Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT

The RAS-activated RAF-->MEK-->extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)-->PDK1-->AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G(1) cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27(Kip1) expression. Repression of p27(Kip1) was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27(Kip1). Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27(Kip1) in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21(Cip1) from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF-->MEK-->ERK and PI3'K-->PDK-->AKT signaling pathways can cooperate to promote G(0)-->G(1)-->S-phase cell cycle progression in both normal and cancer cells.     

Cyclin D1 production in cycling cells depends on ras in a cell-cycle-specific manner.

BACKGROUND: Cellular Ras and cyclin D1 are required at similar times of the cell cycle in quiescent NIH3T3 cells that have been induced to proliferate, but not in the case of cycling NIH3T3 cells. In asynchronous cultures, Ras activity has been found to be required only during G2 phase to promote passage through the entire upcoming cell cycle, whereas cyclin D1 is required through G1 phase until DNA synthesis begins. To explain these results in molecular terms, we propose a model whereby continuous cell cycle progression in NIH3T3 cells requires cellular Ras activity to promote the synthesis of cyclin D1 during G2 phase. Cyclin D1 expression then continues through G1 phase independently of Ras activity, and drives the G1-S phase transition. RESULTS: We found high levels of cyclin D1 expression during the G2, M and G1 phases of the cell cycle in cycling NIH3T3 cells, using quantitative fluorescent antibody measurements of individual cells. By microinjecting anti-Ras antibody, we found that the induction of cyclin D1 expression beginning in G2 phase was dependent on Ras activity. Consistent with our model, cyclin D1 expression during G1 phase was particularly stable following neutralization of cellular Ras. Finally, ectopic expression of cyclin D1 largely overcame the requirement for cellular Ras activity during the continuous proliferation of cycling NIH3T3 cells. CONCLUSIONS: Ras-dependent induction of cyclin D1 expression beginning in G2 phase is critical for continuous cell cycle progression in NIH3T3 cells.     

La(3+)-induced extracellular signal-regulated kinase (ERK) signaling via a metal-sensing mechanism linking proliferation and apoptosis in NIH 3T3 cells

The effects of La(3+) on the extracellular signal-regulated kinase (ERK) signaling were investigated to explore the mechanism by which La(3+) results in cell proliferation associated with apoptosis in mouse embryo fibroblast NIH 3T3 cells. Our data showed that La(3+) ions could induce a pulse of phosphorylation of ERK mainly through an unknown metal-sensing mechanism, which is different from the Ca(2+)-sensing receptor . The putative sensor protein showed one binding site for La(3+) with a dissociation constant of approximately 8 nM. Inductions of c-fos, c-myc, and cyclin D1 and phosphorylation of retinoblastoma protein (pRb) were observed after activation of ERK. These results are consistent with our previous observation that La(3+) promotes proliferation by helping the cells pass through the G1/S restriction point and enter S phase. This La(3+)-induced signaling cascade exhibited abnormally sustained c-myc induction and pRb phosphorylation. Furthermore, a continual increase of the p53 level was observed along with the signal transduction, and a significant decrease of B-cell lymphoma/leukemia-2 gene was observed after approximately 18 h of incubation. All of the results were highly correlated with the increase of S-phase population and apoptotic cells. Therefore, the experimental results suggested that La(3+) induced cell proliferation and apoptosis compatible to a p53-related mechanism in NIH 3T3 cells via an ERK-signaling cascade induced by a metal-sensing mechanism.     

Involvement of fibroblast growth factor receptor 2 isoform switching in mammary oncogenesis

We identified the IIIb C2 epithelial cell-specific splice variant of fibroblast growth factor receptor 2 (FGFR2 IIIb C2) receptor tyrosine kinase in a screen for activated oncogenes expressed in T-47D human breast carcinoma cells. We found FGFR2 IIIb C2 expression in breast carcinoma cell lines and, additionally, expression of the mesenchymal-specific FGFR2 IIIc splice variant in invasive breast carcinomas. FGFR2 IIIc expression was associated with loss of epithelial markers and gain of mesenchymal markers. Although FGFR2 IIIb is expressed in epithelial cells, previous studies on FGFR2 IIIb transformation have focused on NIH 3T3 fibroblasts. Therefore, we compared the transforming activities of FGFR2 IIIb C2 in RIE-1 intestinal cells and several mammary epithelial cells. FGFR2 IIIb C2 caused growth transformation of epithelial cells but morphologic transformation of only NIH 3T3 cells. FGFR2 IIIb C2-transformed NIH 3T3, but not RIE-1 cells, showed persistent activation of Ras and increased cyclin D1 protein expression. NIH 3T3 but not RIE-1 cells express keratinocyte growth factor, a ligand for FGFR2 IIIb C2. Ectopic treatment with keratinocyte growth factor caused FGFR2 IIIb C2-dependent morphologic transformation of RIE-1 cells, as well as cyclin D1 up-regulation, indicating that both ligand-independent and stromal cell-derived, ligand-dependent mechanisms contribute to RIE-1 cell transformation. Our results support cell context distinct mechanisms of FGFR2 IIIb C2 transformation.     

Schlafen-1 causes a cell cycle arrest by inhibiting induction of cyclin D1

Schlafen-1 (Slfn-1), the prototypic member of the Schlafen family of proteins, was described as an inducer of growth arrest in T-lymphocytes and causes a cell cycle arrest in NIH3T3 fibroblasts prior to the G1/S transition. How Slfn-1 exerts its effects on the cell cycle is not currently known. We report that synchronized murine fibroblasts expressing Slfn-1 do not exit G1 when stimulated with fetal calf serum, platelet-derived growth factor BB (PDGF-BB) or epidermal growth factor (EGF). The induction of cyclin D1 by these stimuli was blocked in the presence of Slfn-1 as were all downstream cell cycle processes. Overexpression of cyclin D1 in growth-arrested, Slfn-1-expressing cells induced an increase in cell growth consistent with this protein being the biological target of Slfn-1. Activation of the mitogen-activated protein kinase pathway by EGF or phorbol 12-myristate 13-acetate was unaffected by Slfn-1 expression. PDGF signaling was, however, almost completely blocked. This was due to a lack of PDGF receptor expression in Slfn-1-expressing cells consistent with Slfn-1 blocking the cell cycle in G1 where PDGF receptor expression is normally down-regulated. Finally, overexpression of Slfn-1 inhibited the activation of the cyclin D1 promoter. Slfn-1 therefore causes a cell cycle arrest during G1 by inhibiting induction of cyclin D1 by mitogens.     

DeltaRaf-1:ER* bypasses the cyclic AMP block of extracellular signal-regulated kinase 1 and 2 activation but not CDK2 activation or cell cycle reentry

Elevation of cellular cyclic AMP (cAMP) levels inhibits cell cycle reentry in a variety of cell types. While cAMP can prevent the activation of Raf-1 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) by growth factors, we now show that activation of ERK1/2 by DeltaRaf-1:ER is insensitive to cAMP. Despite this, DeltaRaf-1:ER-stimulated DNA synthesis is still inhibited by cAMP, indicating a cAMP-sensitive step downstream of ERK1/2. Although cyclin D1 expression has been proposed as an alternative target for cAMP, we found that cAMP could inhibit DeltaRaf-1:ER-induced cyclin D1 expression only in Rat-1 cells, not in CCl39 or NIH 3T3 cells. DeltaRaf-1:ER-stimulated activation of CDK2 was strongly inhibited by cAMP in all three cell lines, but cAMP had no effect on the induction of p21(CIP1). cAMP blocked the fetal bovine serum (FBS)-induced degradation of p27(KIP1); however, loss of p27(KIP1) in response to DeltaRaf-1:ER was less sensitive in CCl39 and Rat-1 cells and was completely independent of cAMP in NIH 3T3 cells. The most consistent effect of cAMP was to block both FBS- and DeltaRaf-1:ER-induced expression of Cdc25A and cyclin A, two important activators of CDK2. When CDK2 activity was bypassed by activation of the ER-E2F1 fusion protein, cAMP no longer inhibited expression of Cdc25A or cyclin A but still inhibited DNA synthesis. These studies reveal multiple points of cAMP sensitivity during cell cycle reentry. Inhibition of Raf-1 and ERK1/2 activation may operate early in G(1), but when this early block is bypassed by DeltaRaf-1:ER, cells still fail to enter S phase due to inhibition of CDK2 or targets downstream of E2F1.     

Regulation of apoptotic and growth inhibitory activities of C/EBPalpha in different cell lines

C/EBPalpha is expressed in many tissues and inhibits cell growth. In this paper, we have examined mechanisms which regulate activities of C/EBPalpha in cell lines derived from different tissues. We found that C/EBPalpha possesses strong pro-apoptotic activity in NIH3T3 cells, while this activity is not detected in 3T3-L1, Hep3B2 and HEK293 cells. Micro-array data show that C/EBPalpha activates many genes of apoptosis signaling in NIH3T3 cells. One of these genes, ARL6IP5, is a direct target of C/EBPalpha and is a key mediator of the apoptosis. Using C/EBPalpha mutants which do not cause cell death; we have found that C/EBPalpha does not arrest proliferation of NIH3T3 cells. The lack of growth arrest in NIH3T3 cells correlates with the inhibition of p16INK4 and with low levels of cyclin D3. The limited growth inhibitory activity of C/EBPalpha is also observed in Hep3B2 cells which express low levels of cyclin D3. Elevation of cyclin D3 restores growth inhibitory activity of C/EBPalpha in NIH3T3 and in Hep3B2 cells. These data show that apoptotic and growth inhibitory activities of C/EBPalpha are differentially regulated in different cells and that cooperation of cyclin D3 and C/EBPalpha is required for the inhibition of proliferation.     

Rho GTPase-dependent signaling is required for macrophage migration inhibitory factor-mediated expression of cyclin D1

Our previous studies demonstrated that the proinflammatory peptide, macrophage migration inhibitory factor (MIF), functions as an autocrine mediator of both growth factor- and integrin-dependent sustained ERK MAPK activation, cyclin D1 expression, and cell cycle progression. We now report that MIF promotes the activation of the canonical ERK MAPK cascade and cyclin D1 expression by stimulating the activity of the Rho GTPase and downstream signaling to stress fiber formation. Rho-dependent stress fiber accumulation promotes the sustained activation of ERK and subsequent cyclin D1 expression during G(1)-S phase cell cycle progression. This pathway is reported to be dependent upon myosin light chain (MLC) kinase, integrin clustering, and subsequent activation of focal adhesion kinase, leading to sustained MAPK activity. Our studies reveal that recombinant MIF induces cyclin D1 expression in a Rho-, Rho kinase-, MLC kinase-, and ERK-dependent manner in asynchronous NIH 3T3 fibroblasts. Moreover, MIF(-/-) murine embryonic fibroblasts display aberrant cyclin D1 expression that is linked to defective Rho activity, stress fiber formation, and MLC phosphorylation. These results suggest that MIF is an integral autocrine mediator of Rho GTPase-dependent signaling events and provide mechanistic insight into how MIF regulates proliferative, migratory, and oncogenic processes.     

ERK1/2 and p38 cooperate to delay progression through G1 by promoting cyclin D1 protein turnover

The conditional kinase DeltaMEKK3:ER allows activation of JNK, p38 and ERK1/2 without overt cellular stress or damage and has proved useful in understanding how these pathways regulate apoptosis and cell cycle progression. We have previously shown that activation of DeltaMEKK3:ER causes a sustained G(1) cell cycle arrest which requires p21(CIP1), with ERK1/2 and p38 cooperating to promote p21(CIP1) expression. In cells lacking p21(CIP1), DeltaMEKK3:ER causes only a transient delay in cell cycle re-entry. We now show that this delay in cell cycle re-entry is due to a reduction in cyclin D1 levels. Activation of DeltaMEKK3:ER promotes the proteasome-dependent turnover of cyclin D1; this requires phosphorylation of threonine 286 (T(286)) and expression of cyclin D1T(286)A rescues the delay in G(1)/S progression. DeltaMEKK3:ER-dependent phosphorylation of T(286) does not appear to be mediated by GSK3beta but requires activation of the ERK1/2 and p38 pathways. ERK1/2 can physically associate with cyclin D1 but activation of ERK1/2 alone is not sufficient for phosphorylation of T(286). Rather, cyclin D1 phosphorylation appears to require coincident activation of ERK1/2 and p38. Thus activation of DeltaMEKK3:ER promotes a sustained G(1) cell cycle arrest by a bipartite mechanism involving the rapid destruction of cyclin D1 and the slower more prolonged expression of p21(CIP1). This has parallels with the bipartite response to ionizing radiation and p53-independent mechanisms of G(1) cell cycle arrest in simple organisms such as yeast.     

Complementation of Defective Colony-Stimulating Factor 1 Receptor Signaling and Mitogenesis by Raf and v-Src

Ras-activated signal transduction pathways are implicated in the control of cell proliferation, differentiation, apoptosis, and tumorigenesis, but the molecular mechanisms mediating these diverse functions have yet to be fully elucidated. Conditionally active forms of Raf, v-Src, and MEK1 were used to identify changes in gene expression that participate in oncogenic transformation, as well as in normal growth control. Activation of Raf, v-Src, and MEK1 led to induced expression of c-Myc and cyclin D1. Induction of c-Myc mRNA by Raf was an immediate-early response, whereas the induction of cyclin D1 mRNA was delayed and inhibited by cycloheximide. Raf activation also resulted in the induction of an established c-Myc target gene, ornithine decarboxylase (ODC). ODC induction by Raf was mediated, in part, by tandem E-boxes contained in the first intron of the gene. Activation of the human colony-stimulating factor 1 (CSF-1) receptor in NIH 3T3 cells leads to activation of the mitogen-activated protein (MAP) kinase pathway and induced expression of c-Fos, c-Myc, and cyclin D1, leading to a potent mitogenic response. By contrast, a mutated form of this receptor fails to activate the MAP kinases or induce c-Myc and cyclin D1 expression and fails to elicit a mitogenic response. The biological significance of c-Myc and cyclin D1 induction by Raf and v-Src was confirmed by the demonstration that both of these protein kinases complemented the signaling and mitogenic defects of cells expressing this mutated form of the human CSF-1 receptor. Furthermore, the induction of c-Myc and cyclin D1 by oncogenes and growth factors was inhibited by PD098059, a specific MAP kinase kinase (MEK) inhibitor. These data suggest that the Raf/MEK/MAP kinase pathway plays an important role in the regulation of c-Myc and cyclin D1 expression in NIH 3T3 cells. The ability of oncogenes such as Raf and v-Src to regulate the expression of these proteins reveals new lines of communication between cytosolic signal transducers and the cell cycle machinery.     

Ras transformation results in an elevated level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells.

Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells resulted in cellular transformation and an acceleration of G1 progression of these cells. A shortened G1 phase was found to be associated with an increased level of cyclin D1 but not cyclin E protein. Using an antisense blocking method, reduced synthesis of cyclin D1 in v-H-Ras transformants resulted in a slower G1 progression rate of these cells. Although constitutive overexpression of cyclin D1 in NIH 3T3 cells accelerated G1 progression, cells remained untransformed. 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 G1 phase of these cells. Activation of an additional Ras-independent pathway was suggested to be responsible for the further acceleration of the G1 phase in Src transformants.     

ERK signaling is triggered by hepatitis C virus E2 protein through DC-SIGN

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a binding receptor for hepatitis C virus (HCV). Binding of HCV envelope protein E2 to target cells is a prerequisite to DC-SIGN-mediated signaling. Using cell lines with stable or transient expression of DC-SIGN, we investigated effects of soluble HCV E2 protein on ERK pathway. MEK and ERK are activated by the E2 in NIH3T3 cells stably expressing DC-SIGN. Treatment of the cells with antibody to DC-SIGN results in inhibition of the E2 binding as well as the E2-induced MEK and ERK activation. In HEK293T cells transiently expressing DC-SIGN, activation of MEK and ERK is also induced by the E2. Activation of ERK pathway by HCV E2 through DC-SIGN provides useful information for understanding cellular receptor-mediated signaling.     

Requirement for a hsp90 chaperone-dependent MEK1/2-ERK pathway for B cell antigen receptor-induced cyclin D2 expression in mature B lymphocytes

A requirement for cyclin D2 in G(1)-to-S phase progression has been definitively established in mature B cells stimulated via the B cell antigen receptor (BCR). However, the identity of constituents of the BCR signaling cascade that leads to cyclin D2 accumulation remains incomplete. We report that inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1/2 blocked BCR-induced activation of extracellular signal-regulated kinase (ERK). Inhibition of the MEK1/2-ERK pathway was sufficient to abrogate BCR-induced cyclin D2 expression at the mRNA and protein levels. Disruption of endogenous heat shock protein 90 (hsp90) function with geldanamycin abrogated BCR-induced cyclin D2 expression and proliferation. Geldanamycin effects were attributed to a selective depletion of cellular Raf-1 that interrupted BCR-coupled activation of MEK1/2 and ERK. By contrast, signaling through the phosphatidylinositol 3-kinase and protein kinase C pathways was not affected, suggesting that disruption of hsp90 function did not cause a general impairment of BCR signaling. These results suggest that the MEK1/2-ERK pathway is essential for BCR signaling to cyclin D2 accumulation in ex vivo splenic B lymphocytes. Furthermore, these findings imply that hsp90 function is required for BCR signaling through the Raf-1-MEK1/2-ERK pathway but not through the phosphatidylinositol 3-kinase- or protein kinase C-dependent pathways.     

Enhancement of serum- and platelet-derived growth factor-induced cell proliferation by Necl-5/Tage4/poliovirus receptor/CD155 through the Ras-Raf-MEK-ERK signaling

Necl-5/Tage4/poliovirus receptor/CD155 has been shown to be the poliovirus receptor and to be up-regulated in rodent and human carcinoma. We have found previously that mouse Necl-5 regulates cell motility. We show here that mouse Necl-5 is furthermore involved in the regulation of cell proliferation. Studies using a specific antibody against Necl-5 and a dominant negative mutant of Necl-5 revealed that Necl-5 enhanced the serum-induced proliferation of NIH3T3, Swiss3T3, and mouse embryonic fibroblast cells. Necl-5 enhanced the serum-induced activation of the Ras-Raf-MEK-ERK signaling, up-regulated cyclins D2 and E, and down-regulated p27(Kip1), eventually shortening the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 similarly enhanced the platelet-derived growth factor-induced activation of the Ras-Raf-MEK-ERK signaling and shortened the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 acted downstream of the platelet-derived growth factor receptor and upstream of Ras. Moreover, up-regulated Necl-5 was involved at least partly in the enhanced proliferation of transformed cells including NIH3T3 cells transformed by an oncogenic Ras or v-Src. These results indicate that Necl-5 plays roles not only in cell motility but also in cell proliferation.     

Biphasic estradiol-induced AKT phosphorylation is modulated by PTEN via MAP kinase in HepG2 cells

We reported previously in HepG2 cells that estradiol induces cell cycle progression throughout the G1-S transition by the parallel stimulation of both PKC-alpha and ERK signaling molecules. The analysis of the cyclin D1 gene expression showed that only the MAP kinase pathway was involved. Here, the presence of rapid/nongenomic, estradiol-regulated, PI3K/AKT signal transduction pathway, its modulation by the levels of the tumor suppressor PTEN, its cross-talk with the ERK pathway, and its involvement in DNA synthesis and cyclin D1 gene promoter activity have all been studied in HepG2 cells. 17beta-Estradiol induced the rapid and biphasic phosphorylation of AKT. These phosphorylations were independent of each other, being the first wave of activation independent of the estrogen receptor (ER), whereas the second was dependent on ER. Both activations were dependent on PI3K activity; furthermore, the ERK pathway modulated AKT phosphorylation by acting on the PTEN levels. The results showed that the PI3K pathway, as well as ER, were strongly involved in both G1-S progression and cyclin D1 promoter activity by acting on its proximal region (-254 base pairs). These data indicate that in HepG2 cells, different rapid/nongenomic estradiol-induced signal transduction pathways modulate the multiple steps of G1-S phase transition.     

Muscarinic cholinergic receptors activate both inhibitory and stimulatory growth mechanisms in NIH3T3 cells.

Activation of G(q) protein-coupled receptors can either stimulate or inhibit cell growth. Previously, these opposite effects were explained by differences in the cell models. Here we show that activation of m3 muscarinic acetylcholine receptors ectopically expressed in NIH3T3 cells can cause stimulation and inhibition of growth in the same cell. A clonal cell line was selected from cells that formed foci agonist dependently (3T3/m3 cells). In quiescent 3T3/m3 cells, carbachol stimulated DNA synthesis. In contrast, when 3T3/m3 cells were growing, either due to the presence of serum or after transformation with oncogenic v-src, carbachol inhibited growth. This inhibition was not due to reduction of extracellular signal-regulated kinase activity because carbachol induced extracellular signal-regulated kinase phosphorylation in both quiescent and growing 3T3/m3 cells. Investigating the cell cycle mechanisms involved in growth inhibition, we found that carbachol treatment decreased cyclin D1 levels, increased p21(cip1) expression, and led to hypophosphorylation of the retinoblastoma gene product (Rb). Proteasome inhibitors blocked the carbachol-induced degradation of cyclin D1. Effects on p21(cip1) were blocked by a protein kinase C inhibitor. Thus, m3 muscarinic acetylcholine receptors couple to both growth-stimulatory and -inhibitory signaling pathways in NIH3T3 cells, and the observed effects of receptor activation depend on the context of cellular growth.     

p67/MetAP2 suppresses K-RasV12-mediated transformation of NIH3T3 mouse fibroblasts in culture and in athymic mice

In many tumor cells, the activation and activity of extracellular signal-regulated kinases (ERK1/2) are very high because of the constitutive activation of the Ras-mediated signaling pathway. Here, we ectopically expressed the human homologue of rat eukaryotic initiation factor 2-associated glycoprotein, p67/MetAP2, in EGF-treated mouse embryonic NIH3T3 fibroblasts and C2C12 myoblasts and NIH3T3 cell lines expressing the constitutively active form of MAP kinase kinase (MEK) to inhibit the activation and activity of ERK1/2 MAP kinases. In addition, we also ectopically expressed rat p67/MetAP2 in oncogenic Ras-induced transformed NIH3T3 fibroblasts and inhibited their transformed phenotype both in culture and in athymic nude mice possibly by inhibiting angiogenesis. This inhibition of ERK1/2 MAP kinases is due to the direct binding with rat p67/MetAP2, and this leads to the inhibition of activity of ERK1/2 MAP kinases both in vitro and in vivo. Furthermore, expression of p67/MetAP2 siRNA in both NIH3T3 fibroblasts and C2C12 myoblasts causes activation and activity of ERK1/2 MAP kinases. Our results thus suggest that ectopic expression of rat p67/MetAP2 in transformed cells can inhibit the tumorigenic phenotype by inhibiting the activation and activity of ERK1/2 MAP kinases and, thus, that p67/MetAP2 has tumor suppression activity.