We have previously shown that the persistent activation of p42/p44MAPK is required to pass the G 1 restriction point in fibroblasts (Pagè s, G., Lenormand, P., L'Allemain, G., Chambard, J. C., Meloche, S., and Pouyssé gur, J. Mammalian cells express multiple mitogen-activated protein (MAP) 1 kinases that mediate the effects of extracellular signals on a wide array of biological processes. In eukaryotic cells, three distinct MAPK cascades have been described, which appear to be linked to separate signal transduction pathways resulting in the final activation of either p42/p44 MAPK

The mitogen-activated protein kinases (MAP (25) and that sustained activation of MAP kinase during G1 progression appears to be essential for triggering entry into the S phase ofthe cell cycle (25,26). Here, exploiting the use of either MAP kinase antisense or dominant negative alleles, we directly demonstrate that MAP kinase activation is essential for Go-arrested fibroblasts to enter the cell cycle. MATERIALS AND METHODSMaterials. Highly purified human a-thrombin (3200 NIH units/mg) and human recombinant basic fibroblast growth factor (FGF) were generous gifts of J.

Abstract. Mitogen-activated protein kinases (p42 '~'k and p44 "~k) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45-~-t), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitopetagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatiotemporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45 m~ and MAP kinases (p42 "~pk, p44 m~) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or o~-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as a-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42 m~ and p44-~,k are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44,~,k T192A or Y194F). In marked contrast the p45 "~pa activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42 "~k and IM4 "~ during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.

A Hf-suicide technique based on the reversibility of Na+/H' antiport was developed for the selection of mutants deficient in this membrane-bound activity. The strategy was to use the Na+/H+ antiporter as a Hf-vector killing device. Chinese hamster lung fibroblasts (CCL39) were loaded with LiCi and incubated in Nat, Li'-free choline Cl saline solution (pH 5.5 8-8.3). These findings strongly suggest that the Na+/H+ antiport activity through regulation of intracellular pH plays a crucial role in growth control.The molecular events that control the reversible Go/Gj growth-arrest state of animal cells are largely unknown. A rather striking observation is that a variety of polypeptide growth factors (e.g., epidermal growth factor, platelet-derived growth factor, a-thrombin) acting on distinct membrane receptors (1-5), converge very early for the stimulation of ionic fluxes (6-10) and phosphorylation of a common set of proteins (11,12). Therefore, a complete dissection and analysis of the early and ubiquitous biochemical changes linked to mitogenicity should help the understanding of growth control at a molecular level.

The ubiquitously expressed Na+/H+ exchanger NHE1 is the target of multiple signaling pathways, including those activated by tyrosine kinase receptors, G protein-coupled receptors, and integrins. The intracellular pathways leading to activation of NHE1 are poorly understood. To gain more insight into these activation pathways, we examined the role of mitogen-activated protein kinases (MAPKs) as potential mediators of NHE1 activation by extracellular stimuli such as growth factors and hyperosmotic stress. Whereas p44 MAPK does not appear to phosphorylate NHE1 in vitro, we found that inhibition of the p42/p44 MAPK signaling by expression of a dominant negative form of p44 MAPK, by expression of the MAP kinase phosphatase MKP-1, or by inhibition of MAPK kinase 1 (MKK1) with the PD 98059 compound reduced by 50-60% NHE1 activation in response to growth factors. This inhibitory effect also was observed in C-terminal NHE1 deletion mutants in which the major phosphorylation sites have been deleted. Furthermore, the use of a CCL39-derived cell line expressing an estradiol-regulated form of oncogenic Raf-1 (CCL39-deltaRaf-1:ER) revealed that the exclusive activation of the Raf --> MKK1 --> p42/p44 MAPK cascade was capable of inducing NHE1 activation to the same extent as potent growth factors like thrombin. Together, our findings demonstrate that the p42/p44 MAPK cascade plays a predominant role in the regulation of NHE1 by growth factors, an action that is mediated via accessory proteins that remain to be identified. In contrast, we found no evidence in favor of the contribution of any MAPK, p42/p44, p38 MAPKs, and Jun kinase, in NHE1 activation by osmotic stress.

Progression of eukaryotic cells through the cell cycle is governed by the sequential formation, activation, and subsequent inactivation of a series of cyclin-dependent kinase (Cdk) complexes. p27Kip1 (p27) is a Cdk inhibitor that blocks, in vitro, the activity of cyclin D-Cdk4, cyclin D-Cdk6, cyclin E-Cdk2 as well as cyclin A-Cdk2, a complex active during S phase. The level of p27 protein expression, usually high in G 0 /G 1 resting cells, declines as cells progress toward S phase and enforced expression of p27 in fibroblasts causes G 1 arrest. This situation prevails in CCL39, a Chinese hamster lung fibroblast cell line (this report). However, in addition to p27, several other Cdk inhibitors known to alter G 1 progression coexist in most mammalian cells. To investigate the specific contribution of p27 in the control of the mitogensensitive G 0 /G 1 arrest, we specifically reduced its synthesis by expressing a full-length p27 antisense cDNA in CCL39 cells. Interestingly, reduction of up to 90% of p27 protein expression increased both basal and serumstimulated gene transcription of cyclin D1, cyclin A, dihydrofolate reductase, and DNA synthesis reinitiation. Moreover, overexpression of this antisense allows cells to grow for several generations in a serum-free medium supplemented with insulin and transferrin only, thus suggesting that p27-depleted cells cannot exit the cell cycle. These effects were fully reversed by coexpression of a plasmid encoding p27 sense. We conclude that p27, by setting the level of growth factor requirement, plays a pivotal role in controlling cell cycle exit, a fundamental step in growth control.

The primary action of a family of mitogens including bombesin, bradykinin, vasopressin and alpha-thrombin is to activate the hydrolysis of polyphosphoinositides. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phospholipase C is mediated through coupling of surface receptors to a GTP-binding protein (Gp protein) which, in some cells, is inactivated by the toxin of Bordetella pertussis. It is not known whether this signalling pathway is involved in initiating DNA replication, whereas it has been firmly established that reinitiation of DNA synthesis can be triggered without activation of PtdIns(4,5)P2 hydrolysis by, for example, EGF (epidermal growth factor), FGF (fibroblast growth factor) and insulin/IGF-I (insulin-like growth factor-I), members of a class of mitogens known to activate receptor tyrosine kinases. Taking advantage of the fact that Chinese hamster lung fibroblasts respond to either class of mitogens and that their Gp protein appears to be sensitive to pertussis toxin, we have now analysed the toxin's effect on reinitiation of DNA synthesis and find that it inhibits up to 95% of thrombin-induced mitogenicity without affecting EGF- or FGF-induced DNA synthesis and proliferation. These findings strongly suggest that activation of PtdIns(4,5)P2-phospholipase C has a determinant function in growth control, and confirm the existence of alternative growth factor-signalling pathways independent of polyphosphoinositide breakdown.

In response to growth factors, quiescent tibroblast mutants lacking Na+/H+ exchange activity fail to elevate their cytoplasmic pH OpH,) and to reinitiate DNA synthesis at neutral and acidic pH,. A pH, threshold of -7.2 exists, below which growth factors cannot set in motion the G, to S phase transition. Restoration of the pH, defect in mutant cells restores the wild-type phenotype. These findings, combined with the properties of another class of mutants able to grow at very low pH,, demonstrate that pH,, modulated by growth factor activation of the Na+/H+ antiporter, plays a determinant role in growth control.Na+ffi+ antiporter pff, Fibroblast mz&mt DNA synthesis Growth factor