keyomarsi
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D017398:Alternative Splicing; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D002460:Cell Line; D019927:Cyclin E / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008970:Molecular Weight; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D019927:Cyclin E; D020033:Protein Isoforms; D012333:RNA, Messenger; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases