SERUM-INDUCED EXPRESSION OF THE CDC25A GENE BY RELIEF OF E2F-MEDIATED REPRESSION

Cell growth is a process of progression of cells through the cell cycle, which contains four defined phases: G1, S, G2, and M (reviewed in reference 52). Mammalian cell cycle progression has often been studied by synchronizing cells in a quiescent state, termed G0, at the beginning of the G1 phase. Serum-starved, quiescent cells reenter the cell cycle after serum treatment and enter S phase after about 12 h depending on the cell type. This process of progression from G0 to S phase is marked by specific changes in gene expression. The first genes to be induced, cellular immediate-early genes, are activated rapidly by serum stimulation, with a peak of gene expression within 30 to 60 min (25). Immediate-early genes are typified by the c-fos and c-jun proto-oncogenes, which are themselves transcription factors. A second class of genes, delayed-early response genes, are induced after 3 to 6 h of serum induction and, in contrast to immediate-early genes, require new protein synthesis for induction of expression (36). There is a third class of genes that are activated late in G1 and that seem to herald the G1-to-S transition. Many of these genes are regulated by the E2F transcription factor and retinoblastoma protein (Rb) family members (13). The connection, if any, among the different classes of serum-inducible genes has yet to be established. Since some immediate-early genes are transcription factors, it is possible that they serve to activate subsequent gene expression of the delayed-early or late G1 genes. To begin to investigate this possibility, we have studied the regulation of a delayed-early gene, cdc25A, known to be important for cell cycle progression. cdc25 was originally identified in the fission yeast, Schizosaccharomyces pombe, as a phosphatase that controls cdc2/cyclin B kinase activity and activates mitosis (reviewed in reference 12). cdc2 must be dephosphorylated by cdc25 at a conserved tyrosine residue, tyrosine 15, in order to be activated (40). Mammalian cells have three cdc25 homologues, cdc25A, -B, and -C (16, 42, 47). These phosphatases can dephosphorylate cyclin-dependent kinase (cdk)-related kinases in vitro and induce their activity (16, 19, 23, 27, 51). cdc25A can also activate cdc2 kinase activity by competitively blocking its binding to the p21 cdk inhibitor (48). The three mammalian cdc25 family members are differentially expressed during the cell cycle following serum stimulation. cdc25A is induced in mid-G1, similar to other delayed-early genes (32). cdc25B is expressed later, during S phase, while cdc25C is expressed in G2 phase (32, 34, 47). Besides undergoing transcriptional induction, cdc25A is also activated by cdk2/cyclin E phosphorylation in late G1 and by cyclin B in vitro (16, 26). Immunodepletion of cdc25A in rat cells by microinjection blocked cell entry into S phase, demonstrating that cdc25A is critical for G1 phase progression (16, 26, 32). cdc25A is also a candidate oncogene since it can cooperate with ras in oncogenic transformation of primary rat fibroblasts (18). We have investigated the mechanism of serum induction of cdc25A in order to understand how delayed-early genes are activated and because cdc25A is an important gene for G1 phase progression. It was previously determined that the proto-oncogene and immediate-early gene c-myc can induce cdc25A expression and that there are myc binding sites in the cdc25A gene (17). It was not determined, however, whether c-myc or the myc binding sites were required for G1 phase induction of cdc25A. We have found that the myc sites in the cdc25A gene are not required for serum induction. Instead we found that cdc25A expression is regulated by an E2F site and Rb family members.