The discovery of a 3-phosphomonoesterase that hydrolyzes phosphatidylinositol 3-phosphate in NIH 3T3 cells

Phosphatidylinositol 3-phosphate (PtdIns(3)P), a recently described phospholipid, has been linked to polyoma virus-induced cellular transformation and platelet-derived growth factor-mediated mitogenesis. PtdIns(3)P, in contrast to phosphatidylinositol, phosphatidylinositol 4-phosphate (PtdIns(4)P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), is resistant to hydrolysis by bovine brain phospholipase C gamma. We present here the identification of a phosphomonoesterase activity from the soluble fraction of NIH 3T3 cells which removes the phosphate from the D-3 position of PtdIns(3)P. This enzyme is specific as it has little or no activity on the monoester phosphates of PtdIns(4)P, PtdIns(4,5)P2, or inositol 1,3-bisphosphate and is tentatively designated phosphatidylinositol 3-phosphatase (PtdIns 3-phosphatase). The enzyme does not require added metal ions for activity and is maximally active in the presence of EDTA. It is inhibited by Ca2+, Mg2+, Zn2+, and the phosphatase inhibitor VO4(3-). In addition, there is no phospholipase C activity toward PtdIns(3)P in the soluble fraction of NIH 3T3 cells. In view of the absence of a phospholipase C activity that hydrolyzes PtdIns(3)P, we propose that PtdIns(3)P is not a precursor for a soluble inositol phosphate messenger but that it instead may act directly to control certain cellular processes or as a precursor for other phosphatidylinositols. PtdIns 3-phosphatase may thus terminate a metabolic signal or regulate precursor levels for other phosphatidylinositols that are phosphorylated in the D-3 position.     

New 3'-deoxythymidines bearing a nucleophilic 3'-substituent

New potential cancer-driven as well as HIV-driven nucleoside heteroanalogs, such as 3'-thio- and 3'- as well as 5'-selenosubstituted thymidines, have been synthesized. We also report an effective method for the preparation of novel nucleoside derivatives, bis(deoxynucleoside) diselenides, in nearly quantitative yields. The North conformation is significantly populated in the conformational equilibrium for 3'-alpha-alkylthiothymidines.     

JMJD3 is a histone H3K27 demethylase

Histone methylation is an important epigenetic phenomenon that participates in a diverse array of cellular processes and has been found to be associated with cancer. Recent identification of several histone demethylases has proved that histone methylation is a reversible process. Through a candidate approach, we have biochemically identified JMJD3 as an H3K27 demethylase. Transfection of JMJD3 into HeLa cells caused a specific reduction oftrimethyl H3K27, but had no effect on di-and monomethyl H3K27, or histone lysine methylations on H3K4 and H3K9. The enzymatic activity requires the JmjC domain and the conserved histidine that has been suggested to be important for a cofactor binding. In vitro biochemical experiments demonstrated that JMJD3 directly catalyzes the demethylation. In addition, we found that JMJD3 is upregulated in prostate cancer, and its expression is higher in metastatic prostate cancer. Thus, we identified JMJD3 as a demethylase capable of removing the trimethyl group from histone H3 lysine 27 and upregulated in prostate cancer.     

Identification of a proline-rich Akt substrate as a 14-3-3 binding partner

Akt (also called protein kinase B) is one of the major downstream targets of the phosphatidylinositol 3-kinase pathway. This protein kinase has been implicated in insulin signaling, stimulation of cellular growth, and inhibition of apoptosis as well as transformation of cells. Although a number of cellular proteins have been identified as putative targets of the enzyme, additional substrates may play a role in the varied responses elicited by this enzyme. We have used a combination of 14-3-3 binding and recognition by an antibody to the phosphorylation consensus of the enzyme to identify and isolate one of the major substrates of Akt, which is also a 14-3-3 binding protein. This 40-kDa protein, designated PRAS40, is a proline-rich Akt substrate. Demonstration that it is a substrate of Akt was accomplished by showing that 1) PRAS40 was phosphorylated in vitro by purified Akt on the same site that was phosphorylated in insulin-treated cells; 2) activation of an inducible Akt was alone sufficient to stimulate the phosphorylation of PRAS40; and 3) cells lacking Akt1 and Akt2 exhibit a diminished ability to phosphorylate this protein. Thus, PRAS40 is a novel substrate of Akt, the phosphorylation of which leads to the binding of this protein to 14-3-3.     

DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns. Dnmt3L, a member of the Dnmt3 family, has been reported to be necessary for maternal methylation imprinting, possibly by interacting with Dnmt3a and/or Dnmt3b (Hata, K., Okano, M., Lei, H., and Li, E. (2002) Development 129, 1983-1993). In the present study, the effect of DNMT3L, a human homologue of Dnmt3L, on the DNA methylation activity of mouse Dnmt3a and Dnmt3b was examined in vitro. DNMT3L enhanced the DNA methylation activity of Dnmt3a and Dnmt3b about 1.5-3-fold in a dose-dependent manner but did not enhance the DNA methylation activity of Dnmt1. Although the extents of stimulation were different, a stimulatory effect on the DNA methylation activity was observed for all of the substrate DNA sequences examined, such as those of the maternally methylated SNRPN and Lit-1 imprinting genes, the paternally methylated H19 imprinting gene, the CpG island of the myoD gene, the 5 S ribosomal RNA gene, an artificial 28-bp DNA, poly(dG-dC)-poly(dG-dC), and poly(dI-dC)-poly(dI-dC). DNMT3L could not bind to DNA but could bind to Dnmt3a and Dnmt3b, indicating that the stimulatory effect of DNMT3L on the DNA methylation activity may not be due to the guiding of Dnmt3a and Dnmt3b to the targeting DNA sequence but may comprise a direct effect on their catalytic activity. The carboxyl-terminal half of DNMT3L was found to be responsible for the enhancement of the enzyme activity.     

ARAP3 is a PI3K- and rap-regulated GAP for RhoA

Rho and Arf family small GTPases are well-known regulators of cellular actin dynamics. We recently identified ARAP3, a member of the ARAP family of dual GTPase activating proteins (GAPs) for Arf and Rho family GTPases, in a screen for PtdIns(3,4,5)P(3) binding proteins. PtdIns(3,4,5)P(3) is the lipid product of class I phosphoinositide 3OH-kinases (PI3Ks) and is a signaling molecule used by growth factor receptors and integrins in the regulation of cell dynamics. We report here that as a Rho GAP, ARAP3 prefers RhoA as a substrate and that it can be activated in vitro by the direct binding of Rap proteins to a neighbouring Ras binding domain (RBD). This activation by Rap is GTP dependent and specific for Rap versus other Ras family members. We found no evidence for direct regulation of ARAP3's Rho GAP activity by PtdIns(3,4,5)P(3) in vitro, but PI3K activity was required for activation by Rap in a cellular context, suggesting that PtdIns(3,4,5)P(3)-dependent translocation of ARAP3 to the plasma membrane may be required for further activation by Rap. Our results indicate that ARAP3 is a Rap-effector that plays an important role in mediating PI3K-dependent crosstalk between Ras, Rho, and Arf family small GTPases.     

ik3-1/Cables is a substrate for cyclin-dependent kinase 3 (cdk 3).

p70ik3-1 (a 70-kDa protein) contains a cyclin box, and binds to p35cdk3 in vivo and in vitro [Matsuoka, M., Matsuura, Y., Semba, K. & Nishimoto, I. (2000) Biochem. Biophys. Res. Commun. 273, 442-447]. In spite of its structural similarity to cyclins, p70ik3-1 does not activate cyclin-dependent kinase 3 (cdk3)-mediated phosphorylation of pRb, histone H1, or the C-terminal domain of RNA polymerase II. Here, we report that Ser274 of p70ik3-1 is phosphorylated by cdk2 or cdk3 bound to cyclin A and to cyclin E in vitro. We also found that in COS7 cells in which cyclin E and cdk3 were ectopically overexpressed, the phosphorylation level of Ser274 in coexpressed p70ik3-1 is upregulated. We therefore conclude that p70ik3-1 is a substrate for cdk3-mediated phosphorylation.     

Galectin-3 as a multifunctional protein

Galectin-3 is a 31 kDa member of a growing family of beta-galactoside-binding animal lectins. This protein is expressed in a variety of tissues and cell types and is mainly found in the cytoplasm, although, depending on cell type and proliferative state, a significant amount of this lectin can also be detected in the nucleus, on the cell surface or in the extracellular environment. Galectin-3 is secreted from cells by a novel and incompletely understood mechanism that is independent of the classical secretory pathway through the endoplasmic reticulum/Golgi network. Galectin-3 exhibits pleiotropic biological function, playing a key role in many physiological and pathological processes.     

MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3

The MCM proteins are essential for the initiation of DNA replication. We have isolated an MCM3-associated protein (MCM3AP) in a two-hybrid screen using MCM3. Here we demonstrate that MCM3AP is an acetyltransferase which acetylates MCM3 and that chromatin-bound MCM3 is acetylated in vivo. The MCM3 acetylase, MCM3AP, is also chromatin-bound. This study also indicates that MCM3AP contains putative acetyl CoA binding motifs conserved within the GCN5-related N-acetyltransferase superfamily. Mutation of those motifs significantly inhibits the MCM3 acetylase activity. Over-expression of MCM3AP inhibits DNA replication, whereas mutation of the acetylase motifs abolishes this effect, suggesting that acetylation plays a role in DNA replication. Taken together, we suggest that MCM3 acetylation is a novel pathway which might regulate DNA replication.     

NCD3G: a novel nine-cysteine domain in family 3 GPCRs

The NCD3G [for nine-cysteine domain of family 3 G-protein-coupled receptors (GPCRs)] domain is a novel protein domain that is conserved in family 3 GPCRs, including metabotropic glutamate receptors, calcium-sensing receptors, pheromone receptors and taste receptors, with the exception of GABA(B) receptors. The NCD3G domain contains nine highly conserved cysteine residues. Structural predictions suggest that NCD3G might possess four beta strands and three disulfide bridges. The structural model of NCD3G highlights the conserved residues co-segregated with certain familial diseases.     

Spatial relationship between cytochrome a and a3

We have studied the spatial relationship between cytochromes a and a3 by the enhancement of the spin relaxation of cytochrome a3-NO EPR signals by the paramagnetic a heme at 15 K. An Fe-Fe distance of 12-19A is estimated from the absence of dipolar broadening and from the observation of spin relaxation enhancement in the a3-NO complex. When this result is combined with resonance x-ray diffraction data reported by Blasie et al. (Blasie, J. K., Pachence, J. M., Tavormina, A., Dutton, P. L., Stamatoff, J., Eisenberger, P., and Brown, G. (1982) Biochim. Biophys. Acta 679, 188-197) and the contribution from the exchange interaction is considered, we can limit the iron-iron distance to 12-16 A and estimate the angle between the Fe-Fe vector and mitochondrial membrane normal as 30-60 degrees. We also consider the possible effects of CuA on cytochrome a3-NO.     

Solution conformation of carboxy-terminal fragments of the third component of human complement C3: proton nuclear magnetic resonance study of C3a, des-Arg-C3a, and C3a Arg69

A proton nuclear magnetic resonance (NMR) study is reported of the solution conformation of human C3a, that is released on activation of C3, the third component of complement. The intact C3a was used along with des-Arg-C3a, which is formed on cleavage of Arg-77 at the C terminal of C3a, and C3a Arg69, which is a 69-residue fragment produced on tryptic digestion of C3. A method of carboxypeptidase digestion/difference spectroscopy (Endo & Arata (1985) Biochemistry 24, 1561-1568) was extensively used for the spectral assignments of Ile-43, Ile-60, Leu-63, Tyr-15, and Tyr-59. On the basis of the results of nuclear Overhauser effect (NOE) measurements, we discuss the solution conformation of the C3a molecule. It has been concluded that removal of Arg-77, which is essential for expression of the biological activity of C3a, does not induce any significant change in the solution conformation of the C3a molecule. The C3a molecule is known to consist of a core region that comprises segment Tyr-15-Tyr-59. We conclude that in solution the C terminal segment sticks out of the core and takes on a helix-like conformation. Possible roles of the core region and the N terminal segment in maintaining the conformation of the C terminal segment are briefly discussed.     

3-fluoro-3-deoxycitrate: a probe for mechanistic study of citrate-utilizing enzymes

The interaction of a novel fluorinated analogue of citrate, 3-fluoro-3-deoxycitrate (3-fluorocitrate), with the four known citrate-processing enzymes is described in this report. Three of the citrate-processing enzymes, citrate synthase, ATP citrate lyase, and citrate lyase, catalyze reversible aldol-type condensations. The fate of 3-fluorocitrate with each enzyme is uniquely related to their mechanisms of action. For citrate synthase, 3-fluorocitrate is a competitive inhibitor. 3-Fluorocitrate is a substrate for the carboxylate activation half-reaction catalyzed by ATP citrate lyase and induces a net ATPase action during conversion to 3-fluorocitryl-S-coenzyme A. Because of the unusual mechanism of citrate cleavage catalyzed by bacterial citrate lyase, 3-fluorocitrate is a mechanism-based inhibitor, acting at two points during turnover of the acetyl enzyme. The fourth citrate-processing enzyme, aconitase, does turn over 3-fluorocitrate catalytically. This enzyme, catalyzing a dehydration and rehydration of citrate, also catalyzes the elimination of HF from 3-fluorocitrate, yielding cis-aconitate and fluoride.     

Immuno-PET Imaging of HER3 in a Model in which HER3 Signaling Plays a Critical Role

HER3 is overexpressed in various carcinomas including colorectal cancer (CRC), which is associated with poor prognosis, and is involved in the development of therapy resistance. Thus, an in vivo imaging technique is needed to evaluate the expression of HER3, an important therapeutic and diagnostic target. Here, we report successful HER3 PET imaging using a newly generated anti-human HER3 monoclonal antibody, Mab#58, and a mouse model of a HER3-overexpressing xenograft tumor. Furthermore, we assessed the role of HER3 signaling in CRC cancer tissue-originated spheroid (CTOS) and applied HER3 imaging to detect endogenous HER3 in CTOS-derived xenografts. Cell binding assays of ⁸⁹Zr-labeled Mab#58 using the HER3-overexpressing cell line HER3/RH7777 demonstrated that [⁸⁹Zr]Mab#58 specifically bound to HER3/RH7777 cells (K_d = 2.7 nM). In vivo biodistribution study in mice bearing HER3/RH7777 and its parent cell xenografts showed that tumor accumulation of [⁸⁹Zr]Mab#58 in HER3/RH7777 xenografts was significantly higher than that in the control from day 1 to day 4, tending to increase from day 1 to day 4 and reaching 12.2 ± 4.5%ID/g. Radioactivity in other tissues, including the control xenograft, decreased or remained unchanged from day 1 to day 6. Positron emission tomography (PET) in the same model enabled clear visualization of HER3/RH7777 xenografts but not of RH7777 xenografts. CTOS growth assay and signaling assay revealed that CRC CTOS were dependent on HER3 signaling for their growth. In PET studies of mice bearing a CRC CTOS xenograft, the tumor was clearly visualized with [⁸⁹Zr]Mab#58 but not with the ⁸⁹Zr-labeled control antibody. Thus, tumor expression of HER3 was successfully visualized by PET with ⁸⁹Zr-labeled anti-HER3 antibody in CTOS xenograft-bearing mice, a model that retains the properties of the patient tumor. Non-invasive targeting of HER3 by antibodies is feasible, and it is expected to be useful for cancer diagnosis and treatment.     

The translational regulator eIF3a: The tricky eIF3 subunit!

Regulation of gene expression is a fundamental step in cellular physiology as abnormalities in this process may lead to de-regulated growth and cancer. Translation of mRNA is mainly regulated at the rate-limiting initiation step, where many eukaryotic initiation factors (eIFs) are involved. The largest and most complex initiation factor is eIF3 which plays a role in translational regulation, cell growth and cancer. The largest subunit of eIF3 is eIF3a, although it is not required for the general function of eIF3 in translation initiation. However, eIF3a may play a role as a regulator of a subset of mRNAs and has been demonstrated to regulate the expression of p27^kip1, tyrosinated α-tubulin and ribonucleotide reductase M2 subunit. These molecules have a pivotal role in the regulation of the cell cycle. Moreover, the eIF3a mRNA is ubiquitously expressed in all tissues at different levels and is found elevated in a number of cancer types. eIF3a can modulate the cell cycle and may be a translational regulator for proteins important for entrance into S phase. The expression of eIF3a is decreased in differentiated cells in culture and the suppression of eIF3a expression can reverse the malignant phenotype and change the sensitivity of cells to cell cycle modulators. However, the role of eIF3a in cancer is still unclear. In fact, some studies have identified eIF3a to be involved in cancer development, while other results indicate that it could provide protection against evolution into higher malignancy. Together, these findings highlight the “tricky” and interesting nature of eIF3a.