Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5-m 7 GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.The casein kinase 1 (CK1) 2 and casein kinase 2 (CK2) families represent a group of ubiquitously expressed, constitutively active protein kinases that are involved in many biological processes including DNA repair, cell cycle control, and circadian rhythm entrainment (1, 2). Hundreds of in vitro substrates of CK1 and CK2 have been identified, and this number continues to rise (1-3). CK1 and CK2 preferentially phosphorylate Ser or Thr residues that are flanked by acidic amino acids or phosphorylated residues in the ϩ3 or Ϫ3 position, respectively (1, 2). Thus, in many instances, CK1 and CK2 do not initiate phosphorylation of a particular substrate but instead fulfill a supportive function by phosphorylating adjacent sites. This property of CK1 and CK2 may be particularly important in those cases where a phosphorylation threshold must be surpassed to elicit a biological response.Ribosomal protein S6 is one of 33 proteins that, together with one molecule of 18 S rRNA, comprise the small 40 S ribosomal subunit (4). rpS6 directly interacts with the m 7 GpppG 5Ј-capbinding complex required for translation initiation and represents a point of regulatory convergence for signal transduction pathways controlling translation initiation in response to cell growth and cell proliferation cues. rpS6 undergoes inducible phosphorylation in response to mitogenic and cell growth stimuli, and this phosphorylation is conserved in vertebrates, invertebrates, plants, and fungi (5). In higher eukaryotes, phosphorylation occurs on a cluster of five serine residues at the carboxyl terminus of rpS6: Ser-235, Ser-236, ...

BackgroundTransmissible spongiform encephalopathies (TSEs) affect both domestic sheep (scrapie) and captive and free-ranging cervids (chronic wasting disease; CWD). The geographical range of bighorn sheep (Ovis canadensis; BHS) overlaps with states or provinces that have contained scrapie-positive sheep or goats and areas with present epizootics of CWD in cervids. No TSEs have been documented in BHS, but the susceptibility of this species to TSEs remains unknown.ResultsWe acquired a library of BHS tissues and found no evidence of preexisting TSEs in these animals. The prion protein gene (Prnp) in all BHS in our library was identical to scrapie-susceptible domestic sheep (A136R154Q171 genotype). Using an in vitro prion protein conversion assay, which has been previously used to assess TSE species barriers and, in our study appears to recollect known species barriers in mice, we assessed the potential transmissibility of TSEs to BHS. As expected based upon Prnp genotype, we observed BHS prion protein conversion by classical scrapie agent and evidence for a species barrier between transmissible mink encephalopathy (TME) and BHS. Interestingly, our data suggest that the species barrier of BHS to white-tailed deer or wapiti CWD agents is likely low. We also used protein misfolding cyclic amplification to confirm that CWD, but not TME, can template prion protein misfolding in A136R154Q171 genotype sheep.ConclusionsOur results indicate the in vitro conversion assay used in our study does mimic the species barrier of mice to the TSE agents that we tested. Based on Prnp genotype and results from conversion assays, BHS are likely to be susceptible to infection by classical scrapie. Despite mismatches in amino acids thought to modulate prion protein conversion, our data indicate that A136R154Q171 genotype sheep prion protein is misfolded by CWD agent, suggesting that these animals could be susceptible to CWD. Further investigation of TSE transmissibility to BHS, including animal studies, is warranted. The lack of reported TSEs in BHS may be attributable to other host factors or a lack of TSE surveillance in this species.

Background: cAMP response element-binding protein (CREB) is a transcriptional regulator that undergoes complex phosphoregulation in response many physiologic stimuli. Results: Ser-270/Ser-271 are identified as mitotically regulated phosphorylation sites that diminish CREB DNA binding activity. Conclusion: Carboxyl-terminal phosphorylation of CREB promotes its chromatin eviction during mitosis. Significance: CDK1-mediated chromatin eviction may serve as a global mechanism to mediate transcriptional inhibition observed during mitosis.