Valeriy Filonenko

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.

Preferential Nuclear and Cytoplasmic NY-BR-1 Protein Expression in Primary Breast Cancer and Lymph Node Metastases

Purpose:NY-BR-1 is a recently isolated differentiation antigen, which is expressed in normal mammary tissue and in breast cancer. However, current data are based on RT-PCR analysis and nothing is known about the presence of NY-BR-1 on a protein level. We previously generated a monoclonal antibody to NY-BR-1 to study the protein expression of NY-BR-1. Methods: In our immunohistochemical study, NY-BR-1 was analyzed in normal tissues, various tumor types, 124 primary breast cancers, and 37 paired lymph node metastases. Results: Among normal tissues, NY-BR-1 was present solely in ductal epithelium of the breast. In tumors, carcinoma in situ and invasive carcinoma of the breast were NY-BR-1 positive whereas other tumors and normal tissues were negative. Sixty percent of invasive breast carcinomas were NY-BR-1 positive, displaying cytoplasmic and/or nuclear immunoreactivity. This coexpression was verified by confocal microscopy. Although the monoclonal antibody identified intratumoral heterogeneity, a majority (72%) of NY-BR-1-positive carcinomas revealed immunoreactivity in >50% of the tumor cells. NY-BR-1 expression was more frequent in estrogen receptor–positive and lymph node–negative primary carcinomas (P < 0.05 each) and was more common in grade 1 (77%) than in grade 2 (63%) or grade 3 (50%) carcinomas (P < 0.05). This suggests that NY-BR-1 expression is lost with tumor progression. Forty-nine percent of lymph node metastases were NY-BR-1 positive. Conclusion: This study supports the notion that NY-BR-1 is a differentiation antigen of the breast, which is present in normal and tumorous mammary epithelium. The organ-specific expression of NY-BR-1 and its high prevalence in metastases indicate that it could be a valuable target for cancer immunotherapy.

mTORβ Splicing Isoform Promotes Cell Proliferation and Tumorigenesis

The mTOR (mammalian target of rapamycin) promotes growth in response to nutrients and growth factors and is deregulated in numerous pathologies, including cancer. The mechanisms by which mTOR senses and regulates energy metabolism and cell growth are relatively well understood, whereas the molecular events underlining how it mediates survival and proliferation remain to be elucidated. Here, we describe the existence of the mTOR splicing isoform, TORβ, which, in contrast to the full-length protein (mTORα), has the potential to regulate the G1 phase of the cell cycle and to stimulate cell proliferation. mTORβ is an active protein kinase that mediates downstream signaling through complexing with Rictor and Raptor proteins. Remarkably, overexpression of mTORβ transforms immortal cells and is tumorigenic in nude mice and therefore could be a proto-oncogene.

Regulation of ribosomal protein S6 kinases by ubiquitination

Ribosomal protein S6 kinase (S6K) is a key player in the regulation of cell growth and energy metabolism via the mTOR and PI3K signalling pathways. The activity and subcellular localization of S6K are regulated by multiple S/T phosphorylations in response to diverse extracellular stimuli. Downregulation of S6K signalling occurs through the action of S/T phosphatases (PP2A and PP1) and tumor suppressors (TSC1/2 and PTEN). We report here that, in addition to phosphorylation, S6Ks are ubiquitinated in cells. The pattern of ubiquitination and the effect of proteasomal inhibitors on the steady-state level of transiently overexpressed and endogenous S6Ks point to proteasome-mediated degradation of ubiquitinated S6Ks. Furthermore, we found that the site(s) of ubiquitination are located in the kinase domain and that the N- and C-terminal regulatory regions modulate the efficiency of S6K ubiquitination. This study suggests that S6K signalling also could be regulated through the proteasome-mediated turnover of S6Ks.

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.

Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings.

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin 1 cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins.

NY-CO-58/KIF2C is overexpressed in a variety of solid tumors and induces frequent T cell responses in patients with colorectal cancer

NY‐CO‐58/KIF2C has been identified as a tumor antigen by screening antibody responses in patients with colorectal cancer. However, expression had not consequently been examined, and nothing was known about its ability to induce spontaneous T cell responses, which have been suggested to play a role in the development of colorectal cancer. We analyzed 5 colorectal cancer cell lines, and tumor samples and adjacent healthy tissues from 176 patients with epithelial cancers for the expression of NY‐CO‐58/KIF2C by RT‐PCR and Western Blot. T cell responses of 43 colorectal cancer patients and 35 healthy donors were evaluated by ELISpot following stimulation with 30mer peptides or full‐length protein. All cell lines and tumor samples from colorectal cancer patients expressed NY‐CO‐58/KIF2C on the protein and RNA level, and expression levels correlated strongly with Ki‐67 expression (r = 0.69; p = 0.0003). Investigating NY‐CO‐58/KIF2C‐specific T cell responses, CD8+ T cells directed against 1 or more peptides were found in less than 10% of patients, whereas specific CD4+ T cells were detected in close to 50% of patients. These T cells were of high avidity, recognized the naturally processed antigen and secreted IFN‐γ and TNF‐α. Depletion of CD4+CD25+ T cells before stimulation significantly increased the intensity of the preexisting response. NY‐CO‐58/KIF2C is significantly overexpressed in colorectal and other epithelial cancers and expression levels correlate with the proliferative activity of the tumor. Importantly, NY‐CO‐58/KIF2C was able to induce spontaneous CD4+ T cell responses of the Th1‐type, which were tightly controlled by peripheral T regulatory cells.

Development of Monoclonal Antibodies Specific for the Human Sodium-dependent Phosphate Co-transporter NaPi2b

Homeostasis of inorganic phosphate in the human body is maintained by regulated absorption, metabolism, and excretion. Sodium-dependent phosphate transporters (NaPi) mediate the transport of inorganic phosphate (P(i)) in cells in response to dietary phosphate consumption, hormones, and growth factors. NaPi2b is a member of the sodium-dependent phosphate transporter family, with a distinct pattern of expression and regulation. Signaling pathways activated by mitogens, glucocorticoids, and metabolic factors have been implicated in regulating P(i) transport via NaPi2b. Inactivation of NaPi2b function by mutations has been linked to human pathologies, such as pulmonary alveolar microlithiasis. In this study, we describe the generation and characterization of monoclonal antibodies against human NaPi2b. The monoclonal antibodies were shown to recognize specifically transiently overexpressed and endogenous NaPi2b in commonly used immunoassays, including Western blotting, immunoprecipitation, and immunohistochemistry. These properties make them particularly valuable reagents for elucidating NaPi2b function in health and disease.