Post-translational modification and stability of low molecular weight cyclin E.
Our laboratory has previously described the presence of five tumor-specific low molecular weight isoforms of cyclin E in both tumor cell lines and breast cancer patient biopsies. We have also shown that one of these low forms arises from an alternate start site, whereas the other four appear as two sets of doublets following cleavage through an elastase-like enzyme. However, the origin of both sets of doublets was unknown. Here, we demonstrate that the larger isoform of each doublet is the result of phosphorylation at a key degradation site. Through site-directed mutagenesis of different phosphorylation sites within the cyclin E protein, we discovered that phosphorylation of threonine 395 is responsible for generating the larger isoform of each doublet. Because phosphorylation of threonine 395 has been linked to the proteasome-mediated degradation of full length cyclin E, we examined the stability of T395A phospho-mutants in both non-tumorigenic mammary epithelial cells and tumor cells. The results revealed that the low molecular weight isoforms appear to be stable in both a tumor cell line and a non-tumor forming cell line regardless of the presence of this critical phosphorylation site. The stability of low molecular weight cyclin E may have implications for both tumorigenesis and treatment of tumors expressing them.
Mull
B B
BB
Department of Experimental Radiation Oncology, University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA.
Cox
J
J
Bui
T
T
Keyomarsi
K
K
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001943:Breast Neoplasms / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D019927:Cyclin E / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D006656:Protamine Kinase / Q000032:analysis / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014162:Transfection; D054462:p21-Activated Kinases / Q000378:metabolism
D019927:Cyclin E; D020033:Protein Isoforms; D050760:Cyclin-Dependent Kinase Inhibitor p27; D006656:Protamine Kinase; D054462:p21-Activated Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D000409:Alanine