Signaling pathways that operate through tightly controlled protein phosphorylation cascades transduce extracellular signals to various intracellular targets. Some of these targets regulate the transcriptional and translational machinery to ensure proper cell cycle progression, cell growth, or differentiation. Phosphorylation of the S6 protein of 40 S ribosomal subunits is a highly conserved response of animal cells to treatment with growth factors, steroid hormones, phorbol esters, and oncogenes(1) . Inhibition of S6 phosphorylation by exposure of cells to the immunosuppressant rapamycin selectively suppresses the translation of certain mRNAs that contain a polypyrimidine tract at the 5` end(2) . These mRNAs encode ribosomal proteins and protein synthesis elongation factors, whose production is required for efficient transit through the G phase of the cell cycle.

Two families of mitogen-stimulated S6 kinases have been identified: the rsk-encoded M 85,000-92,000 S6 kinases ((3) , referred to as p90) ()and the M 70,000 and 85,000 S6 kinases (Refs. 4 and 5, referred to as p70 and p85). A variety of evidence indicates that p90 and p70/p85 lie on different signaling pathways (6, 7, 8, 9, 10) . Unlike p70(8) , p90 is phosphorylated and activated by the erk-encoded M 42,000 and 44,000 mitogen-activated protein (MAP) kinases ((6) , referred to as p42 and p44) in response to signals transmitted through p21, p74, and p47(11) . Once activated, p90 and p42/p44 can be translocated to the nucleus(12, 13) , where they are thought to phosphorylate nuclear transcription factors, thus promoting the transcription of genes required for the growth response.

p70 is the physiological S6 kinase activity in mammalian cells(14) . p85 is a minor species that is identical to p70 except for the presence of a 23-amino acid extension at the amino terminus that carries features of a nuclear targeting sequence(15, 16) . Indeed, p85 is localized in the nucleus (17, 18) , where it might phosphorylate a nuclear pool of S6 protein (19) or the cAMP-response element modulator (CREM), a transcription factor which was recently identified as a substrate of p70(20) . Activation of p70 in response to mitogens is associated with phosphorylation of three serines and one threonine located at the carboxyl terminus of the kinase(21) . p70also contains additional phosphate groups that become dephosphorylated upon rapamycin treatment, leading to inactivation of p70(22) . Each set of phosphorylation sites might be modified by distinct kinases; however, direct activators of p70/p85are so far unknown. Recent experiments based on the use of rapamycin (23, 24) , specific phosphatidylinositol 3-kinase inhibitors such as wortmannin (25, 26) and platelet-derived growth factor receptor mutants (27) have suggested that phosphatidylinositol 3-kinase and the structurally related enzyme RAFT/FRAP are involved in upstream signaling to p70/p85.

Two lines of evidence have suggested that the function of p70/p85 during G is important for cell cycle progression. First, inhibition of p70/p85 by treatment of cells with rapamycin leads to cell cycle arrest in G or a delay of entry into S phase, depending on the cell type(9, 14) . Second, microinjection of rat embryo fibroblasts with antibodies that inhibit p70/p85 abolishes the serum-induced entry into S phase(17, 28) . Activation of p42/p44 is also thought to be essential for triggering the proliferative response in fibroblasts(29) . To gain further insight into how p70/p85 and p42/p44activity is regulated and what role the enzymes might have in cell growth and cell cycle control, we have examined the behavior of these enzymes during the cell cycle. We show here that p70 and p42/p44 activities are regulated in a cell cycle-dependent manner. Furthermore, we present evidence that the cell cycle regulation of p70/p85activity might involve compartmentation and a regulatory mechanism that is independent of the four carboxyl-terminal phosphorylation sites. Finally, our observations of the behavior of p70 and p42/p44 during the cell cycle suggest that there is cross-talk between these signaling molecules and the cell cycle machinery.

MATERIALS AND METHODS

Synchronization of Cells

To arrest cells in metaphase, cells were first presynchronized in G by serum starvation and then stimulated with EGF, insulin, and FCS as described above. Nocodazole (0.4 µg/ml, Sigma) was added 20 h after release from G, before cells entered M phase. Mitotic cells were collected 4 h later by gentle pipetting and were reseeded into DMEM plus 10% FCS at 0.5 10 cells per 10-cm plate. These conditions have been shown to result in an efficient and reversible M phase block that prevents cells from entering a polyploid state(30) . Mitotic cells were isolated without nocodazole by multiple rounds of mechanical shake-off alone (31) from subconfluent plates of cells which were synchronized in G and stimulated for 22 h with mitogens as described above. Cells collected from early rounds were stored in DMEM plus 10% FCS at 4 °C during the collection process.

Cell Extraction, Protein Determination and Kinase Assays

S6 kinase activity was measured using 40 S ribosomal subunits as substrate as described earlier(26) . The reactions were stopped and the samples were subjected to polyacrylamide gel electrophoresis and scintillation counting as described previously(33) . A unit is defined as the amount of kinase incorporating 1 pmol of P into S6 per min.

Polyclonal antibodies to a His-tagged fragment of rat p70 (amino acids 258-469; (4) ) were produced and affinity purified as described previously(34) . These purified antibodies show no cross-reaction with p90 on Western blots or in immunoprecipitation kinase assays (data not shown). For immunocomplex S6 kinase assays, Triton X-100 (to 1%), phenylmethylsulfonyl fluoride (to 0.1 mM), and purified S6 kinase antibody (1 µl) were added to 50 µl of cell extract supernatant and kept on ice for 3 h. Then 25 µl of 50% (v/v) protein A-agarose (preincubated with 1% bovine serum albumin in extraction buffer plus 1% Triton X-100) was added and incubated for 1 h at 4 °C. The beads were washed twice with extraction buffer plus 1% Triton X-100 and twice with S6 kinase assay buffer without dithiothreitol. The kinase assays were performed essentially as described above. Immunocomplex S6 kinase assays with hemagglutinin (HA) antibody 12CA5 (35) were performed essentially as described above except that cell extract supernatants containing 800 µg of protein were used per sample and more extensive washes of the beads were done.

For MAP kinase immunocomplex assays, immunoprecipitations with antibody 122 against p42 were performed essentially as described above, except that the last two washes were done with MAP kinase assay buffer (30 mM Tris, pH 8, 20 mM MgCl, 2 mM MnCl, 0.1% Triton X-100, and 0.1 mM dithiothreitol). MAP kinase assays were initiated by adding 15 µl of MAP kinase assay buffer containing 10 µM ATP, 2 µM of the peptide inhibitor of cAMP-dependent protein kinase (Sigma), 10 µg of myelin basic protein (Sigma), and 0.33 µl [-P]ATP. After 30 min at 37 °C the reactions were stopped and the samples were subjected to electrophoresis on SDS-20% polyacrylamide gels, autoradiography and scintillation counting.

Flow Cytometry

Immunofluorescence

Western Blotting

Fibroblasts Stably Expressing p70 Constructs

()

RESULTS

S6 Kinase and MAP Kinase Activity in Cells Released from G

Figure 1: Behavior of p70 and p42/p44 in synchronized cells released from G. Subconfluent fibroblasts were serum starved and released into the cell cycle by adding mitogens at t = 0 h (see ``Materials and Methods''). A, synchrony of the cells was followed by analyzing their DNA content by flow cytometry. B, at the indicated times after release, extract supernatants were prepared from a parallel set of cells and immunocomplex kinase assays were performed to measure p70() and p42 () activity. C, proteins in cell extract supernatants were separated by polyacrylamide gel electrophoresis and transferred onto nitrocellulose. p70(upper panel) and MAP kinases (lower panel) were detected with polyclonal antibodies (see ``Materials and Methods'').

To measure p70 activity in extracts of synchronized cells, immunocomplex kinase assays were performed using 40 S ribosomal subunits as a substrate. In these experiments an antibody that recognizes both p70 and p85 was used. However, since p85 is much less abundant than p70 in these cells(17) , most of the activity measured in the immunocomplexes is contributed by p70. p70 activity measured 20 min after mitogen stimulation was more than 20 times higher than the activity measured in serum-starved cells (Fig. 1B, closed circles). The kinase lost 40% of its activity by the end of G (Fig. 1B, 16 h), consistent with published data(28) . During S and G the activity continued to decrease, so that during M phase a relatively low level of activity was measured (Fig. 1B). It was shown earlier that p70 is activated at the first G/M boundary during meiotic maturation of Xenopus laevis oocytes(39) . However, we saw no increase in p70 activity in fibroblasts cycling from G into M (Fig. 1B). As the nuclear transcription factor CREM has been shown to be phosphorylated by p70(20) , we also performed immunocomplex kinase assays with recombinant CREM protein as a substrate. The pattern of p70 activity toward CREM during the cell cycle was virtually identical to that seen toward S6 (data not shown).

All evidence obtained so far indicates that p70 is activated by phosphorylation(21, 40) . To determine if the gradual decrease in p70 activity observed in Fig. 1B could be due to dephosphorylation, the phosphorylation state of p70 was assessed by its appearance as multiple bands on Western blots. The active, phosphorylated form of p70 migrates more slowly in SDS-polyacrylamide gels than the dephosphorylated, inactive species (40) . After 20 min of stimulation the most highly phosphorylated form of p70 was detected (Fig. 1C, upper panel), which correlated to the highest S6 kinase activity (Fig. 1B, closed circles). The hyperphosphorylated form was seen throughout G, and by the end of G partially dephosphorylated forms of the kinase appeared (Fig. 1C, upper panel). As p70 activity continued to decrease, the hypophosphorylated forms of the kinase became predominant. The Western analysis also showed that the amount of p70 protein remained constant during the cell cycle (Fig. 1C). Together, these data demonstrate that p70 activity is cell cycle regulated and that the changes in activity appear to be mediated by phosphorylation-dephosphorylation.

In parallel to p70, we also examined the cell cycle regulation of MAP kinases. Immunocomplex kinase assays showed that p42 was strongly but only transiently activated early in G/G (Fig. 1B, open squares). During S and G, p42 activity was at basal levels and during G/M the kinase activity increased to a small extent. MAP kinases were also visualized on Western blots that were probed with an antibody that recognizes both p42 and p44 (Fig. 1C, lower panel). The mitogen-dependent activation of p42/p44 was clearly seen as a shift of the proteins to the phosphorylated, more slowly migrating forms after 20 min of stimulation (Fig. 1C, lower panel). Phosphorylated species of p42/p44 were also seen at 4 h but thereafter the dephosphorylated forms were predominant. Because p42 gives a relatively faint signal on Western blots, no up-shifted band that would represent active p42 was seen in G/M extracts (Fig. 1C, lower panel). However, a minor upper band representing active p44 was visible at later stages in the cell cycle (Fig. 1C, lower panel).

S6 Kinase and MAP Kinase Activity in Cells Released from Metaphase

Figure 2: p70 and p42/p44 in cells progressing from M phase into G. Mitotic cells were collected after nocodazole treatment as described under ``Materials and Methods'' and reseeded into drug-free medium at t = 0 h. A, entry into G was followed by analyzing cellular DNA content by flow cytometry. B, at the indicated times, cell extract supernatants were made and p70 () and p42() activity was measured in immunocomplex kinase assays. C, the amount of expressed protein and the phosphorylation state of p70 (upper panel) and MAP kinases (lower panel) were determined by Western analysis.

p42 also became activated in G cells after release from nocodazole but the activity returned very rapidly to near-basal levels (Fig. 2B, open squares). Western analysis showed that p42 and p44 were phosphorylated 1.5 h after release from nocodazole and extensively dephosphorylated 3 h after release (Fig. 2C, lower panel). These results show that p70 and p42/p44 are not only activated at the G/G boundary, but also in fibroblasts cycling from M into G after release from a nocodazole block.

One could argue that the low p70 activity measured in metaphase-arrested cells might be an artifact of nocodazole treatment. To exclude this possibility, we compared the amount of p70 activity in mitotic cells that were collected by shake-off with or without nocodazole treatment and found that there was no significant difference (Fig. 3A). In addition, there was no difference in kinase activity in the drug-treated or non-treated cells that were left on the plates after mitotic shake-off (Fig. 3A). We also tested whether nocodazole inhibits the stimulation of p70 in resting cells upon addition of EGF. Pretreatment of cells for 30 min with different concentrations of nocodazole did not significantly inhibit the EGF-induced activation of p70 (Fig. 3B). Thus, these control experiments show that the low p70 activity measured in nocodazole-treated cells in Fig. 2B is not an artifact of drug treatment.

Figure 3: Effect of nocodazole on S6 kinase activity. A, mitotic cells were collected by shake-off after treatment with (+) or without(-) nocodazole (see ``Materials and Methods''). Total S6 kinase activity was measured in extract supernatants prepared from mitotic cells and from cells that were left on the plates after the shake-off (non-mitotic cells). B, confluent cells were treated with (+) or without(-) different concentrations of nocodazole for 30 min. Then the cells were treated with or without 5 nM EGF for 20 min. Total S6 kinase activity was measured.

Additional control experiments were performed to determine whether kinase activation might be due to withdrawal of nocodazole rather than to a specific cell cycle change. Removal of nocodazole from cycling cells, resting cells, or cells in S phase after 4 h of treatment did not significantly increase p70 or p42/p44 activity (data not shown). Furthermore, release of cells from a hydroxyurea-induced S phase arrest did not lead to kinase activation (data not shown). Therefore, the activation of p70 and p42/p44 seen in Fig. 2B is probably not a general phenomenon seen whenever cells are released from a cell cycle block.

To further pinpoint when during M or G p70 and p42/44 become activated, shorter time points after release from the metaphase block were examined (Fig. 4A). Tubulin staining (red) and DAPI staining of DNA (blue) were done to follow the cells through the different stages of mitosis and to determine when cells entered G. The immunofluorescence pictures show that the metaphase-arrested cells had condensed DNA but no mitotic spindles (Fig. 4A, t = 0 min). Fifteen min after release from the nocodazole block the mitotic spindles had reformed (Fig. 4A). Thirty min after release the sister chromatids started to separate and 15 min later the cells were in anaphase. One hour after release the cells had gone through telophase and cytokinesis and had entered G.

Figure 4: Activation of p70 and MAP kinases during M/G. Nocodazole-arrested cells were induced to enter G (see ``Materials and Methods''). A, at the indicated times after release, cells were fixed with paraformaldehyde and microtubules were stained with a monoclonal antibody against tubulin (red; see ``Materials and Methods''). DNA was stained with DAPI (blue). B, cell extract supernatants were prepared at the indicated times and p70 (upper panel) and MAP kinases (lower panel) were examined on Western blots (see ``Materials and Methods'').

Examination of p70 on Western blots showed that a minor fraction of p70 was phosphorylated during anaphase (Fig. 4B, t = 45 min), but most of the enzyme became hyperphosphorylated after cells had gone through cytokinesis and entered G (Fig. 4B, t = 60 min). In contrast to p70, a substantial fraction of p42/p44 was already highly phosphorylated during anaphase of mitosis (Fig. 4B, t = 45 min). Together, these analyses show that p70 and p42/p44 display different kinetics of activation in cells progressing from M into G. p70 is activated very early in G and remains active throughout G, whereas p42/p44 are activated at the end of mitosis and remain active during very early G ( Fig. 2and 4).

Localization of p70/p85 during the Cell Cycle

Figure 5: Localization of S6 kinase during the cell cycle. A, immunofluorescence of fibroblasts which were synchronized in G and stimulated as described in the legend to Fig. 1. At the indicated times cells were fixed and incubated with p70 antibody (green) and DAPI (blue). Resting, t = 0 h; stimulated, t = 45 min; late G, t = 13 h; S, t = 17 h; G, t = 21 h; M, t = 24 h. B, immunofluorescence of fibroblasts which were released after nocodazole treatment as described in the legend to Fig. 2.

We also examined the distribution of p70/p85 in cells released from a nocodazole block. As was seen in Fig. 5A, the kinase colocalized with DNA during mitosis (Fig. 5B). In very early G, after cytokinesis had occurred and the DNA had decondensed, the speckled S6 kinase pattern appeared in the nucleus (Fig. 5B, t = 2 h) and then disappeared 1 h later. Thus, p70/p85 is localized in the cytoplasm at all times during the cell cycle but becomes enriched at certain locations in the nucleus during S/G phase and early G in cells released from a metaphase block.

Constitutive Signaling to p70 during the Cell Cycle

Figure 6: Effect of serum withdrawal on S6 kinase activity. A, fibroblasts were synchronized in G by serum starvation as described under ``Materials and Methods.'' At t = 0 h 20% FCS was added to induce cell cycle entry. Synchronized or asynchronously cycling fibroblasts received no further treatment (control), or at various times were incubated for 10 min without FCS or for 10 min without FCS and then 20 min with FCS. Total S6 kinase activity was determined in cell extract supernatants. B, Western blot analysis of p70 in cell extract supernatants prepared in A. Numbers refer to treatments in A.

Involvement of Carboxyl-terminal Phosphorylation Sites in Regulating p70 Activity during the Cell Cycle

Figure 7: Regulation of mutant p70 during the cell cycle. A, structure of expressed S6 kinase molecules. Mutant S6 kinase contains three Ser Asp and one Thr Glu changes in the carboxyl-terminal phosphorylation sites. Both wild-type and mutant proteins have an HA epitope at the amino terminus. B, S6 kinase activity in extract supernatants from fibroblasts transfected with wild-type p70 (pMV7+ wt S6K), mutant p70 (pMV7+ mutant S6K), or the pMV7 vector alone (pMV7) was measured in HA immunoprecipitates (see ``Materials and Methods''). Mitotic cells (M) were collected after nocodazole treatment as described under ``Materials and Methods'' and G cells were obtained 2 h after reseeding mitotic cells into drug-free medium. Contact-inhibited cells were treated for 15 min without (G) or with (EGF) 5 nM EGF. Contact-inhibited cells were also pretreated for 15 min with either 400 nM wortmannin (EGF+ WM) or 100 nM rapamycin (EGF+ Rapa) before addition of EGF. S phase cells were collected 16 h after addition of mitogens to serum-starved cells as described under ``Materials and Methods'' (S+ FCS) and were incubated for 10 min with DMEM in the absence of FCS (S+ DMEM). The autoradiograph shows incorporation of P into S6 during the kinase assay. Similar results were obtained in several independent experiments (data not shown). The P in S6 was quantitated by scintillation counting; results from two experiments were averaged and values for pMV7 were subtracted as background. For pMV7+ wt S6K: 486 cpm (M), 1720 cpm (G), 635 cpm (G), 2519 cpm (EGF), 62 cpm (EGF+ WM), 76 cpm (EGF+ Rapa), 6948 cpm (S+ FCS) and 1910 cpm (S + DMEM). For pMV7+ mutant S6K: 225 cpm (M), 469 cpm (G), 175 cpm (G), 482 cpm (EGF), 10 cpm (EGF + WM), 128 cpm (EGF+ Rapa), 855 cpm (S + FCS), and 154 cpm (S + DMEM). A portion (one-fifth volume) of the supernatants that had been subjected to precipitation with HA antibodies were then immunoprecipitated with antibodies to p70. Endogenous S6 kinase was assayed in the p70 immunoprecipitates (see ``Material and Methods'') and quantitated by scintillation counting. For pMV7+ wt S6K: 2010 cpm (G) and 10,350 cpm (EGF). For pMV7+ mutant S6K: 5745 cpm (G) and 12,525 cpm (EGF). For pMV7: 4000 cpm (G) and 12,540 cpm (EGF).

If the four carboxyl-terminal phosphorylation sites are responsible for regulating the activity of p70 during the cell cycle, the mutant kinase should display the same level of activity at all times. To test this prediction, mitotic and G populations of fibroblasts were collected and the tagged wild-type and mutant p70 were assayed in HA immunoprecipitates. As expected, the activity of recombinant wild-type p70 was low in mitotic cells and increased when cells entered G (Fig. 7B). However, the mutant enzyme also became more active as cells moved from M phase into G. No p70 activity was immunoprecipitated from cells transfected with the empty vector (Fig. 7B). To further characterize the behavior of the mutant p70, we examined its ability to respond to mitogens in G cells. Confluent fibroblasts were treated with or without EGF and the tagged kinases were assayed in HA immunoprecipitates. Both the wild-type and mutant kinases were activated upon addition of EGF (Fig. 7B). The degree of EGF-induced activation of the HA-tagged kinases (2.8-4.0 fold; Fig. 7B) was significantly lower than that seen with endogenous p70 in the parental cells (Fig. 3B). We therefore measured the activity of endogenous p70 in the transfected cells by adding p70 antibodies to supernatants that had been precleared with HA antibody. Assay of these immunoprecipitates showed that activation of the endogenous p70 in pMV7-transfected cells was also reduced (2.2-5.1 fold; see legend to Fig. 7B).

Having established that the mutant p70 could still be activated during M/G and G/G, we asked if the mutant enzyme was also sensitive to negative regulators of the p70 pathway such as wortmannin (27) and rapamycin(14) . Indeed, treatment of transfected fibroblasts with wortmannin or rapamycin completely abolished the EGF-induced activation of both mutant and wild-type p70 (Fig. 7B). In addition, similar to the results obtained with endogenous p70 in the parental cells (Fig. 6), withdrawal of FCS from transfected cells in S phase led to a rapid decline in S6 kinase activity of the mutant protein (Fig. 7B). Thus, the apparently normal regulation of the p70 mutant suggests that the carboxyl-terminal phosphorylation sites alone do not regulate p70 activity at the G/G boundary or at other stages during the cell cycle.

DISCUSSION

Cell Cycle Regulation of p70 and p42/p44 Activity

Our data suggest that there is a difference in the way that p70 and p42/p44 are activated in G/G cells as compared to M/G cells. The kinases in G are able to respond immediately to signals generated by external growth factors. By contrast, the enzymes in mitotic cells are maintained in an inactive state despite the presence of abundant growth factors in the medium, implying the existence of a negative regulatory mechanism. We have found that when metaphase-arrested cells are released into serum-free medium, mitosis is completed but p70 is not activated (data not shown). Thus, activation of p70 at M/G is triggered by the presence of external growth factors and not by the completion of mitosis per se. Completion of mitosis appears to suppress the negative regulatory mechanism, thus creating an environment that is permissive for kinase activation. This negative regulation of p70 and p42/p44 might be mediated by a component of the cell cycle machinery such as a cyclin, which accumulates until the end of M phase and is then rapidly degraded.

p70 and p42/p44 were activated more strongly during the G/G transition (Fig. 1B) than during M/G (Fig. 2B). This difference is probably linked to the differential protein synthesis requirement of these cells. Quiescent cells contain fewer ribosomes and synthesize proteins at a lower rate than cycling cells(41) . A sustained increase in protein synthesis is required for resting cells to synthesize new ribosomes and other proteins essential for entry into S phase(42) , and as a result the G phase is approximately 4 h longer than G of cycling cells. Since S6 phosphorylation enhances the synthesis of certain proteins involved in translation(2) , it seems consistent that cells moving from a quiescent state into S phase would require higher levels of S6 kinase activity than M/G cells. In addition, an essential function of MAP kinases in G might be to phosphorylate an inhibitory subunit of translation initiation factor eIF-4E, thereby stimulating the overall rate of protein synthesis in response to mitogens(43) .

Similar to our results, Tamemoto et al.(44) found that p42/p44 activity is low in nocodazole-arrested Chinese hamster ovary cells and that the enzymes become active upon entry into G. Furthermore, p42/p44 were re-activated at around M phase of the next cell cycle(44) . The interpretation of these results was that p42/p44 are activated biphasically, first in G and then in G/M before the nocodazole arrest point. However, loss of synchrony by the end of the first cell cycle would make it impossible to determine whether activation occurred in G/M or M/G. It has been proposed that activation of MAP kinases might be a one-time event involved in releasing cells from an arrested state, rather than a recurring event required for progression through each cell cycle(45) . This hypothesis is based mainly on experiments done with oocytes and early embryos. However, MAP kinase activation at M/G in fibroblasts ( Fig. 2and Fig. 4) and Chinese hamster ovary cells (44) suggests that the requirement for MAP kinase activity during G might be different in rapidly dividing embryonic cells and in established cell lines.

Nuclear Localization of p70/p85

Ribosomal protein S6 is present in both the cytoplasm and nucleus, where it is found in nucleoli and in association with chromatin(19) . The nucleolar pool of S6 has been shown to become phosphorylated in response to treatment of cells with phorbol esters(19) . Since protein synthesis does not take place in the nucleus, the function of phosphorylated S6 protein in this compartment remains obscure. Although it has been shown that p85 activity in the nucleus is required for entry into S phase(17) , it is not known whether the essential function of this enzyme is to phosphorylate S6 or another substrate.

Constitutive Signaling to p70

Involvement of Carboxyl-terminal Phosphorylation Sites in the Cell Cycle Regulation of p70

We have shown that the behavior of p70/p85 and p42/p44 is cell cycle-regulated. Because the activities of these enzymes are sensitive to changes in the growth factor supply, one might imagine that the kinases are part of a sensory system that evaluates growth conditions and makes the decision to exit or remain in the cell cycle. A tightly controlled interplay between these signal transduction molecules and the cell cycle machinery might be important for ensuring proper cell growth and proliferation.

FOOTNOTES

*

This work was supported in part by a grant from the Austrian Industrial Research Promotion Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: International Centre for Genetic Engineering and Biotechnology, Area de Ricerca, 34012 Trieste, Italy.

¶

To whom correspondence should be addressed. Tel.: 1-797-30; Fax: 1-798-7153.

()

The abbreviations used are: p90, rsk-encoded ribosomal S6 kinase; p70/p85, M = 70,000 and 85,000 S6 kinases; MAP, mitogen-activated protein; p42/p44, M = 42,000 and 44,000 MAP kinases; CREM, cAMP-response element modulator; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; EGF, epidermal growth factor; HA, hemagglutinin; PBS, phosphate-buffered saline; DAPI, 4`,6`-diamidino-2-phenylindole.

()

C. Kühne, unpublished data.

ACKNOWLEDGEMENTS

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