Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G 1 ͞S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E͞cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1͞cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin Ddependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107͞E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D͞cdk4-cdk6͞p16͞pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.Progression through the eukaryotic cell cycle is mediated both positively and negatively by a variety of growth regulatory proteins (1-3). Cyclins and their catalytic cyclin-dependent kinase (cdk) partners act positively to propel a cell through the proliferative cycle (4, 5). Activation of cyclin-cdk complexes results in a cascade of protein phosphorylations that ultimately induce cell cycle progression (1, 4). Although the identity of downstream substrates and effectors of cyclin-cdks remains to be firmly established, it is commonly believed that cdk-mediated phosphorylations manifest cell cycle regulation via inhibition of growth inhibitory signals and activation of proteins necessary for each stage of the cell cycle (6). A putative, well-characterized substrate for the G1 cyclins is retinoblastoma susceptibility gene product (pRb; refs. 7 and 8). This protein is sequentially phosphorylated during the cell cycle presumably through the concerted activity of different cyclin-cdk complexes (9-11). This phosphorylation is required for cell cycle progression, and the hypophosphorylated form of pRb inhibits cell cycle progression by tethering and inactivating transcription factors of the E2F family, which are required for the transactivation of S phase-specific proteins, including dihydrofolate reductase, cyclin A, and thymidyla...

No abstract

No abstract

No abstract