Richard B. Pearson

Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k.

Treatment of mammalian cells with the immunosuppressant rapamycin, a bacterial macrolide, selectively suppresses mitogen‐induced translation of an essential class of mRNAs which contain an oligopyrimidine tract at their transcriptional start (5′TOP), most notably mRNAs encoding ribosomal proteins and elongation factors. In parallel, rapamycin blocks mitogen‐induced p70 ribosomal protein S6 kinase (p70s6k) phosphorylation and activation. Utilizing chimeric mRNA constructs containing either a wild‐type or disrupted 5′TOP, we demonstrate that an intact polypyrimidine tract is required for rapamycin to elicit an inhibitory effect on the translation of these transcripts. In turn, a dominant‐interfering p70s6k, which selectively prevents p70s6k activation by blocking phosphorylation of the rapamycin‐sensitive sites, suppresses the translation of the chimeric mRNA containing the wild‐type but not the disrupted 5′TOP. Conversion of the principal rapamycin‐sensitive p70s6k phosphorylation site, T389, to an acidic residue confers rapamycin resistance on the kinase and negates the inhibitory effects of the macrolide on 5′TOP mRNA translation in cells expressing this mutant. The results demonstrate that the rapamycin block of mitogen‐induced 5′TOP mRNA translation is mediated through inhibition of p70s6k activation.

Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations.

Publisher Summary This chapter presents a table of phosphorylation site sequences for protein-serine/threonine and protein-tyrosine kinases. Over 240 phosphorylation site sequences along with the phosphorylated residue(s) are included in this table. The chapter presents another table containing consensus phosphorylation site motifs for each enzyme. The frequencies listed in this table are derived from the first table, unless indicated otherwise. The S : T ratio is for the total number of phosphorylation sites.

Protein kinase recognition sequence motifs

Protein kinases play a crucial role in the regulation of many cellular processes. They alter the functions of their target proteins by phosphorylating specific serine, threonine and tyrosine residues. Identification of phosphorylation site sequences and studies with corresponding model peptides have provided clues to how these important enzymes recognize their substrate proteins. This knowledge has made it possible to identify potential sites of phosphorylation in newly sequenced proteins as well as to construct specific model substrates and inhibitors.

Mutation of the PIK3CA gene in ovarian and breast cancer.

Phosphatidylinositol 3′-kinases are lipid kinases with important roles in neoplasia. Recently, a very high frequency of somatic mutations in PIK3CA has been reported among a large series of colorectal cancers. However, the relevance of PIK3CA mutation in other cancer types remains unclear because of the limited number of tumors investigated. We have screened a total of 284 primary human tumors for mutations in all coding exons of PIK3CA using a combination of single stranded conformational polymorphism and denaturing high-performance liquid chromatography analysis. Among 70 primary breast cancers, 40% (28 of 70) harbored mutations in PIK3CA, making it the most common mutation described to date in this cancer type. Mutations were not associated with histologic subtype, estrogen receptor status, grade or presence of tumor in lymph nodes. Among the primary epithelial ovarian cancers only 11 of 167 (6.6%) contain somatic mutations, but there was a clear histologic subtype bias in their distribution. Only 2 of 88 (2.3%) of serous carcinomas had PIK3CA mutations compared with 8 of 40 (20.0%) endometrioid and clear cell cancers, which was highly significant (P = 0.001). In contrast, PIK3CA gene amplification (>7-fold) was common among all histologic subtypes (24.5%) and was inversely associated with the presence of mutations. Overall, PIK3CA mutation or gene amplification was detected in 30.5% of all ovarian cancers and 45% of the endometrioid and clear cell subtypes. Our study is the first direct evidence that PIK3CA is an oncogene in ovarian cancer and greatly extends recent findings in breast cancer.

The Akt kinase signals directly to endothelial nitric oxide synthase

Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain.

The immunosuppressive agent rapamycin induces inactivation of p70s6k with no effect on other mitogen‐activated kinases. Here we have employed a combination of techniques, including mass spectrometry, to demonstrate that this effect is associated with selective dephosphorylation of three previously unidentified p70s6k phosphorylation sites: T229, T389 and S404. T229 resides at a conserved position in the catalytic domain, whose phosphorylation is essential for the activation of other mitogen‐induced kinases. However, the principal target of rapamycin‐induced p70s6k inactivation is T389, which is located in an unusual hydrophobic sequence outside the catalytic domain. Mutation of T389 to alanine ablates kinase activity, whereas mutation to glutamic acid confers constitutive kinase activity and rapamycin resistance. The importance of this site and its surrounding motif to kinase function is emphasized by its presence in a large number of protein kinases of the second messenger family and its conservation in putative p70s6k homologues from as distantly related organisms as yeast and plants.

mTOR-Dependent Regulation of Ribosomal Gene Transcription Requires S6K1 and Is Mediated by Phosphorylation of the Carboxy-Terminal Activation Domain of the Nucleolar Transcription Factor UBF†

ABSTRACT Mammalian target of rapamycin (mTOR) is a key regulator of cell growth acting via two independent targets, ribosomal protein S6 kinase 1 (S6K1) and 4EBP1. While each is known to regulate translational efficiency, the mechanism by which they control cell growth remains unclear. In addition to increased initiation of translation, the accelerated synthesis and accumulation of ribosomes are fundamental for efficient cell growth and proliferation. Using the mTOR inhibitor rapamycin, we show that mTOR is required for the rapid and sustained serum-induced activation of 45S ribosomal gene transcription (rDNA transcription), a major rate-limiting step in ribosome biogenesis and cellular growth. Expression of a constitutively active, rapamycin-insensitive mutant of S6K1 stimulated rDNA transcription in the absence of serum and rescued rapamycin repression of rDNA transcription. Moreover, overexpression of a dominant-negative S6K1 mutant repressed transcription in exponentially growing NIH 3T3 cells. Rapamycin treatment led to a rapid dephosphorylation of the carboxy-terminal activation domain of the rDNA transcription factor, UBF, which significantly reduced its ability to associate with the basal rDNA transcription factor SL-1. Rapamycin-mediated repression of rDNA transcription was rescued by purified recombinant phosphorylated UBF and endogenous UBF from exponentially growing NIH 3T3 cells but not by hypophosphorylated UBF from cells treated with rapamycin or dephosphorylated recombinant UBF. Thus, mTOR plays a critical role in the regulation of ribosome biogenesis via a mechanism that requires S6K1 activation and phosphorylation of UBF.

Inhibition of RNA Polymerase I as a Therapeutic Strategy to Promote Cancer-Specific Activation of p53

Increased transcription of ribosomal RNA genes (rDNA) by RNA Polymerase I is a common feature of human cancer, but whether it is required for the malignant phenotype remains unclear. We show that rDNA transcription can be therapeutically targeted with the small molecule CX-5461 to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population. The therapeutic effect is a consequence of nucleolar disruption and activation of p53-dependent apoptotic signaling. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. These results identify selective inhibition of rDNA transcription as a therapeutic strategy for the cancer specific activation of p53 and treatment of hematologic malignancies.

STIM1: a novel phosphoprotein located at the cell surface

STIM1 is a novel candidate growth suppressor gene mapping to the human chromosome region 11p15.5 that is associated with several malignancies. STIM1 overexpression studies in G401 rhabdoid tumour, rhabdomyosarcoma and rodent myoblast cell lines causes growth arrest, consistent with a potential role as a tumour growth suppressor. We used highly specific antibodies to show by immunofluorescence and cell surface biotinylation studies that STIM1 is located at the cell surface of K562 cells. Western blot analysis revealed that the 90-kDa STIM1 protein is ubiquitously expressed in various human primary cells and tumour cell lines. STIM1 is not secreted from cells and does not appear to undergo proteolytic processing. We show evidence of post-translational modification of STIM1, namely phosphorylation and N-linked glycosylation. Phosphorylation of STIM1 in vivo occurs predominantly on serine residues. Thus, STIM1, the putative tumour growth suppressor gene is ubiquitously expressed and has features of a regulatory cell-surface phosphoprotein.

Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function

Current understanding of the mechanisms by which cell growth is regulated lags significantly behind our knowledge of the complex processes controlling cell cycle progression. Recent studies suggest that the mammalian target of rapamycin (mTOR) pathway is a key regulator of cell growth via the regulation of protein synthesis. The key mTOR effectors of cell growth are eukaryotic initiation factor 4E-binding protein 1 (4EBP-1) and the ribosomal protein S6 kinase (S6K). Here we will review the current models for mTOR dependent regulation of ribosome function and biogenesis as well as its role in coordinating growth factor and nutrient signaling to facilitate homeostasis of cell growth and proliferation. We will place particular emphasis on the role of S6K1 signaling and will highlight the points of cross talk with other key growth control pathways. Finally, we will discuss the impact of S6K signaling and the consequent feedback regulation of the PI3K/Akt pathway on disease processes including cancer.