S6K1^−/−/S6K2^−/− Mice Exhibit Perinatal Lethality and Rapamycin-Sensitive 5′-Terminal Oligopyrimidine mRNA Translation and Reveal a Mitogen-Activated Protein Kinase-Dependent S6 Kinase Pathway

Abstract

Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Here we show that mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1 ؊/؊ mice are significantly smaller, whereas S6K2 ؊/؊ mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1 ؊/؊ /S6K2 ؊/؊ mice, cell cycle progression and the translation of 5-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1 ؊/؊ /S6K2 ؊/؊ cells, S6 phosphorylation persisted at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1 ؊/؊ , and S6K2 ؊/؊ cells, this step was catalyzed by a mitogenactivated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens.Recent studies showed that the 40S ribosomal protein S6 kinase (S6K) p70 S6K /p85 S6K , termed S6K1 (51), is a major effector of cell growth. This conclusion stems from gene deletion studies with Drosophila (39) and with mice (51) as well as recent studies with cell cultures (11). The loss of the Drosophila S6K (dS6K) gene is semilethal, with the few surviving adults having a severely reduced body size. The larvae of such flies exhibit a long developmental delay, consistent with a twofold increase in cell cycle doubling times. The few surviving adults are quite lethargic, living no longer than 2 weeks, and females are sterile. Surprisingly, the reduction in mass is strictly due to a decrease in cell size rather than to a decrease in cell number (39).In mice, removal of this kinase is not lethal, but the mice are approximately 20% smaller at birth (51). Such mice exhibit normal fasting glucose levels but are mildly glucose intolerant due to markedly reduced levels of circulating insulin (42).Reduced insulin levels are caused by a reduction in pancreatic endocrine mass and an impairment of insulin secretion, which can be traced to a selective reduction in ␤-cell size. Unexpectedly, the effects on body mass and hypoinsulinemia do not appear...

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

“…Although it is tempting to implicate mTORC1 activity in the response to this stress, as mTORC1 has been shown to act as a sensor for various cellular insults, we did not see strong effects on direct mTORC1 targets such as S6K T389 or 4E-BP1 phosphorylation. Nor is it clear whether S6K is responsible for the effects seen on S6 S235/S236 phosphorylation, as measurement of more specific sites of S6K phosphorylation, namely S6 S240/S244 [34] showed that these sites were not affected by 3,4-DMB-PP1 or 1-NM-PP1 in PDK1 −/− +WT ES cells. A third possibility is that the bulky analogues inhibit WT PDK1 to a small extent, and that S6 phosphorylation is a very sensitive readout for this minor inhibition.…”

“…However, none of the available phospho-specific antibodies worked reliably enough to obtain interpretable results. We therefore assessed S6K activity indirectly by analyzing its phosphorylation at T389 as well as phosphorylation of S6 at S6K specific sites, namely S240/S244 [34]. We also further analyzed mTORC1 activity by assessing phosphorylation of 4E-BP1 at the mTORC1 sites S37/S46 and S65 [35].…”

Stress and mTORture signaling

“…Specific transcripts with polypyrimidine tracts at their 5 0 end named 5 0 terminal oligopyrimidine tract (TOP) mRNAs largely encode for RPs and elongation factors (Meyuhas, 2000;Thomas, 2002). The original assumption that S6Ks stimulate protein synthesis via S6 phosphorylation by upregulating the translation of 5 0 TOP mRNAs has lost attraction, because in cells lacking both S6K1 and S6K2, translation of these types of mRNAs was not attenuated (Pende et al, 2004). Replacement of the endogenous S6 protein with an S6 mutant form where all five S6K phosphorylation sites were mutated to unphosphorylatable residues (rpS6 PÀ/À ) showed that phosphorylation of S6 positively regulates cell size but not translation.…”

Phosphorylated S6 as an immunohistochemical biomarker of vulvar intraepithelial neoplasia

“…33 Phosphorylation at S235 and S236 in normal keratinocytes and squamous cell carcinoma cells is accomplished by the kinase p90RSK, which is activated by the EGFR/ERK signal pathway, whereas the S240 and S244 phosphorylations appear to be accomplished exclusively by S6K1, a kinase activated by the PI3K/AKT/mTOR signaling pathway. 29,[34][35][36] An antibody specific for p-S6(S235/ 236) works well as a immunohistochemical staining reagent, whereas antibodies specific for the phosphorylated, activated states of other proteins in the EGFR/ERK and PI3K/mTOR signal pathways, which work well for western blotting, do not identify their antigens in neoplastic basal epithelial cells in formalin-fixed, paraffin-embedded tissue. 29 Therefore, p-S6(S235/236) immunostaining provides the best read-out of EGFR/ERK signal pathway hyperactivity in pathology tissue specimens.…”