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...

Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins’ intrinsic reactivity, yet paradoxically eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase, RSK. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.

The active sites of 491 human protein kinase domains are highly conserved, which makes the design of selective inhibitors a formidable challenge. We used a structural bioinformatics approach to identify two selectivity filters, a threonine and a cysteine, at defined positions in the active site of p90 ribosomal protein S6 kinase (RSK). A fluoromethylketone inhibitor, designed to exploit both selectivity filters, potently and selectively inactivated RSK1 and RSK2 in mammalian cells. Kinases with only one selectivity filter were resistant to the inhibitor, yet they became sensitized after genetic introduction of the second selectivity filter. Thus, two amino acids that distinguish RSK from other protein kinases are sufficient to confer inhibitor sensitivity.Phosphorylation of serine, threonine, and tyrosine residues is a primary mechanism for regulating protein function in eukaryotic cells. Protein kinases, the enzymes that catalyze these reactions, regulate essentially all cellular processes and have thus emerged as therapeutic targets for many human diseases (1). Small-molecule inhibitors of the Abelson tyrosine kinase (Abl) and the epidermal growth factor receptor (EGFR) have been developed into clinically useful anticancer drugs (2, 3). Selective inhibitors can also increase our understanding of the cellular and organismal roles of protein kinases. However, nearly all kinase inhibitors target the adenosine triphosphate (ATP) binding site, which is well conserved even among distantly related kinase domains. For this reason, rational design of inhibitors that selectively target even a subset of the 491 related human kinase domains continues to be a daunting challenge.Structural and mutagenesis studies have revealed key determinants of kinase inhibitor selectivity, including a widely exploited selectivity filter in the ATP binding site known as the "gatekeeper." A compact gatekeeper (such as threonine) allows bulky aromatic substituents, such as those found in the Src family kinase inhibitors, PP1 and PP2, to enter a deep hydrophobic pocket (4-6). In contrast, larger gatekeepers (methionine, leucine, isoleucine, or phenylalanine) restrict access to this pocket. A small gatekeeper provides only partial discrimination between kinase active sites, however, as ∼20% of human kinases have a threonine at this position. Gleevec, a drug used to treat chronic myelogenous leukemia, exploits a threonine gatekeeper in the Abl kinase domain, yet it also potently inhibits the distantly related tyrosine kinase, c-KIT, as well as the platelet-derived growth factor receptor (PDGFR) (7).We therefore sought a second selectivity filter that could be discerned from a primary sequence alignment. Among the 20 amino acids, cysteine has unique chemical reactivity and is commonly targeted by electrophilic inhibitors. In the case of cysteine protease inhibitors, the reactive cysteine is not a selectivity filter, because it is found in every cysteine protease * To whom correspondence should be addressed. taunton@cmp.ucsf.edu. and is ...

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner.Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.