RAS/ERK Signaling Promotes Site-specific Ribosomal Protein S6 Phosphorylation via RSK and Stimulates Cap-dependent Translation

Abstract

Converging signals from the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) pathways are well established to modulate translation initiation. Less is known regarding the molecular basis of protein synthesis regulated by other inputs, such as agonists of the Ras/extracellular signal-regulated kinase (ERK) signaling cascade. Ribosomal protein (rp) S6 is a component of the 40S ribosomal subunit that becomes phosphorylated at several serine residues upon mitogen stimulation, but the exact molecular mechanisms regulating its phosphorylation and the function of phosphorylated rpS6 is poorly understood. Here, we provide evidence that activation of the p90 ribosomal S6 kinases (RSKs) by serum, growth factors, tumor promoting phorbol esters, and oncogenic Ras is required for rpS6 phosphorylation downstream of the Ras/ERK signaling cascade. We demonstrate that while ribosomal S6 kinase 1 (S6K1) phosphorylates rpS6 at all sites, RSK exclusively phosphorylates rpS6 at Ser 235/236 in vitro and in vivo using an mTORindependent mechanism. Mutation of rpS6 at Ser 235/236 reveals that phosphorylation of these sites promotes its recruitment to the 7-methylguanosine cap complex, suggesting that Ras/ERK signaling regulates assembly of the translation preinitiation complex. These data demonstrate that RSK provides an mTORindependent pathway linking the Ras/ERK signaling cascade to the translational machinery.In eukaryotic cells, the main rate-limiting step of translation is initiation, which is controlled by an array of proteins that respond to signaling cascades activated by extracellular signals (reviewed in Refs. 1-3). The mammalian target of rapamycin, mTOR, 4 is a conserved serine/threonine kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate mammalian cell growth (reviewed in Refs. 4, 5-8).Under conditions of nutrient and energy sufficiency and insulin or mitogen stimulation, mTOR stimulates two important translational regulators, the ribosomal S6 kinases (S6K1 and S6K2) and the eukaryotic initiation factor 4E (eIF4E). eIF4E is crucial for ribosome recruitment as it binds to the 7-methylguanosine cap structure (m7GpppN, where N is any nucleotide) at the 5Ј-end of nearly all transcribed mRNAs to initiate cap-dependent translation (reviewed in Ref. 7). When mTOR is active, eIF4E nucleates the assembly of the translation preinitiation complex through recruitment of numerous initiation factors, resulting in association of the ribosomal subunits to the mRNA. S6K1 and S6K2 are serine/threonine kinases directly stimulated by mTOR which in turn, phosphorylate substrates involved in cell and body size (5, 6). S6K1 phosphorylates several substrates located in the cytoplasm and the nucleus, including the ribosomal protein (rp) S6 (reviewed in Ref. 9).Ribosomal protein S6 is one of 33 proteins that comprise the 40 S ribosomal subunit and represents the most extensively studied substrate of S6K1 (10). Because the initial discovery that liver-derived rpS6 was phosphoryla...

The transcriptional induction of PIK3CA in tumor cells is dependent on the oncoprotein Y-box binding protein-1

“…We suspect this is because RSK remains activated. In support of this idea, RSK has been reported to phosphorylate S6RP on its S235/236 residue (Roux et al, 2007). We showed that P-RSK levels decreased with the loss of p110a following YB-1 or PIK3CA knockdown (Figures 5b and c), demonstrating that the phosphorylation of this protein is also PI3K dependent.…”

“…Therefore, we queried if loss of p110a affected P-S6RP levels in an AKTindependent manner. Alternatively, RSK is also known to phosphorylate S6RP on its Ser235/236 residue (Roux et al, 2007). Indeed, similar to P-S6RP, P-RSK (S380) levels decreased after reduction in p110a subsequent to silencing YB-1 or PIK3CA.…”

mTOR signaling response to resistance exercise is altered by chronic resistance training and detraining in skeletal muscle

“…The ERK1/2 signaling pathway has the ability to regulate proteins involved in the initiation and elongation stages of mRNA translation in an mTOR-dependent and -independent manner (28,35,43,44). Recent studies indicated that ERK signaling is involved in later phase anabolic responses after resistance exercise (5,6,15).…”

Identification of cAMP-Dependent Kinase as a Third in Vivo Ribosomal Protein S6 Kinase in Pancreatic β-Cells

“…However, the translation of 5'TOP mRNAs is unaffected in hepatocytes from both S6K1 -/-/2 -/-knock-out mice 9 and rpS6 P-/-mice 10 . A recent study provided evidence that site specific phosphorylation of rpS6 on Ser(235/236) via Rsk promotes cap-dependent translation by facilitating the formation of pre-initiation translation complexes 11 .…”