Regulation of cellular proliferation, differentiation and cell death by activated Raf
© Thiel et al; licensee BioMed Central Ltd. 2009
Received: 20 February 2009
Accepted: 21 April 2009
Published: 21 April 2009
The protein kinases Raf-1, A-Raf and B-Raf connect receptor stimulation with intracellular signaling pathways and function as a central intermediate in many signaling pathways. Gain-of-function experiments shed light on the pleiotropic biological activities of these enzymes. Expression experiments involving constitutively active Raf revealed the essential functions of Raf in controlling proliferation, differentiation and cell death in a cell-type specific manner.
Raf connects cellular stimulation with intracellular signaling pathways. Raf translocates to the plasma membrane as a result of receptor tyrosine kinase stimulation that leads to a subsequent activation of Ras. Following activation, Raf phosphorylates and activates mitogen-activated protein kinase (MAP) kinase (MEK) which in turn phosphorylates and activates the MAP kinases extracellular signal-regulated protein kinases ERK1 and ERK2 . Raf functions therefore as a vital link between activated Ras and ERK. The activated protein kinases ERK1/2 are able to translocate into the nucleus and change the gene expression pattern via phosphorylation of gene regulatory proteins. Thus, activation of Raf is essential for activating the Raf/MEK/ERK signaling pathway and many functions attributed to Raf activation are executed by the subsequent activation of MEK and ERK. A microarray analysis confirmed that the transcriptional response to Raf activation almost completely depends on MEK activation . In line with this, MEK is the only generally acknowledged substrate for Raf [1, 3].
Lessons from Raf-deficient mice
Gene ablation experiments involving the genes encoding the Raf isoforms Raf-1, A-Raf, and B-Raf revealed divergent phenotypes, indicating that Raf isoforms are not always able to compensate for each other. In particular, distinct essential functions are served by Raf-1 and B-Raf in embryonic development . Nevertheless, a functional redundancy among the Raf family proteins exists and only phenotypes requiring the activity of a distinct Raf isoform are found. Inactivation of the Raf-1 and B-Raf-encoding genes revealed that Raf-1 and B-Raf play essential anti-apoptotic roles [5, 6]. B-Raf is necessary for survival of embryonic motoneurons and sensory neurons . Several review articles have been published that discuss these mouse models in detail [4, 8–10]
Gain-of-function mutants of Raf
Two strategies have been used to express constitutively active Raf. The translocation of Raf to the plasma membrane via binding to Ras-GTP is the key event in Raf activation . Thus, a method to express a constitutively active Raf-1 relies in the tethering of Raf to the plasma membrane. This Raf-1 mutant termed Raf-CAAX carries at the C-terminus an isoprenylation sequence derived from K-Ras [11, 12]. The artifical targeting of Raf-1 to the plasma membrane leads to an activation of the enzyme in a Ras-independent manner and shows 30-fold higher kinase activity in growth-factor-deprived cells.
Role of Raf in the regulation of proliferation
The fact that Raf is activated following stimulation of the cells with mitogens (i.e. EGF, PDGF, IGF) indicates that these enzymes are involved in the regulation of cell growth and proliferation. Accordingly, expression of the hormone-regulated form of Raf-1, ΔRaf-1:ER, induced cell proliferation in NIH 3T3 fibroblasts that was accompanied by an upregulation of cyclin D1 and a repression of p27KIP, a cyclin-dependent protein kinase inhibitor . Expression of conditionally active forms of A-Raf and B-Raf in NIH 3T3 cells revealed differences between the individual Raf isoforms. While the activation of both ΔA-Raf:ER and ΔB-Raf:ER induced the activation of MEK and ERK protein kinases, ΔB-Raf:ER activated MEK with the highest efficiency . A microarray analysis performed with human epithelial cells underlined the importance of MEK activation by Raf . Moreover, the fact that activation of ΔRaf:ER strongly induced the expression of growth factors of the EGF growth factor family suggests the existence of an autocrine loop through the activation of the EGF receptor: Activation of ΔRaf:ER triggers the stimulatation of the EGF receptor. As a result, the Raf-MEK-ERK signaling pathway is activated, leading to further synthesis of EGF growth factors [2, 18].
In a human breast epithelial cell line, activation of ΔRaf:ER triggered the expression of genes encoding regulators of cell proliferation, including cyclin D1, and induced a transient increase in S phase cells. However, Raf activation did not induce growth factor-independent proliferation , in contrast to the situation encountered with ΔA-Raf:ER expressing keratinocytes. These data indicate that cell-type specific variations are important for the biological outcome of Raf activation.
Although the activation of a conditional form of Raf can promote DNA synthesis and cellular proliferation, other reports show that it can also provoke cell cycle arrest. Expression of ΔRaf-1:ER in small lung cancer cells induced a growth inhibitory pathway that is accompanied by the induction of the cyclin-dependent protein kinase inhibitor p27KIP and a decrease in cdc2 protein kinase activity . In prostate cancer cells, activation of ΔRaf-1:ER induced expression of the cyclin-dependent protein kinase inhibitor p21KIP and an accumulation of the cells in G1, thus leading to growth suppression . Likewise, ΔRaf-1:ER and ΔB-Raf:ER elicited a G1 arrest in NIH 3T3 cells that was accompanied by an upregulation of the cyclin-dependent protein kinase inhibitor p21KIP. In contrast, activation of ΔA-Raf:ER promoted the entry of quiescent NIH 3T3 cells into the S-phase of the cell cycle. A catalytically potentiated form of ΔA-Raf:ER, however, induced cell cycle arrest and enhanced p21KIP expression, similarly to ΔB-Raf:ER or ΔRaf-1:ER . These data suggest that the catalytical activity and the duration of the signaling of Raf might determine the role of these enzymes in the progression of the cell cycle. In addition, cell type-specific differences are essential for Raf induction and impairment of the growth capacity of the cells.
Anti-apoptotic role of Raf
Raf-1-deficient embryos are growth retarded and apoptotic cells are found in different tissues [6, 7]. Raf-1-deficient fibroblasts are hypersensitive to apoptotic stimuli such as serum withdrawal or Fas/Fas ligand interaction. Thus, it was concluded that the major function of Raf-1 is to counteract apoptosis . Also B-Raf-deficient embryos die because of vascular defects due to apoptotic death of differentiated endothelial cells .
The activation of the MEK/ERK signaling pathway by Raf has been correlated with inhibition of programmed cell death. The ERK signaling pathway has been described to play an important role as a main antagonist of various apoptosis-inducing challenges [[24, 25]; reviewed in ref ]. Activation of the ERK signaling pathway suppresses the proapoptotic activity of stress-activated JNK/p38 protein kinases in PC12 pheochromocytoma cells, thus protecting the cells from NGF withdrawal-induced cell death . In line with this, BDNF-elicited ERK activation protects cortical neurons against a challenge with the topoisomerase I inhibitor campthothecin . In addition, it has been shown that activation of the ERK signaling pathway via treatment of the cells with either EGF or 12-O-tetradecanoylphorbol-13-acetate may lead to an inactivation of caspase-9 due to a direct phosphorylation of Thr125 of caspase-9 by ERK. This phosphorylation blocks caspase-9 processing and the subsequent activation of caspase-3 .
The survival of cells requires the presence of survival factors, and the lack of this trophic support is one of the best-studied signals for induction of cell death. In Rat-1 fibroblasts, overexpression of B-Raf protected the cells from apoptosis, induced by growth factor withdrawal. Treatment with the MEK inhibitor PD98059 blocked the anti-apoptotic activity of B-Raf, indicating that the activation of the Raf-MEK-ERK signaling pathway is necessary for the anti-apoptotic role of B-Raf in Rat-1 fibroblasts .
The activation of an estrogen-inducible activated Raf-1 mutant ΔRaf-1:ER also prevented apoptosis induced by loss of matrix contact (anoikis), cytoskeletal integrity and serum removal in lung fibroblasts . In these cells it has been shown that activation of ΔRaf-1:ER prevented the upregulation of Bim, a proapoptotic BH3-only protein of the Bcl-2 family, in serum-starved cells. This rescue relies on the activation of the ERK pathway and was independent of the JNK → c-Jun and PI3 kinase → PDK → AKT pathway . In human breast epithelial cells, the expression of genes encoding growth factors of the EGF family as a result of ΔRaf-1:ER activation protected the cells from detachment-induced apoptosis . Activation of ΔRaf-1:ER also blocked programmed cell death induced by TGFβ in MLCK epithelial cells . However, activation of ΔRaf-1:ER did not provide protection against oxidative glutamate toxicity in HT22 hippocampal cells . These data indicate that the anti-apoptotic function of Raf is restricted to particular apoptotic signaling pathways.
In addition to the well-established target MEK, Raf may use other effectors to inhibit programmed cell death. It has been shown that Raf-1 promotes cell survival in a MEK/ERK-independent manner via antagonizing apoptosis signal-regulating kinase-1 (ASK-1) . Raf-1 is also targeted to the mitochondria by Bcl-2 that leads to cell survival without ERK activation, probably by phosphorylating substrates other than MEK, such as Bcl-2 family members [37, 38].
Role of Raf in cellular differentiation
Results obtained with constitutively active Raf mutants has been questioned, since the lack of the regulatory domains in the Raf mutants may compromise the substrate specificity and the dynamic regulation of activity . Nevertheless, the many data obtained using these mutants have improved our knowledge of the functions of Raf in growth control, apoptosis and differentiation.
extracellular signal-regulated protein kinase
We thank Martin McMahon, UCSF, for providing ΔRaf:ER constructs and Libby Guethlein for critical reading of the manuscript. The research of the laboratory concerning intracellular signaling cascades underlying neuronal cell death and survival is supported by the Deutsche Forschungsgemeinschaft (grant SFB 530, C14).
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