The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of ⌬A-Raf:ER promoted cell cycle progression, activation of ⌬Raf-1:ER and ⌬B-Raf:ER elicited a G 1 arrest that was not overcome by exogenously added growth factors. Activation of all three ⌬Raf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27 Kip1. However, activation of ⌬B-Raf:ER and ⌬Raf-1:ER induced the expression of p21 Cip1 , whereas activation of ⌬A-Raf:ER did not. A catalytically potentiated form of ⌬A-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to ⌬B-Raf:ER and ⌬Raf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of ⌬B-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of ⌬Raf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1؊/؊ MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to ␣-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.Biochemical and genetic strategies have implied that the Ras-activated extracellular ligand-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase pathway is a key regulator of cell proliferation and differentiation in metazoan organisms (3, 4, 17-19, 21, 32, 46, 66-68). The binding of a variety of ligands to their cognate cell surface receptors elicits the activation of members of the Ras family of GTPases. Activation of Ras leads to the sequential activation of Raf, MEK, and p42 and p44 MAP-ERK kinases (16,27,35,55,(103)(104)(105). Nuclear translocation of MAP kinases leads to the phosphorylation of transcription factors, such as Elk-1 and Ets-2, which regulate the expression of immediate-early genes, such as the c-Fos and HB-EGF genes, respectively (33,34,39,53,57,58,102). The loss of function of components of this pathway has severe developmental consequences for the organism (28, 50, 68, 77). Furthermore, acti...

The oncogenes RAS and RAF came to view as agents of neoplastic transformation. However, in normal cells, these genes can have effects that run counter to oncogenic transformation, such as arrest of the cell division cycle, induction of cell differentiation, and apoptosis. Recent work has demonstrated that RAS elicits proliferative arrest and senescence in normal mouse and human fibroblasts. Because the Raf/MEK/MAP kinase signaling cascade is a key effector of signaling from Ras proteins, we examined the ability of conditionally active forms of Raf-1 to elicit cell cycle arrest and senescence in human cells. Activation of Raf-1 in nonimmortalized human lung fibroblasts (IMR-90) led to the prompt and irreversible arrest of cellular proliferation and the premature onset of senescence. Concomitant with the onset of cell cycle arrest, we observed the induction of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p16 Ink4a. Normal cells proliferate in vitro for a finite number of cell divisions after which they enter a state known as senescence (Hayflick and Moorhead 1961;Campisi 1996 Campisi , 1997. Senescent cells withdraw irreversibly from the cell division cycle, but remain viable indefinitely, develop a distinctive morphology, and display characteristic phenotypic markers, such as the senescence-associated, acidic ␤-galactosidase activity (SA-␤-gal; Dimri et al. 1995). Although the biochemical mediators of senescence in human cells remain uncertain, candidates include the p53 tumor suppressor protein, the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p16 Ink4a, and regulators of telomere length and function, such as telomerase and TRF1 and TRF2. Increased expression of p53, p16 Ink4a, and p21Cip1 is detected in a variety of senescent cells, and overexpression of telomerase leads to immortalization of human cells in culture (Kulju and Lehman 1995;Alcorta et al. 1996;Hara et al. 1996;Reznikoff et al. 1996;Shay and Bacchetti 1997;Bodnar et al. 1998;van Steensel et al. 1998). Furthermore primary human cells that are deficient in the expression of p21Cip1 have increased replicative capacity in vitro (Brown et al. 1997). p53, p16 Ink4a , and p21Cip1 can arrest the cell division cycle: p21Cip1 and p16 Ink4a do so by inhibiting CDKs required for progression through the cell cycle (Lees 1995), and p53 does so by inducing the expression of p21Cip1 (El-Deiry et al. 1993). Cell lines derived from most tumors are capable of extended proliferation as if the capability to become senescent has been somehow repressed or lost. Accordingly, either p53, p16 Ink4a , or both are frequently defective in tumor cells (Hall and Peters 1996;Hainaut et al. 1997), and restoration of p53 to deficient tumor cells elicits prompt senescence (Sugrue et al. 1997). Furthermore, tumor cells frequently express telomerase activity, which may contribute to the extended proliferation of these cells in culture (Meyerson et al. 1997). Clearly the extension of proliferative life span might make an important contribution to tumorigenesi...

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The growth inhibitory functions of p53 are controlled in unstressed cells by rapid degradation of the p53 protein. One of the principal regulators of p53 stability is MDM2, a RING finger protein that functions as an E3 ligase to ubiquitinate p53. MDM2 promotes p53 nuclear export, and in this study, we show that ubiquitination of the C terminus of p53 by MDM2 contributes to the efficient export of p53 from the nucleus to the cytoplasm. In contrast, MDM2 did not promote nuclear export of the p53-related protein, p73. p53 nuclear export was enhanced by overexpression of the export receptor CRM1, although no significant relocalization of MDM2 was seen in response to CRM1. However, nuclear export driven by CRM1 overexpression did not result in the degradation of p53, and nuclear export was not essential for p53 degradation. These results indicate that MDM2 mediated ubiquitination of p53 contributes to both nuclear export and degradation of p53 but that these activities are not absolutely dependent on each other.The p53 tumor suppressor protein plays an important role in preventing malignant development, and p53 function is lost or compromised in most human cancers (37). One of the principal functions of p53 is to inhibit cell growth, and p53 shows strong cell cycle arrest and apoptotic activities (43). While these functions play an important role in preventing the growth of abnormal or damaged cells, p53 activity must be tightly regulated in normal tissue to allow growth and development. One of the principal regulators of p53 is the MDM2 protein (26), and loss of MDM2 results in early embryonic lethality associated with deregulated p53-mediated apoptosis (10). MDM2 expression is transcriptionally activated by p53, establishing a feedback loop in which p53 controls expression of its own negative regulator (4, 45).MDM2 shows several functions that contribute to the inhibition of p53 activity. p53 is a transcription factor, and the activation of cell cycle arrest and apoptotic responses to p53 are dependent, at least in part, on the expression of p53 target genes (43). The p53 protein contains domains for sequencespecific DNA binding and an N-terminal transactivation domain that forms direct contacts with a number of proteins that are involved in transcriptional control (22). Since the MDM2 binding site is also within the N-terminal region of p53 (7), one of the consequences of MDM2 binding is to inhibit p53-mediated transcriptional activity by blocking the p53-transcriptional coactivator interactions (31,33,44). This effect may be further enhanced by MDM2-mediated inhibition of the acetylation of p53 by factors such as p300 (21, 23) and an ability of MDM2 to function directly as a transcriptional repressor (42).Another function of MDM2 that efficiently abolishes all p53 activity is the ability of MDM2 to target p53 for degradation through the ubiquitin-dependent proteasome pathway (17,24). Interaction between MDM2 and p53 leads to the ubiquitination and degradation of p53, and this is likely to play a key role in maint...

The HDM2 protein is a key regulator of the tumour suppressor, p53. Control of HDM2 function is critical for normal cell proliferation and stress responses, and it is becoming evident that multiple modifications of HDM2 can regulate its function within cells. In this study we show that HDM2 associated with the serine-threonine kinase, Akt, in response to growth factor stimulation of human primary cells. This association was concurrent with phosphorylation of Akt (at Ser 473), and resulted in elevated expression of HDM2 and enhanced nuclear localization. However, analysis of HDM2 proteins mutated at the consensus Akt recognition sites at serines 166 and 186 indicated that modification at these residues was not sufficient for the increased expression of the protein, which was blocked by the PI3 kinase inhibitor LY294002. Tryptic peptide and mutational analyses revealed evidence for an Akt phosphorylation site in HDM2 additional to the two consensus sites.

Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. Here, we show that the mdm2 gene is also regulated by the Ras-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdm2 induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19ARF. This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19ARF. Levels of p53 are therefore determined by opposing effects of Raf-induced p19ARF and Mdm2.

ABSTRACT

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