Alexander Zhyvoloup

Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase βII and Regulates Its Subcellular Localization

ABSTRACT The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G1/S transition of the cell cycle. There are two forms of S6K, termed S6Kα and S6Kβ, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kα, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kβ, but not S6Kα, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KβII by PKC-mediated phosphorylation.

Subcellular Localization and Regulation of Coenzyme A Synthase

CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4′-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1–29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.

Nuclear export of S6K1 II is regulated by protein kinase CK2 phosphorylation at Ser-17.

Ribosomal S6 kinases (S6Ks) are principal players in the regulation of cell growth and energy metabolism. Signaling via phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways mediates the activation of S6K in response to various mitogenic stimuli. The family of S6Ks consists of two forms, S6K1 and -2, that have cytoplasmic and nuclear splicing variants, S6K1 II and S6K1 I, respectively. Nuclear-cytoplasmic shuttling of both isoforms induced by mitogenic stimuli has been reported recently. Here we present the identification of protein kinase CK2 (CK2) as a novel binding and regulatory partner for S6K1 II. The interaction between S6K1 II and CK2β regulatory subunit was initially identified in a yeast two-hybrid screen and further confirmed by co-immunoprecipitation of transiently expressed and endogenous proteins. The interaction between S6K1 II and CK2 was found to occur in serum-starved and serum-stimulated cells. In addition, we found that S6K1 II is a substrate for CK2. The localization of the CK2 phosphorylation site was narrowed down to Ser-17 in S6K1 II. Mutational analysis and the use of phosphospecific antibody indicate that Ser-17 is a major in vitro and in vivo phosphorylation site for CK2. Functional studies reveal that, in contrast to the wild type kinase, the phosphorylation-mimicking mutant of S6K1 II (S17E) retains its cytoplasmic localization in serum-stimulated cells. Treatment of cells with the nuclear export inhibitor leptomycin B revealed that the S17E mutant accumulates in the nucleus to the same extent as S6K1 II wild type. These results indicate that nuclear import of the S17E mutant is not affected, although the export is significantly enhanced. We also provide evidence that nuclear export of S6K1 is mediated by a CRM1-dependent mechanism. Taken together, this study establishes a functional link between S6K1 II and CK2 signaling, which involves the regulation of S6K1 II nuclear export by CK2-mediated phosphorylation of Ser-17.

Specific interaction between S6K1 and CoA synthase: a potential link between the mTOR/S6K pathway, CoA biosynthesis and energy metabolism

Ribosomal protein S6 kinase (S6K) is a key regulator of cell size and growth. It is regulated via phosphoinositide 3‐kinases (PI3K) and the mammalian target of rapamycin (mTOR) signaling pathways. We demonstrate for the first time that CoA synthase associates specifically with S6K1. The association was observed between native and transiently overexpressed proteins in vivo, as well as by BIAcore analysis in vitro. The sites of interaction were mapped to the C‐terminal regions of both CoA synthase and S6K1. In vitro studies indicated that the interaction does not affect their enzymatic activities and that CoA synthase is not a substrate for S6 kinase. This study uncovers a potential link between mTor/S6K signaling pathway and energy metabolism through CoA and its thioester derivatives, but its physiological relevance should be further elucidated.

Evidence for the ability of L10 ribosomal proteins of Salmonella typhimurium and Klebsiella pneumoniae to regulate rplJL gene expression in Escherichia coli

Genes rplJ, coding for ribosomal protein L10 of Salmonella typhimurium and Klebsiella pneumoniae, have been cloned on pUC plasmid. The resultant multicopy recombinant plasmids were detrimental for the growth of normal JM101 E. coli host cells and harmless for the mutant JF3029 host. This negative effect is the evidence for the ability of heterologous L10 proteins to regulate expression of rplJL genes in E. coli. Nucleotide sequence was determined completely for S. typhimurium rplJL ‘DNA portion and partially for rplJL’ genes of K. pneumoniae. According to the nucleotide sequence data obtained three amino acid substitutions differ L10 proteins of S. typhimurium and E. coli and the long range, providing for the coupled translations of L10 and L7/L12 cistrons in E. coli mRNA is also valid for S. typhimurium and K. pneumoniae.

Large-scale yeast transformation in low-percentage agarose medium.

BENCHMARKS The use of yeast as a research tool became especially popular with the development of the yeast two-hybrid (Y2H) assay, which is a powerful tool for the detection of specific protein-protein interactions. Several versions of the Y2H system have been developed. All variations of this method demand the large-scale transformation of yeast with cDNA library plasmids (1). Transformation of yeast with plas-mid DNA can be achieved by several methods, such as agitation with glass beads (2), spheroplast preparation (3), treatment with lithium acetate (4), or electroporation (5). The most popular and commonly used is lithium acetate transformation was developed by Ito et al. (6) and modified by Gietz and Woods (7). The standard Y2H library protocols for an average mammalian cDNA library (10 6 primary independent clones) and maximal colony density 5/mm 2 require plating of more than fifty 150-mm plates (8). In addition to being laborious and having problems in maintaining sterility, the inevitable overgrowth of marginal colonies near the plate edge causes loss of uniformity across the library. Here we present a protocol for the large-scale transformation of yeast in a semi-solid agarose medium that does not require plating. The protocol allows for the growth of transformed cells as separate colonies in the volume of semi-solid medium. The protocol is optimized for lithium acetate transformation , gives high transformation efficiency , and provides an equal growth environment for each colony, thereby faithfully preserving the integrity of the primary library. It also allows yeast library propagation with a significantly higher colony density. The protocol was developed on the basis of several published techniques (7–10) and requires fewer reagents when compared to other approaches. Working with DupLEX-A™, a LexA-based Y2H system (OriGene Technologies, Rockville, MD, USA), we successfully used this protocol for the EGY48 strain (MATα, his3, trp1, ura3, 6 LexAop-LEU2) transformed with different pEG202-based bait plas-mids and a reporter plasmid pSH18-34 (10). Using this protocol, we managed to achieve transformation efficiencies of 0.25–1 × 10 6 /µg of library DNA or 2–8 million independent colonies/400 mL of the semi-solid agarose medium. Therefore, this protocol allowed us to cover an average cDNA library with just one or two bottles of semi-solid medium (400 mL each). All reagents used were from Sigma (St. Louis, MO, USA) except the low melting point agarose). Culture media reagents as well as carrier DNA were from Bio101 (Vista, CA, USA). Procedures were performed according …