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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.35 no.2 Santiago  2002

http://dx.doi.org/10.4067/S0716-97602002000200022 

Biol Res 35: 305-313, 2002

The S6 kinase Signaling Pathway In The Control of
Development and Growth

GEORGE THOMAS

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

ABSTRACT

The discussion will focus on the role of the ribosomal protein S6 kinase (S6K) signaling pathway in the regulation of cell growth and proliferation. Although 40S ribosomal protein S6 phosphorylation was first described 25 years ago (Gressner and Wool, 1974), it only recently has been implicated in the translational up-regulation of mRNAs coding for the components of protein synthetic apparatus (Fumagalli and Thomas, 2000). These mRNAs contain an oligopyrimidine tract at their 5' transcriptional start site, termed a 5'TOP, which has been shown to be essential for their regulation at the translational level (Meyuhas et al., 1996). In parallel, a great deal of information has accumulated concerning the identification of the signaling pathway and the regulatory phosphorylation sites involved in controlling S6K activation (Dufner and Thomas, 1999). Despite this knowledge we are only beginning to identify the direct upstream elements involved in growth factor-induced kinase activation (Dennis et al., 2001; Pullen et al., 1998). Use of the immunosuppressant rapamycin, a bacterial macrolide, in conjunction with dominant interfering and activated forms of S6K1 has helped to establish the role of this signaling cascade in the regulation of growth and proliferation (Dennis and Thomas, 2002). In addition, current studies employing the mouse as well as Drosophila melanogaster have provided new insights into physiological function of S6K in the animal (Montagne et al., 1999; Pende et al., 2000). Loss of dS6K function in Drosophila melanogaster demonstrated its paramount importance in development and growth control (Montagne et al., 1999), whereas deletion of the S6K1 gene in the mouse led to an animal of reduced size and the identification of the S6K1 homologue, S6K2 (Shima et al., 1998). Such mice are significantly smaller during fetal development (Shima et al., 1998) and hypoinsulinemic in the adult, conditions known to lead to type 2 diabetes (Pende et al., 2000).

Key terms: Drosophila / ßcells / Diabetes

INTRODUCTION

Our group has had a long-term interest in the underlying mechanisms which control cell growth, how these mechanisms are linked to those that control cell cycle progression and their involvement in development and human disease, especially diabetes and cancer. Our interest in cell growth rapidly led us to protein synthesis, the most energy consuming anabolic process in a growing cell (Schmidt, 1999). However, what largely goes unrecognised is that the major product being generated by the protein synthetic apparatus is more translational machinery, especially ribosomes. Indeed an exponentially growing epithelial carcinoma produces 7500 ribosomes per minute and if one considers there are approximately eighty distinct ribosomal proteins which make-up the 40S and 60S subunits of the 80S ribosome, each present in one copy per 80S ribosome, this means the translational machinery is producing 600,000 ribosomal proteins a minute (Lewis and Tollervey, 2000). In addition, there is a large number of enzymes involved in the biogenesis process, including rRNA helicases, pseudouridylating enzymes, sno rRNAs and exo- and endo-nucleases (Lewis and Tollervey, 2000). In the clinics, the increase in nucleolar size, the site of ribosome biogenesis, is used as prognostic indicator of tumor progression, with increased size and number being a negative indication of survival (Derenzini et al., 2000).

Our entrée into this field has been the S6 kinase signalling pathway, which in many cell types, is triggered by the recruitment of phosphatidylinositide-3OH kinase (PI3K) to the activated receptor and the production of phosphatidylinositol-3,4,5-trisphosphate (Blume-Jensen and Hunter, 2001). Activation of S6K is brought about by the phosphorylation in a hierarchical manner of key residues, which reside within distinct regulatory domains (Pullen and Thomas, 1997). The kinase has a short amino terminus, containing an acidic domain, which confers rapamycin sensitivity (see below). The catalytic domain, containing the eleven conserved domains found in all Serine and Threonine protein kinases, follows this segment. The catalytic domain is followed by a linker domain, which couples the catalytic domain with an auto-inhibitory domain (Pullen and Thomas, 1997). The linker domain was first noted by our group and found to be conserved in all the kinases of the Protein A, G and C (AGC) family of Serine/Threonine kinases (Moser et al., 1997). Immediately, downstream of the auto-inhibitory domain is the amino terminus, which has been implicated in the interaction of the kinase with other proteins, such as neurabin or spinophilin (see below) (Burnett et al., 1998).

SUMMARY OF LECTURE

Activation of S6K1 requires phosphorylation of four S/T-P sites, which reside in the auto inhibitory domain (Dennis et al., 1998; Han et al., 1995). This event, in combination with phosphorylation of S371 in the linker domain, facilitates T389 phosphorylation, which also resides in the linker domain, and provides a docking site for the phosphoinositide-dependent protein kinase, (PDK1) (Biondi et al., 2001). PDK1 docks on T389 and phosphorylation of S371 in the linker domain, and provides a docking site for the phosphoinositide-dependent protein kinase, (PDK1) (Biondi et al., 2001). PDK1 docks on T389 and phosphorylates T229 in the activation loop of the kinase, leading to kinase activation. The critical event then in activating S6K is phosphorylation of T389 (Dennis et al., 1998). A number of kinases have been suggested as potential T389 kinases, including protein kinase B (Burgering and Coffer, 1995), PDK1(Balendran et al., 1999) and more recently the NIMA related kinases NEK6 and 7 (Belham et al., 2001). However, in our hands we find that the mammalian Target of Rapamycin, mTOR, is a potent T389 kinase (Dennis et al., 2001). mTOR is a member of the PI3K related family of protein kinases, which also includes ATM, ATR and DNA dependent kinases. The latter three are all involved in DNA damage repair (Gingras et al., 2001), whereas mTOR acts as an amino acid and energy effector (Dennis and Thomas, 2002; Gingras et al., 2001). If either amino acids, especially branch-chain amino acids, or ATP levels drop, mTOR activity decreases leading to inhibition of S6K1 (Dennis et al., 2001; Hara et al., 1998). mTOR itself is the inhibitory target of rapamycin, a bacterial macrolide, which was initially discovered in a screen of natural products from Rapa Nui, Easter Island (Dennis and Thomas, 2002). Rapamycin forms with FKBP-1 a gain-of-function-inhibitory-complex, which binds to mTOR, abolishing mTOR activity, and S6K activity. Rapamycin and its analogues are emerging as powerful therapeutic agents in the treatment of allograft rejection, cancer, restenosis and rheumatoid arthritis (Forre and Hassfeld, 2000; Huang, 2001; Marx, 2001; Schwarz , 2002).

Downstream of S6K, recent studies employing alleles of the kinase which are either rapamycin resistant or dominant interfering have provided evidence that the kinase regulates the expression of a family of mRNAs at the translational level (Fumagalli and Thomas, 2000). This family is small, maybe representing one to two hundred gene products. However, they can account for up to 20-30% of the mRNA in the cell and they largely encode for components of the translational apparatus, particularly ribosomal proteins (Meyuhas et al., 1996). These mRNAs are characterized by having an oligopyrimidine tract at their 5' transcriptional start site, termed a 5'TOP, which invariably begins with a cytosine (Meyuhas et al., 1996). This sequence normally acts as a repressor, such that by mutating the first residue to an adenosine, which most mammalian transcripts begin with, these mRNAs relocate to actively translating polysomes and are refractile to mitogenic stimulation and resistant to rapamycin treatment (Meyuhas et al.1996).

The data described above is summarized in the model shown in Figure 1. This is roughly where we stood two years ago, and what we were concerned with was whether S6K was involved in growth in the animal. To test this possibility we decided to delete the S6K gene both in Drosophila melanogaster and in the mouse. We chose to employ Drosophila because we wanted to take advantage of its powerful genetics, conservation of signalling pathways, and its small genome size, containing less redundant genes. By way of introduction, I will first discuss the results obtained in Drosophila melanogaster and then turn to the mouse.

Fig. 1: PI3K signaling pathway, generic model (reprinted from Kozma and Thomas, 2002).

Initially we cloned the cDNA orthologue of S6K from a Drosophila melanogaster cDNA library (Stewart et al., 1996), termed dS6K, and then mapped its chromosomal location. We then turned to FlyBase, a powerful database, to learn if any mutations had been mapped near this locus (Montagne et al., 1999). One such P-element insertional mutant mapped near the dS6K locus (Karpen and Spradling, 1992), and we found that the P-element was inserted 48 base pairs upstream of the predicted AUG translational initiation start site (Montagne et al., 1999). This mutant, termed fs(3)07084, had been partially characterized as semi-lethal and female sterile; we found that in addition they were severely delayed in development (Montagne et al., 1999). To determine whether the P-element was responsible for the observed phenotype we excised the gene using standard Drosophila protocols. This restored S6K expression and rescued all the phenotypes described above. To determine whether the mutation was in fact due to the reduction in S6K expression we crossed these flies with transgenic flies harbouring an extra copy of the Drosophila kinase, dS6K, or the mammalian kinase, S6K. In both cases, the phenotypes were rescued showing that the effects were due to a reduction in dS6K expression (Montagne et al., 1999). Given that P-element insertions lead to hypomorphic phenotypes we reasoned that we might be able to obtain a more severe phenotype by deleting the gene. When one excises the P-element on rare occasions one also removes the gene and these events can be captured genetically. In this way we obtained five lines which were all non-complementary and exhibited late larval lethality. However, after carrying these flies over balancer chromosomes for six months we begun to obtain a few escapers. These adults were severely reduced in size, approximately half the size of wild type flies. They only lived for two weeks, were very lethargic, could not fly and females were sterile. Despite these phenotypic lesions all of the extra skeletal structures of the dS6K deficient flies were reduced to approximately the same extent, including the head, eyes, thorax, wings and legs (Montagne et al., 1999).

Given the seminal observations of Hartwell and Zetterberg that a cell must grow to a certain size before it can progress through the cell cycle (Johnston et al., 1977; Killander and Zetterberg, 1965), we reasoned these flies were smaller because they had less cells. To test this possibility we counted the number of cells in a fixed area of the wing or the eye. In both cases, it was obvious that the cells were smaller and denser. More importantly, when we corrected for the total area of the wing or eye it was apparent that there were the same number of cells (Montagne et al., 1999). Thus, contrary to dogma (Conlon and Raff, 1999), these cells appeared to be passing through the cell cycle at a reduced cell size.

However, the dS6K deficient flies which did emerge as adults emerged after a long delay. Thus the cells could be smaller because, they simply did not complete their last round of cell growth before becoming adults. To test this possibility, we generated clones of B-galactosidase marked cells during larval development by taking advantage of the Flp/frt recombination system under the control of the heat shock promoter (Neufeld et al., 1998). This allowed us to induce recombination, then, after a period of approximately fifty hours, we could count the number of cells which had arisen from single recombination events. By knowing the number of cells as well as length of time following the heat shock one could roughly predict the cell cycle time. In good agreement with the literature, we found that wild type cells had a cell cycle time of approximately twelve and half hours, whereas for dS6K deficient cells, the cell cycle was almost twice as long, twenty-four hours. Thus, not only are the cells progressing though the cell cycle at a smaller size, but also a slower rate.

At this point, it was important to establish that the effect on cell size was a cell autonomous event, as dS6K has been implicated in the expression of a number of growth factors and hormones, including insulin and insulin-like growth factor 2 (see below). To test the possibility, we again took advantage of the Flp/frt system, to generate genetically marked homozygous dS6K deficient cells by somatic recombination in either a hetero or homozygous wild type cell background (Xu and Rubin, 1993). Recombination was induced through the utilization of the heat shock promoter during larval development and the genetically marked homozygous dS6K deficient cells were examined in the adult extra skeletal structures. The results showed in the wing and in the eye that dS6K homozygous deficient cells were significantly reduced in size, whereas surrounding hetero- and homozygous cells were unaffected (Montagne et al., 1999). These findings demonstrate that the effect of the dS6K deficiency on cell size is a cell autonomous effect. Given that removal of dS6K led to reduced cell size it was reasoned that increasing dS6K expression would increase cell size. To test this possibility, transgenic flies were generated where a copy of dS6K was linked to UAS response element. These flies were then crossed with transgenic flies, which harboured the GAL4 transcription factor under the control of distinct promoters, whose expression was confined to a specific compartment. One of the promoters tested was the apterouse promoter (Gorfinkiel et al., 1997) which is only expressed in the dorsal compartment of the imaginal wing disc giving rise to the dorsal epithelial sheet of the wing blade following morphogenesis. Given that the cells on the dorsal, versus the ventral surface, grow larger this should cause the wing to turn down in an umbrella shape. Indeed, this is what is observed (Montagne et al., 1999) (see Figure 2). This phenotype turns out to be a «sensitised» phenotype and has been extremely valuable in a screen looking for upstream and downstream effectors in the S6K signalling pathway (Radimerski et al., 2002). Thus, removal or over expression of dS6K has a pronounced effect on cell growth consistent with models derived from biochemical and tissue culture studies.


Fig. 2: Comparison of GAL4-apterous flies in the absence or presence of a transgene harboring a UAS response element coupled to an extra copy of dS6K (reprinted from Montagne et al., 1999).

In the mouse the story became more complicated than in the fly. The S6K gene was deleted by CRE-loxp technology. Unlike the fly, the mice were viable and fertile, although they were 15 to 20 % smaller at birth (Shima et al., 1998). More striking was the fact that all the biological responses associated with S6K were intact, including S6 phosphorylation, the upregulation of oligopyrimidine tract mRNAs and the sensitivity of both these responses to rapamycin (Shima et al., 1998). These findings suggested the existence of a second S6K. To test this possibility, we analysed mouse embryo fibroblasts from wild type and S6K deficient animals for S6K activity and began to search EST's for the existence of a related kinase. In brief, these two approaches led to the discovery of a second S6K, which we termed S6K2 whereas we renamed S6K, S6K1 (Shima et al., 1998). In parallel, two other groups also identified S6K2, and referred to the kinases as S6Ka (S6K1) and S6Kß (S6K2) (Gout et al., 1998; Saitoh et al., 1998). S6K2 is very homologous to S6K1 except at the carboxy tail, which seems to be involved in the differential targeting of the two proteins. We have set-up strategies for generating S6K2 deficient mice and these studies are under way.

Closer analysis of S6K1 deficient mice revealed that in all the tissues examined S6K2 appeared to be compensatorily upregulated (Shima et al., 1998). Despite this observation, the S6K1 mice were smaller and leaner at birth, and were retarded in development during embryogenesis, suggesting a potential metabolic deficiency (Shima et al., 1998). To test this possibility we measured glucose and insulin levels in fasted animals (Pende et al., 2000). Although glucose levels were normal we found that circulating insulin levels were dramatically reduced. This observation suggested that these animals might be glucose intolerant. To examine this, wild type and S6K1 deficient mice were given a bolus of glucose, and their ability to dispose of the sugar in their peripheral tissues was monitored over time. The results showed that the S6K1 deficient animals were impaired in their ability to clear glucose from the blood (Pende et al., 2000). Consistent with this finding, the ability of S6K1 deficient mice to secrete insulin into the blood following the administration of the bolus of glucose was also impaired (Pende et al., 2000). In parallel, we analysed glucose uptake and glycogen synthesis in isolated soleus and EDL muscle, but could distinguish no differences. Taken together, the results argued that glucose intolerance was not due to insulin resistance in peripheral tissues, but due to hypoinsulinemia.

The findings above would suggest that ß cell function of the islets of Langerhans the site of insulin production was impaired. To examine this possibility, islets were handpicked following collagenase digestion of exocrine pancreas. Insulin secretion was measured by high glucose perifusion, which induced a characteristic first and second phase of insulin release in wild-type islets. Although the kinetics of insulin release were similar in S6K1 deficient islets (Pende et al., 2000), the amount of insulin secreted per cell was significantly reduced (see Figure 3). Similarly, the release of insulin by K+ induced depolarisation of the membrane instead of glucose treatment, also led to a strong reduction in insulin secretion in S6K1 deficient islets versus wild type islets. Analysis of total insulin, both in isolated islets and in total pancreas revealed a 30-40% reduction in S6K1 deficient mice (Pende et al., 2000). Thus, not only insulin secretion is impaired but insulin content is also reduced.


Fig. 3: Insulin release was measured from fifteen islets of S6K1+/+ or S6K1-/- mouse pancreas, during perifusion with a Krebs buffer solution containing 2.8 mM (low) or 16.7 mM (high) glucose for the indicated time (reprinted from Pende et al., 2000).

The finding above suggested that either impairment in insulin production or in endocrine mass was responsible for the reduction in the amount of insulin. In support of the first possibility, others had demonstrated that insulin induction of ß cells leads to the increased synthesis of insulin to replenish the lost pools insulin following a glucose challenge (Leibiger et al., 1998). This step was found to be rapamycin sensitive and protected from the bacterial macrolide when rapamycin resistant alleles of S6K1 were employed (Leibiger et al., 1998). However, we could detect no difference in the synthesis, the processing or the degradation of insulin within the islets (Pende et al., 2000). Therefore, we examined ß cell mass. Preliminary analysis revealed that the ten largest islets in S6K1 deficient mice were twice as small as the ten largest from wild type mice. A careful morphometric analysis of a large number of sections following hematoxyline-eosine staining of pancreatic sections revealed a 30% reduction in endocrine mass, consistent with the reduction observed in insulin content (Pende et al., 2000). To determine whether this effect was selective, we compared by immunohistochemistry the ratio of a secreting glucagon cell mass to ß cell mass. The results showed that the effect on cell mass was selective for ß cells. To determine whether this selective reduction in ß cells mass was due to a reduction in cell number or cell size, the number of nuclei was counted in a fixed area of the islet. The results showed that effect could be accounted for by a reduction in cell size, whereby volume of the cell decreased approximately 24 per cent as compared to wild type cells. This was an exciting finding, as a decrease ß cell size is known to have a pronounced effect on insulin secretion of ß cells (Giordano et al., 1993; Swenne et al., 1988). Thus, the reduction in cell size can account for both the reduction in insulin content and in insulin secretion.

Type two diabetes mellitus, is a polygenic, multifactorial disease and hypoinsulinemia and ß cell dysfunction are usually associated with the late on set of the disease (DeFronzo, 1997). However, these factors are thought to be the primary ones associated with diabetes induced by protein malnutrition during gestation or early childhood (Phillips, 1996). Consistent with this model when the diet protein content of newborn rats is reduced, the rats stop growing. After a short period, if the normal diet is restored they catch-up in size with their control littermates which have been fed a normal diet (Swenne et al., 1992). However, in contrast to other cell types, ß cell size is reduced, as is insulin secretion. These animals also exhibit glucose intolerance, an effect partially attenuated by hypersensitivity to insulin in peripheral tissues (Swenne et al., 1992). An issue which arises from these studies is why ß cells are selectively smaller. ß cells are exquisitely sensitive to both nutrients and growth factors, as is S6K1 (Hugl et al., 1998; Swenne, 1992). Thus, a failure to integrate these two pathways, may lead to the observed selective effects on ß cells. Taken together these findings suggest that mutation in S6K1 or downstream effectors can contribute together with genetic and environmental factors to specific forms of diabetes.

ACKNOWLEDGMENTS

The work presented in this study was supported by the Swiss Cancer League and the Novartis Research Foundation.

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Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66. CH-4058, Basel, Switzerland
Telephone: +41-61-6973012. Fax: +41-61-6973976. e-mail: gthomas@fmi.ch

Received: May 17, 2002. In revised form: June 26, 2002. Accepted: July 15, 2002

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