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mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) signaling pathways and plays an important role in the viability response of these cytokines. In this study, we demonstrated that cytokine stimulation of mcl-1 mRNA and protein expression were attenuated by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-K) inhibitors. Reporter gene assays further showed that the PI3-K/Akt signaling pathway was involved in IL-3 activation of mcl-1 gene transcription. Analysis of the mcl-1 promoter revealed that both promoter elements, SIE at position ؊87 and CRE-2 at ؊70, contribute to IL-3 stimulation of mcl-1 gene expression. Although either the SIE site or the CRE-2 site alone was sufficient to confer IL-3 inducibility on a heterologous promoter, only IL-3 activation of the CRE-2 reporter was mediated via the PI3-K/Akt pathway. The SIE binding activity was constitutively high in cells deprived of or stimulated by IL-3. In contrast, the CRE-2 binding activity was low in cytokine-starved cells and was strongly induced within 1 h following cytokine treatment of cells. In addition, cytokine induction of the CRE-2 but not of the SIE binding activity was dependent on activation of the PI3-K/Akt signaling pathway. Lastly, we showed that CREB was one component of the CRE-2 binding complex and played a role in IL-3 activation of the mcl-1 reporter gene. Taken together, our results suggest that both PI3-K/Aktdependent and -independent pathways contribute to the IL-3 activation of mcl-1 gene expression. Activation of mcl-1 by the PI3-K/Akt-dependent pathway is through a transcription factor complex containing CREB.

. Furthermore, inhibition of cyclin D1 synthesis greatly impaired the soft-agar cloning efficiency of v-H-Ras transformants. These results suggest that increased expression of cyclin D1 is necessary but not sufficient for the transforming activity of v-H-Ras. Similar effect on cell cycle progression was also observed in Raf-transformed cells. In addition to cyclin D1, cyclin E protein was found to be elevated in Src transformants. This may account for the further shortening of the G 1 phase of these cells. Activation of an additional Ras-independent pathway was suggested to be responsible for the further acceleration of the G 1 phase in Src transformants.The ras gene product, p21, is among those oncoproteins whose structure and function are most thoroughly characterized. By cycling between inactive GDP-bound and active GTPbound forms, membrane-bound Ras protein has been found to be involved in transducing various extracellular signals into cells, for example, (i) growth factor stimulation of fibroblast cells; (ii) signaling pathway in T-cell activation; (iii) cytokineinduced proliferation and differentiation of hematopoietic cells; (iv) nerve growth factor (NGF)-induced, neuronal differentiation of PC12 cells; and (v) inhibition of epithelial cell proliferation by transforming growth factor ␤1 (TGF-␤1) (reviewed in reference 34 and references therein). Although the immediate target of Ras protein is not clear, many cytoplasmic factors are known to function downstream of the Ras signaling pathway, such as Raf kinase, mitogen-activated protein (MAP) kinase kinase, and MAP kinase (reviewed in references 6 and 24). Recent reports by several groups suggest further that Ras and Raf may interact directly (25,(39)(40)(41)44).In addition to the activities of the above noted cytoplasmic proteins, the activity of c-Jun, one component of the nuclear transcription factor AP1 has also been shown to be modulated by the Ras signaling pathway (3). This suggests that Ras modulates cell growth by transducing signals from the outer membrane into the nucleus. Earlier studies (27) demonstrate that microinjection of Ras protein into quiescent cells induces DNA synthesis inside the nucleus. These findings suggest that the Ras signaling pathway is linked directly to the G 1 /S phase transition of the cell cycle.The purpose of this work was to identify those downstream targets of Ras whose activities are important for the G 1 /S phase transition. In this report, we provide evidence that cyclin D1 is one of the major G 1 /S transition control factors whose level is modulated by the Ras signaling pathway. We also demonstrate that increased expression of cyclin D1 accounts for the shortening of the G 1 phase associated with v-H-Ras transformation of NIH 3T3 cells. MATERIALS AND METHODSPlasmids. v-H-Ras (pZIP-v-H-ras) expression vectors carrying a neo selectable marker was kindly provided by Channing Der. cDNA-containing plasmids for cyclins A, D1, and E and cdk4 were generous gifts from Christian Brechot, Andrew Arnold, James Roberts and Ch...