Raf-Induced Proliferation or Cell Cycle Arrest Is Determined by the Level of Raf Activity with Arrest Mediated by p21^Cip1

Abstract

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...

Post-transcriptional silencing of RET occurs, but is not required, during raf-1 mediated differentiation of medullary thyroid carcinoma cells

“…On the other hand, EGF treatment of PC12 cells leads to transient ERK activation and cell proliferation (Heasley and Johnson, 1992;Nguyen et al, 1993;Traverse et al, 1992). Similarly, in NIH3T3 cells, two groups have recently demonstrated that high level activation of raf-1 correlates with growth arrest and induction of p21 WAF1/CIP1 , while low level activity has a mitogenic e ect (Sewing et al, 1997;Woods et al, 1997). These studies are consistent with the observation that moderate ERK1/2 activity is insu cient to reach the threshold required to di erentiate TT:DRaf-1:ER cells.…”

“…It is also possible that the dose of MAPK activity generated by the mutant RET kinase might be su cient to elicit inappropriate proliferation (leading to C-cell hyperplasia and/or MTC), but not high enough to trigger terminal di erentiation, unlike the MAPK activity generated by DRaf-1:ER. Other groups have demonstrated that low level raf-1 activity, such as might be caused by activated RET, is mitogenic, while high level raf-1 activity, like that caused by DRaf-1:ER induction, induces growth arrest (Sewing et al, 1997;Woods, et al, 1997). An additional possibility is that the DRaf-1:ER construct and/or mutant RET protein target not just MEK and MAPK, but additional di erentiation or proliferation speci®c substrates, respectively, as has been suggested by Kuo et al (1996Kuo et al ( , 1997.…”

Pirin Inhibits Cellular Senescence in Melanocytic Cells

“…Human diploid fibroblasts (TIG3-hTERT) expressing a conditional form of constitutively activated BRAF fused to the ligand-binding domain of the estrogen receptor (ER) rapidly undergo oncogene-induced senescence on treatment with 4-hydroxytamoxifen (OHT). 28,29 PIR protein and mRNA levels were measured in TIG3-BRAF-ER cells at different time points of treatment with 800 nmol/L OHT. PIR expression was significantly repressed both at the mRNA and at the protein level after BRAF activation ( Figure 6A), and remained at low levels after 120 hours, suggesting that a significant reduction of PIR expression is associated with the establishment of oncogene-induced senescence in different cell types.…”

“…It has been shown that low levels of BRAF expression (induced by low doses of OHT) elicit a mitogenic response due to induction of cyclins D1 and E, 28 while only a modest activity of mitogen-activated protein kinase (MAPK) is detected. In contrast, higher levels of BRAF result in high MAPK activity, and both conditions are associated with increased expression of cyclin-dependent kinase inhibitors and cell cycle arrest.…”

Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-independent cell survival and neuronal differentiation

“…In this regard, sustained activation of the MAPK pathway has been suggested as an essential component of this process Kaplan and Miller, 1997). Sustained MAPK activity, in turn, stimulates the expression of the p21 CIP/Waf-1 inhibitor of cell cycle progression (Woods et al, 1997) providing a functional link between TrkA signaling, mitotic arrest and neuronal di erentiation. Presently, NGF-induced activation of Rap1, via Crk/C3G, is thought to be essential for prolonged MAPK activation while activation of Ras, via Grb/Sos, is thought to regulate an immediate-early phase of activation (York et al, 1998).…”