We have previously shown that cyclin E can malignantly transform primary rat embryo fibroblasts in cooperation with constitutively active Ha-Ras. In addition, we demonstrated that high level cyclin E expression potentiates the development of methyl-nitroso-urea-induced T-cell lymphomas in mice. To further investigate the mechanism underlying cyclin E-mediated malignant transformation, we have performed a mutational analysis of cyclin E function. Here we show that cyclin E mutants defective to

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... rm an active kinase complex with Cdk2 are unable to drive cells from G 1 into S phase but can still malignantly transform rat embryo fibroblasts in cooperation with Ha-Ras. In addition, Cdk2 activation is not a prerequisite for the ability of cyclin E to rescue yeast triple cln mutations. We also find that the oncogenic properties of cyclin E did not entirely correspond with its ability to interact with the negative cell cycle regulator p27 Kip1 or the pocket protein p130. These findings suggest that the oncogenic activity of cyclin E does not exclusively rely on its ability as a positive regulator of G 1 progression. Rather, we propose that cyclin E harbors other functions, independent of Cdk2 activation and p27 Kip1 binding, that contribute significantly to its oncogenic activity. E-type cyclins (cyclin E and cyclin E2) and their associated catalytic subunits Cdk2 constitute part of the regulatory proteins that control progression through the G 1 /S border and the entry of cells into S phase of the cell cycle. Both cyclins were found to be expressed in the late G 1 phase through S phase, and it has been shown that the level of cyclin E is the limiting factor for G 1 phase progression and S phase entry (1-11). One of the important events in this phase of the cell cycle is the phosphorylation of the tumor suppressor protein Rb, which is one of the substrates of E-type cyclin-Cdk complexes (12-14). The Rb protein is active in its nonphosphorylated or hypophosphorylated form and becomes hyperphosphorylated and inactivated by cyclin-Cdk complexes during G 1 phase (12) (13) (14) . This inactivation by phosphorylation leads to the release of E2F family transcription factors from a complex with the Rb protein. E2F transcription factors in turn activate a set of target genes that are essential for entry into S phase (15-17). Cyclin-Cdk complexes are regulated by a number of inhibitors called Cdk inhibitors, among them the proteins p16 Ink4a , p21 Waf1 , and p27 Kip1 (18 -24). Both p21 Waf1 and p27 Kip1 bind to cyclin E-Cdk2 complexes and block their activities, whereas p16 Ink4a specifically inhibits the catalytic subunits of D-type cyclins Cdk4 and Cdk6 (25). A link between cyclin E and malignant transformation has been established by a number of independent studies. Cyclin E was found to be expressed at high levels in a number of human tumors (for an overview see Refs. 26 and 27), and the overexpression of the cyclin E protein is in many cases the result of an amplification of the locus of the cyclin E gene at chromosome 19q12-13 (27-31). In particular, high levels of cyclin E and low levels of p27 Kip1 have been correlated with poor prognosis in breast cancer (32-34), and one study with transgenic mice suggested a functional role of elevated cyclin E levels in malignant transformation of mammary tissue (35). In addition, in several types of leukemias, in chronic lymphocytic leukemia, Hodgkin's and non-Hodgkin's lymphoma cyclin E overexpression was detected and appeared to correlate with low rates of complete remission and disease free survival (36, 37). We have previously shown that cyclin E can cooperate with constitutively active Ha-Ras in the malignant transformation of primary rat embryo fibroblasts (REFs) 1 (38). Moreover, we have established a transgenic mouse model in which high levels of ectopically expressed cyclin E was targeted to the T-lymphoid lineage. These animals were significantly more susceptible to the induction of T-cell lymphomas upon treatment with methyl-nitroso-urea (39). To explore in more detail the molecular mechanisms by which cyclin E overexpression can lead to malignant transformation, we have used cyclin E mutants and the REF transformation assay. We demonstrate here that the oncogenic activity of cyclin E relies on different domains of the protein, which are distinct in their function to mediate binding and/or activation of Cdk2, p27 Kip1 , and p130 and their ability to drive cells into S phase. EXPERIMENTAL PROCEDURES Plasmid Construction-cDNAs for cyclin E, cyclin E mutants, and Myc were subcloned into a pLTR⌬7bp vector (40), which was cut, PstI/T4-DNA-polymerase-blunted, and modified with a part of the multiple cloning site of pBluescript (Stratagene), that was cut NotI/Asp 718I/Klenow-blunted, to gain pLTR-MCSϩ. The vector pEGFP-N3 (CLONTECH) was cut with BamHI/XbaI, and oligonucleotides encoding the FLAG epitope (GATCCATGGACTACAAAGACGATGACGATA-AATAGTCTAGAGGC and CTAGGACTATTTATCGTCATCGTCTTTG-TAGTCCATG) were inserted into these sites to replace the enhanced green fluorescent protein gene and gain a new vector, pFLAG-N3, which expresses the gene of interest with a C-terminal FLAG epitope under the control of the cytomegalovirus promoter. Cyclin E mutants