Ribosomal protein S6 kinase: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Ribosomal protein S6 kinase

Synonyms -

Cytological map position - 64F1--2

Function - signaling

Keywords - growth, insulin signaling pathway, TOR pathway

Symbol - S6k

FlyBase ID: FBgn0283472

Genetic map position -

Classification - ribosomal protein S6 kinase

Cellular location - cytoplasmic and possibly nuclear



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Mitchell, N. C., Tchoubrieva, E. B., Chahal, A., Woods, S., Lee, A., Lin, J. I., Parsons, L., Jastrzebski, K., Poortinga, G., Hannan, K. M., Pearson, R. B., Hannan, R. D. and Quinn, L. M. (2015). S6 kinase is essential for MYC-dependent rDNA transcription in Drosophila. Cell Signal 27: 2045-2053. PubMed ID: 26215099
Summary:
Increased rates of ribosome biogenesis and biomass accumulation are fundamental properties of rapidly growing and dividing malignant cells. The MYC oncoprotein drives growth predominantly via its ability to upregulate the ribosome biogenesis program, in particular stimulating the activity of the RNA Polymerase I (Pol I) machinery to increase ribosomal RNA (rRNA) transcription. Although MYC function is known to be highly dependent on the cellular signalling context, the pathways interacting with MYC to regulate transcription of ribosomal genes (rDNA) in vivo in response to growth factor status, nutrient availability and cellular stress are only beginning to be understood. To determine factors critical to MYC-dependent stimulation of rDNA transcription in vivo, a transient expression screen for known oncogenic signalling pathways was performed in Drosophila. Strikingly, from the broad range of pathways tested, ribosomal protein S6 Kinase (S6K) activity, downstream of the TOR pathway, was the only factor rate-limiting for the rapid induction of rDNA transcription due to transiently increased MYC. Further, one of the mechanism(s) by which MYC and S6K cooperate was shown to be through coordinate activation of the essential Pol I transcription initiation factor TIF-1A (RRN 3). As Pol I targeted therapy is now in phase 1 clinical trials in patients with haematological malignancies, including those driven by MYC, these data suggest that therapies dually targeting Pol I transcription and S6K activity may be effective in treating MYC-driven tumours.

Beck, K., Ehmann, N., Andlauer, T. F., Ljaschenko, D., Strecker, K., Fischer, M., Kittel, R. J. and Raabe, T. (2015). Loss of the Coffin-Lowry syndrome associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons. Dis Model Mech 8(11):1389-400. PubMed ID: 26398944
Summary:
Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling (see Drosophila Rolled) has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry Syndrome, which is characterized by severe mental retardation and low IQ scores in male patients. Knockout of RSK2 in mice or the RSK ortholog in Drosophila result in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, this study shows that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses it is concluded that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, a novel function of RSK in anterograde axonal transport was uncovered. These results emphasize the importance of fine tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling.

Acevedo, S. F., Peru, Y. C. d. P. R. L., Gonzalez, D. A., Rodan, A. R. and Rothenfluh, A. (2015). S6 kinase reflects and regulates ethanol-induced sedation. J Neurosci 35: 15396-15402. PubMed ID: 26586826
Summary:
Individuals at risk for Alcohol use disorders (AUDs) are sensitive to alcohol's rewarding effects and/or resistant to its aversive and sedating effects. The molecular basis for these traits is poorly understood. This study shows that p70 S6 kinase (S6k), acting downstream of the insulin receptor (InR) and the small GTPase Arf6, is a key mediator of ethanol-induced sedation in Drosophila. S6k signaling in the adult nervous system determines flies' sensitivity to sedation. Furthermore, S6k activity, measured via levels of phosphorylation (P-S6k), is a molecular marker for sedation and overall neuronal activity: P-S6k levels are decreased when neurons are silenced, as well as after acute ethanol sedation. Conversely, P-S6k levels rebound upon recovery from sedation and are increased when neuronal activity is enhanced. Reducing neural activity increases sensitivity to ethanol-induced sedation, whereas neuronal activation decreases ethanol sensitivity. These data suggest that ethanol has acute silencing effects on adult neuronal activity, which suppresses InR/Arf6/S6k signaling and results in behavioral sedation. In addition, activity of InR/Arf6/S6k signaling was shown to determine flies' behavioral sensitivity to ethanol-induced sedation, highlighting this pathway in acute responses to ethanol.
Kakanj, P., Moussian, B., Grönke, S., Bustos, V., Eming, S.A., Partridge, L. and Leptin, M. (2016). Insulin and TOR signal in parallel through FOXO and S6K to promote epithelial wound healing. Nat Commun 7: 12972. PubMed ID: 27713427
Summary:
The TOR and Insulin/IGF signalling (IIS) network controls growth, metabolism and ageing. Although reducing TOR or insulin signalling can be beneficial for ageing, it can be detrimental for wound healing, but the reasons for this difference are unknown. This study shows that IIS is activated in the cells surrounding an epidermal wound in Drosophila melanogaster larvae, resulting in PI3K activation and redistribution of the transcription factor FOXO. Insulin and TOR signalling are independently necessary for normal wound healing, with FOXO and S6K as their respective effectors. IIS is specifically required in cells surrounding the wound, and the effect is independent of glycogen metabolism. Insulin signalling is needed for the efficient assembly of an actomyosin cable around the wound, and constitutively active myosin II regulatory light chain suppresses the effects of reduced IIS. These findings may have implications for the role of insulin signalling and FOXO activation in di
BIOLOGICAL OVERVIEW

The phosphorylation of ribosomal protein S6 (RPS6 or simply S6) is a rapid and highly conserved cellular growth response that is observed during development and/or in response to a variety of extracellular stimuli. This phosphorylation is correlated with regulation of mRNA translation which, in turn, may influence cell size, cell proliferation or differentiation. The kinase responsible for the phosphorylation of RPS6 in mammalian cells is the serine/threonine kinase p70S6k (S6k).

Increased RPS6 phosphorylation has been found to be paralleled by the selective translational up-regulation of a class of mRNAs containing an oligopyrimidine tract at their transcriptional start site, termed the 5'TOP. The genes representing this family of mRNAs are small in number, containing no more than 200 members, but can account for up to 20% of total cellular mRNA. 5'TOP mRNAs encode for a number of components of the translational apparatus, including ribosomal proteins and translation elongation factors. Recent studies have shown that the up-regulation of these mRNAs is largely suppressed by the antibiotic rapamycin, a bacterial macrolide. In parallel, rapamycin also abolishes S6K activation, and subsequent mitogen-induced S6 phosphorylation. More importantly, the suppressive effects of rapamycin on 5'TOP translation can be rescued by coexpression of recombinant S6K1 rapamycin-resistant mutants, providing a causal link between S6K1 activation and translational up-regulation of 5'TOP mRNAs. The effects of S6K activation on the translation of 5'TOP mRNAs are thought to be mediated through the increased phosphorylation of S6. S6 itself has been mapped by protein-protein and RNA-protein cross-linking studies to the mRNA-tRNA binding site of the 40S ribosome, suggesting that it might have a role in the regulation of mRNA translation. Following mitogenic stimulation, up to 5 mol of phosphate is incorporated in an ordered fashion into mammalian S6. In mammals, the phosphorylation sites have been mapped to a short sequence at the carboxy terminus of the molecule, with phosphorylation progressing in an ordered fashion from Ser236, Ser235, Ser240, Ser244 and Ser247 (Radimerski, 2000 and references therein).

Like RPS6 phosphorylation, activation of S6k, the protein that targets RPS6, is a highly conserved mitogenic response. Activation of S6k is regulated by phosphatidylinositol 3-kinase (PI3K: see Drosophila Phosphotidylinositol 3 kinase 92E). The PI3K inhibitor wortmannin abrogates the mitogen-stimulated activation of S6k. A separate pathway contributing to S6k activation involves the FKBP12-rapamycin (RAP)-associated protein (FRAP, also known as mTOR, RAFT, and RAPT) and has been demonstrated using the immunosuppressant RAP. RAP, through complex formation with FKBP12 and FRAP/mTor causes G1-phase arrest in T lymphocytes and other hematopoietic tissues. Many of the effects of RAP correlate with the inactivation of S6k and inhibition of protein synthetic activities. FRAP/mTor activity is regulated directly by Protein phosphatase 2A (Drosophila homolog: Twins) which also interacts directly with the S6k. S6k is activated by PP2A inhibition of FRAP/mTor (Peterson, 1999).

Inhibition of p70S6k activation results in the reduction of RPS6 phosphorylation that apparently leads to the selective inhibition of protein synthesis and a delay or arrest at the G1/S phase of the cell cycle in certain cell types. The requirement for p70S6k activity during G1 phase was shown in experiments using neutralizing p70S6k antibodies that prevented both the activation of protein synthesis and the serum-induced entry of cells into S phase. S6k (RPS6-p70-protein kinase) has been identified in Drosophila (Stewart, 1996 and Watson, 1996), and has been found to regulate cell size in a cell-autonomous manner without impinging on cell number (Montagne, 1999). One of the activating kinases of mammalian p70S6k has been identified: mammalian target of rapamycin (mTOR). The Drosophila mTor homolog, dTor, similarly acts to activate Drosophila S6k (Zhang, 2000).

Drosophila deficient in the S6k gene exhibit an extreme delay in development and a severe reduction in body size. These flies have smaller cells rather than fewer cells. The effect is cell-autonomous, displayed throughout larval development, and distinct from that of ribosomal protein mutants (Minute mutants). A female sterile mutant, fs(3)07084, was found to contain a P-element insertion in the 5' noncoding region of the Drosophila S6k gene. Only 25% of the expected number of homozygous flies emerge as adults, with a 3-day delay and reduced body size. This phenotype is rescued either by excision of the P element or by a Drosophila S6K or mammalian S6K transgene. Northern (RNA) blot analysis and sequencing of a reverse transcriptase polymerase chain reaction product (RT-PCR) from homozygous mutant flies reveals the presence of anomalous transcripts, suggesting that S6k expression may persist in homozygous mutant flies. More severe alleles were generated by imprecise P-element excisions, removing part of the S6k gene. Most of these flies die as larvae, with the lethality rescued by expression of Drosophila S6k or mammalian S6K transgenes. One of the excisions, dS6Kl-1, removes part of the first exon, including a portion of the catalytic domain. The few surviving dS6Kl-1 homozygous flies emerge after a 5-day delay, live no longer than 2 weeks, and display a severe reduction in body size, with all body parts apparently affected to the same extent. Thus, loss of S6k function induces female sterility, a strong developmental delay, a severe reduction in growth, and often death (Montagne, 1999).

Since mammalian S6Ks control the synthesis of ribosomal proteins, it was hypothesized that the dS6Kl-1 phenotype might be equivalent to that of Minutes. The Minute M(3)95A, harbors a P-element insertion that severely reduces the expression of ribosomal protein S3. However, analysis of M(3)95A and two other Minutes shows no effect on size, although all display a developmental delay and slender bristles. In contrast, the bristles of homozygous dS6Kl-1 flies are proportional to body size. To determine whether the reduction in body size of homozygous dS6Kl-1 flies is due to a decrease in cell number, cells in wings and ommatidia in eyes of wild-type and dS6K mutant flies were compared. The cell density is greater in wings of homozygous dS6Kl-1 flies (as represented by each hair) than in wild-type flies. The difference in cell size is almost 30%, and flies homozygous for partial loss of function dS6K07084 display an intermediate cell size. However, the total number of cells in wings remain constant. Analysis of eyes reveals a similar phenotype with reduced size but no effect on the number of ommatidia. Thus, in S6k mutants the decrease in the rate of proliferation is probably attributable to a reduction in ribosomal protein synthesis, whereas the effect on cell size may be due to the absence of S6 phosphorylation and an altered pattern of translation (Montagne, 1999).

The reduction in cell size of dS6Kl-1 flies indicates either that cells are proliferating at a smaller size or that flies emerge from the extensive developmental delay before completion of the last round of cell growth. To examine these possibilities, proliferating epithelial cells from the imaginal wing disc of larvae were analyzed at the end of the third instar. Imaginal discs give rise to the adult structures. At the end of the third instar, wing disc cells still require two mitotic cell cycles before they differentiate. Comparison of wing discs from homozygous dS6Kl-1 and wild-type larvae reveals that mutant discs are substantially smaller in size. Analysis of single cells from discs with a fluorescence-activated cell sorter (FACS) confirms that, on average, cells derived from S6k mutants are smaller than wild-type cells. There was no apparent difference between the distributions of S6k mutant and wild-type cells within each phase of the cell cycle, implying that the dS6Kl-1 loss-of-function mutation affects all stages of the cell cycle. Analysis of disc cells during puparium formation, when proportionally more cells are present in G2 phase, also shows no detectable difference in the cell cycle distribution of mutant and wild-type cells. In addition, the number of wing disc cells present in somatically induced clones, marked by ectopic expression of beta-galactosidase, is reduced in mutant versus wild-type larvae. Consistent with this, cell cycle times are 12.5 ±1 hours and 24 ± 4 hours for wild-type and mutant wing disc cells, respectively. Thus, loss of Drosophila S6k function leads to cell proliferation at a smaller size and at a reduced rate, without affecting any specific stage of the cell cycle (Montagne, 1999).

S6k mutants may affect cell size through the loss of a humoral factor that regulates cell growth. To examine this possibility, genetically marked homozygous mutant cells were generated in a heterozygous mutant background by somatic recombination. At the wing margin, homozygous S6k mutant sensory bristles, identified by a yellow (y minus) marker, are reduced in size compared with their neighbors. In eyes, homozygous S6k mutant photoreceptor and pigment cells are marked by a white (w minus) mutation and recognized by the absence of red pigment, appearing as dark spots in photoreceptor cells. Again, only mutant cells are reduced in size, indicating that S6k acts in a cell-autonomous manner. Because dS6K mutations affect size in a cell-autonomous manner, expression of an extra copy of the wild-type gene in a specific compartment might positively affect growth. A compartment represents an independent unit of growth and size control, thought to be analogous to a mammalian organ. The wing disc is composed of a dorsal compartment and a ventral compartment that fold in an apposed manner at the wing margin to generate the flattened wing blade. Because the apterous promoter is only functional in the dorsal compartment of the wing disc, it was coupled to the GAL4 transcription factor to induce an extra copy of the S6k gene linked to a UAS responsive element. An increase in cell size of less than 1% should alter the morphology of the adult wing blade. In all UAS S6k lines examined, S6k protein expression was increased and the wing was convex and bent downward. The phenotype can be explained by an increase in the size of the dorsal versus the ventral wing surface, forcing the wing blade to curve down to accommodate the greater surface. Therefore, increased expression of S6k positively affects growth in a cell-autonomous and compartment-dependent manner (Montagne, 1999).

S6Ks appear to be downstream effectors of the phosphatidylinositide-3OH kinase (PI3K) signaling pathway. However, activated or dominant interfering alleles of PI3K affect cell number and cell size. This would imply that S6Ks reside on a branch of the PI3K signaling pathway that controls cell growth and size but not cell number. Overexpression of the cell cycle regulator E2F in the posterior compartment of the wing disc increases cell number without affecting final compartment size. These findings are consistent with the hypothesis that compartments, like organs, adjust their final mass independent of cell number. However, the Drosophila S6k phenotypes suggest that cell size participates in the control of compartment size. Indeed, S6Ks are thought to play a critical role in organ hypertrophy, where the organ increases in size as a function of demand (Montagne, 1999).

It is concluded that flies deficient in S6k exhibit an extreme delay in development and a severe reduction in body size. These flies have smaller cells rather than fewer cells. The effect is cell-autonomous, displayed throughout larval development, and distinct from that of ribosomal protein mutants. Thus, the S6k gene product regulates cell size in a cell-autonomous manner without impinging on cell number (Montagne, 1999).


GENE STRUCTURE

Three S6k cDNA classes were isolated from embryonic and third-instar larval libraries. Two classes of cDNA clones contain sequences predicting identical open reading frames (ORFs) encoding a polypeptide of 637 residues with a calculated molecular mass of 76 kDa. The first cDNA class (class 1) had a 530- nt 5' untranslated region (UTR) whereas the second class (class 2) had an overlapping but shorter 5' UTR of approximately 90 nt. The third cDNA class has divergent 5'-end coding sequences but contains an identical ORF starting from residue 52 through to the C terminus, suggesting a second isoform. The significance of the two putative isoforms is unknown. Two forms, p85 and p70, are found in mammalian cells, with the former containing an N-terminal extension with a nuclear localization signal. The mammalian p85S6k isoform is primarily nuclear whereas the p70S6k isoform is both nuclear and cytoplasmic (Watson, 1996).


PROTEIN STRUCTURE

Amino Acids - 637

Structural Domains

The conceptually translated S6K has all of the characteristic motifs of a serine/threonine protein kinase including the 12 canonical subdomains of the catalytic domain. The catalytic domain shows the highest degree of amino acid identity (~78%) with mammalian p70S6k. The N-terminal domain has 23% identity and 80% overall similarity with the human protein sequence suggesting that its general structure is important in the regulation or function of these enzymes. The C-terminal region contains sequences known as the SKAIPS (S6 kinase autoinhibitory pseudosubstrate) domain, which may regulate activity through interactions with the catalytic domain and the acidic N-terminal domain. The Drosophila S6K SKAIPS region has 26% sequence identity with Drosophila RPS6 (Watson, 1992), its proposed substrate. This is similar to the sequence relationship between mammalian p70S6k SKAIPS and the region of RPS6 that contains all of the known phosphorylation sites (Watson, 1996).

Mitogen stimulation results in the phosphorylation and activation of mammalian S6k. In cultured cells, S6k shows a basal level of phosphorylation but additional phosphorylation is observed following mitogenic stimulation. Phosphorylation at multiple sites, including T229 and T389, appears to be critical for p70S6k activation, as demonstrated through the use of kinase mutants and two inhibitors (wortmannin and RAP), which act by eliminating the phosphorylation of these residues. The high degree of sequence similarity between S6K and p70S6k, including the conservation of residues T229 and T389, suggests that they may have conserved biochemical functions and upstream regulators (Watson, 1996 and references therein).

Analysis of the predicted Drosophila S6k amino acid sequence shows 57% overall identity to the mammalian s6k sequence with the highest homology in the catalytic domain where the identity is 78% and the similarity is 86%. The acidic N-terminal domain of mammalian S6k confers rapamycin sensitivity on the kinase. In Drosophila S6k this domain is also highly acidic, displaying 55% similarity with its mammalian counterpart, suggesting it may play an equivalent role in Drosophila S6k. Immediately adjacent to the catalytic domain, Drosophila S6k contains a 68-amino acid long sequence, which has a striking 72% identity with its mammalian homolog. In mammalian S6k, this domain couples the catalytic and autoinhibitory domains and is termed the linker region. It has recently been noted that this region is conserved in many members of the second messenger subfamily of SeryThr kinases and may play a critical role in regulating kinase activity. This linker region is immediately followed by the putative autoinhibitory domain that contains four SeryThr-Pro sites whose phosphorylation is thought to relieve the inhibitory impact of this domain as well as contribute to full kinase activation. Consistent with this model it has been shown that peptides covering this domain, residues 400-432, inhibit kinase activity in the low mM range. Strikingly, in Drosophila S6k the sequence R417SPRRTPR425 immediately following the linker region is very similar in context to a piece of the mammalian autoinhibitory domain, R410SPRRFIGSPR419. Although this peptide only represents a fragment of the autoinhibitory domain found in mammalian 6k, in recent studies it has been defined as the sequence within the larger peptide that is responsible for exerting the inhibitory effect on S6k. Thus in Drosophila S6k this sequence may be involved in autoregulation of kinase activity in a manner similar to that predicted in mammalian p70 s6k. The final stretch of amino acids, residues 425-637, display no significant homology with the mammalian S6k nor with known proteins from data base searches, indicating that this C-terminal domain is unique to Drosophila S6k. However, this domain contains a highly basic stretch of lysine residues beginning at amino acid 525, which is separated by 35 amino acids from a long stretch of acidic amino acids nearer the C terminus. Given that there is no detectable nuclear targeting sequence in the Drosophila S6k equivalent to that found in the mammalian p85 S6k isoform, it would be of interest to test whether these sequences perform a similar function in Drosophila S6k (Stewart, 1996).


Ribosomal protein S6 kinase: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 November 2000

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