BBA - Molecular Cell Research (v.1589, #2)

Insulin induces PFKFB3 gene expression in HT29 human colon adenocarcinoma cells by Lluı́s Riera; Anna Manzano; Aurea Navarro-Sabaté; Jose C. Perales; Ramon Bartrons (89-92).
Fructose 2,6-bisphosphate is present at high concentrations in many established lines of transformed cells. It plays a key role in the maintenance of a high glycolytic rate by coupling hormonal and growth factor signals with metabolic demand. The concentration of fructose 2,6-bisphosphate is controlled by the activity of the homodimeric bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). We report here the PFKFB-3 gene expression control by insulin in the human colon adenocarcinoma HT29 cell line. The incubation of these cells with 1 μM insulin resulted in an increase in the PFK-2 mRNA level after 6 h of treatment, this effect being blocked by actinomycin D. Furthermore, insulin induced ubiquitous PFK-2 protein levels, that were evident after a lag of 3 h and could be inhibited by incubation with cycloheximide.
Keywords: Glycolysis; Fructose 2,6-bisphosphate; 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Gene expression;

Changes in endothelial cell (EC) shape result in inter-EC gap formation and subsequently regulate transendothelial passage. In this work, we investigated the effects of protein phosphorylation (induced by inhibition of protein phosphatases) on EC shape changes. Treatment of bovine pulmonary artery endothelial cells (BPAEC) with calyculin A (100 nM, an inhibitor of protein Ser/Thr phosphatases 1 and 2A) resulted in cell retraction, surface bleb formation and cell rounding. Trypan blue and electrophysiological experiments suggested that the plasma membrane of these rounded cells maintained functional integrity. Calyculin A-induced morphological changes were strongly inhibited by staurosporine, but not affected by specific inhibitors of the myosin light chain (MLC) kinase, protein kinases A, C and G, and tyrosine kinases. The calyculin A effects were not mimicked by phorbol myristate acetate, dibutyryl cAMP, 8-bromo-cGMP or ionomycin. Cytochalasin B (an inhibitor of actin polymerization) almost completely abolished such shape changes while colchicine (an inhibitor of microtubule polymerization) had no inhibitory effect at all. Ca2+ imaging experiments showed that the morphological changes were not associated with any global or local cytosolic Ca2+ concentration ([Ca2+]i) elevation. The results suggest that calyculin A unmasked the basal activities of some protein Ser/Thr kinases other than MLC kinase and protein kinases A, C and G; these unknown kinases might cause BPAEC shape changes by a mechanism involving actin polymerization but not [Ca2+]i elevation.
Keywords: Calyculin A; Endothelial cell; Cell rounding; Cytosolic Ca2+; Actin;

Assessment of the microbody luminal pH in the filamentous fungus Penicillium chrysogenum by Ted R van der Lende; Pieter Breeuwer; Tjakko Abee; Wil N Konings; Arnold J.M Driessen (104-111).
The enzymes of the penicillin biosynthetic pathway in Penicillium chrysogenum are located in different subcellular compartments. Consequently, penicillin pathway precursors and the biologically active penicillins have to cross one or more membranes. The final enzymatic step that is mediated by acyltransferase takes place in a microbody. The pH of the microbody lumen in penicillin producing cells has been determined with fluorescent probes and mutants of the green fluorescent protein and found to be slightly alkaline.
Keywords: Organelle; Microbody; Peroxisome; Vacuole; Penicillin; Penicillium chrysogenum;

WTH3, a new member of the Rab6 gene family, and multidrug resistance by Jidong Shan; Liming Yuan; Daniel R. Budman; Hao-peng Xu (112-123).
The WTH3 gene was obtained by a DNA fragment isolated by the methylation-sensitive representational difference analysis technique due to its hypermethylation in the human multidrug resistant (MDR) breast cancer cell line MCF7/AdrR. The WTH3 gene product is 89% and 91% identical to the human Rab6 and Rab6c proteins, but possesses an elongated C-terminal region which contains 46 extra amino acids. Nevertheless, we consider the WTH3 gene a new member of the Rab6 gene family. Semi-quantitative reverse transcriptase–polymerase chain reaction results showed that WTH3 was 15 and 4 times downregulated in MCF7/AdrR and MES-SA/Dx5, a human MDR uterine sarcoma cell line, as compared to their non-MDR parental cell lines. Permanent expression of the WTH3 transgene in MDR cell lines increased to varying degrees their sensitivity to several anticancer drugs, which included doxorubicin, taxol, vinblastine, vincristine, and etoposide, as compared to the control sublines transfected with the empty vector. Flow cytometry and fluorescence microscope experiments suggest that the WTH3 transgene stimulated the host’s uptake and retention of DOX. Our results imply that the WTH3 gene plays a role(s) in MDR phenotype development in vitro.
Keywords: Methylation-sensitive representational difference analysis; G protein; Gene transfection; Drug resistance;

Retrograde transport of protein toxins under conditions of COPI dysfunction by Alice Chen; Tonghuan Hu; Carole Mikoryak; Rockford K. Draper (124-139).
Retrograde transport dependent on coat protein I (COPI) was impaired using two different approaches and the effects on the retrograde transport of protein toxins were investigated. One approach was to study ldlF cells that express a temperature-sensitive defect in the ϵ-COP subunit of COPI. The second approach was to treat cells with 1,3-cyclohexanebis(methylamine) (CBM), a drug that interferes with the binding of COPI to Golgi membranes. With both approaches, cells remained sensitive to a variety of protein toxins regardless of whether the toxins contained a KDEL motif. Moreover, cholera toxin, which contains a KDEL sequence, was observed by immunofluorescence microscopy to enter the endoplasmic reticulum of Vero cells in the presence of CBM. These data support published evidence indicating the presence in cells of a COPI- and KDEL receptor-independent pathway of retrograde transport from the Golgi complex to the endoplasmic reticulum. In addition, the results suggest that certain toxins containing a KDEL motif may use either the COPI-dependent or COPI-independent pathway of retrograde transport.
Keywords: Golgi; Retrograde transport; Coat protein I; Ricin; Cholera toxin;

The oxygen-substituted palmitic acid analogue, 13-oxypalmitic acid, inhibits Lck localization to lipid rafts and T cell signaling by Ibrahim Y. Hawash; X.Eric Hu; Adiam Adal; John M. Cassady; Robert L. Geahlen; Marietta L. Harrison (140-150).
Palmitoylation of cysteines 3 and 5 is necessary for targeting Lck to lipid rafts and is needed for Lck function in T cell receptor (TCR) signaling. Point mutations of cysteines 3 and 5 result in a form of Lck that fails to associate with the plasma membrane, which limits the usefulness of this genetic approach to address the role of palmitoylation in the distribution of Lck within the plasma membrane. To circumvent this problem, we sought to identify a palmitic acid analogue that would enable plasma membrane association of Lck, but not facilitate its localization within lipid rafts. Here we examined the effects of the heteroatom-substituted analogue of palmitic acid, 13-oxypalmitic acid (13-OP), on Lck subcellular localization and function. 13-OP is similar in chain length to palmitic acid, but possesses reduced hydrophobicity. We found that treatment of cells with 13-OP inhibited incorporation of ω-[125I]iodopalmitate into Lck. 13-OP inhibited localization of Lck to lipid rafts without altering its membrane localization. Consistent with the dissociation of Lck from rafts, treatment with 13-OP abolished Lck association with the GPI-anchored protein, CD48, but not the transmembrane glycoprotein CD4. Jurkat T cells treated with 13-OP showed marked reduction in tyrosine phosphorylation and activation of mitogen-activated protein kinase upon TCR stimulation. In conclusion, the less hydrophobic analogue of palmitate, 13-OP, alters the normal acylation of Lck that provides Lck with the necessary hydrophobicity and tight packing order required for inclusion in lipid rafts.
Keywords: Protein palmitoylation; Lck; Signal transduction; Acylation; Lipid raft;

Diverse effects of RacV12 on cell transformation by Raf: partial inhibition of morphological transformation versus deregulation of cell cycle control by Eugen Kerkhoff; Cornelia B. Leberfinger; Gudula Schmidt; Klaus Aktories; Ulf R. Rapp (151-159).
Activated Raf kinases and Rac GTPases were shown to cooperate in the oncogenic transformation of fibroblasts, which is characterised by the disassembly of the cellular actin cytoskeleton, a nearly complete loss of focal adhesion complexes and deregulated cell proliferation. This is surprising since the Rac GTPase induces actin structures and the adhesion of suspended cells to extracellular matrix proteins. NIH 3T3 cells expressing a hydroxytamoxifen-inducible oncogenic c-Raf-1–oestrogen receptor fusion protein (c-Raf-1-BxB-ERTM, N-BxB-ERTM cells) undergo morphological transformation upon stimulation of the Raf kinase. We show that treatment with the Rac, Rho and Cdc42 activating Escherichia coli toxin CNF1 or coexpression of an activated RacV12 mutant partially inhibits and reverses the disassembly of cellular actin structures and focal adhesion complexes by oncogenic Raf. Activation of the Rac GTPase restores actin structures and focal adhesion complexes at the cellular boundary, leading to spreading of the otherwise spindle-shaped Raf-transformed cells. Actin stress fibres, however, which are regulated by the function of the Rho GTPase, are disassembled by oncogenic Raf even in the presence of activated Rac and Rho. With respect to the RacV12-mediated spreading of Raf-transformed cells, we postulate an anti-oncogenic function of the activated Rac. Another feature of cell transformation is the deregulation of cell cycle control. NIH 3T3 cells expressing high levels of the c-Raf-1-BxB-ERTM protein undergo a cell cycle arrest upon stimulation of the oncogenic Raf kinase. Our results show that in N-BxB-ERTM-RacV12 cells the expression of the activated RacV12 mediates cell proliferation in the presence of high-intensity Raf signals and high levels of the Cdk inhibitor p21Cip1. These results indicate a pro-oncogenic function of the Rac GTPase with respect to the deregulation of cell cycle control.
Keywords: Raf; Rac; Morphological transformation; Actin cytoskeleton; Adhesion;

Transcriptome changes during intestinal cell differentiation by Mehrdad Tadjali; Jakob B. Seidelin; Jørgen Olsen; Jesper T. Troelsen (160-167).
The expression of 18 149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change in the transcriptome was observed during the differentiation of the Caco-2 cells. 8762 of the 18 149 genes analysed were expressed above background level in the undifferentiated Caco-2 cells, whereas only 5767 genes were expressed above background in differentiated Caco-2 cells. This pattern of expression was caused by a general down-regulation of genes in the low abundance class. Similar results were found using mouse small intestinal crypt and villus cells, suggesting that the phenomenon also occurs in the intestine in vivo. The expression data were subsequently used in a search for markers for subsets of epithelial cells by performing reverse transcriptase–polymerase chain reaction on RNA extracted from laser dissected intestinal crypt and villi. In a screen of eight transcripts one – SART3 – was identified as a marker for human colonic crypts.
Keywords: Caco-2; Gene expression; DNA array; Differentiation; Intestine;

Gastric irritant-induced apoptosis in guinea pig gastric mucosal cells in primary culture by Shinji Tsutsumi; Wataru Tomisato; Tatsunori Takano; Kazuhito Rokutan; Tomofusa Tsuchiya; Tohru Mizushima (168-180).
When the gastric mucosa is exposed to various irritants, apoptosis and subsequent gastric mucosal lesion can result in vivo. We here show that gastric irritants induced apoptosis in gastric mucosal cells in primary culture and examined its molecular mechanism. Ethanol, hydrogen peroxide, and hydrochloric acid all induced, in a dose-dependent manner, cell death, apoptotic DNA fragmentation, and chromatin condensation, suggesting that each of these gastric irritants induced apoptosis in vitro. Since each of these irritants decreased the mitochondrial membrane potential and stimulated the release of cytochrome c from mitochondria, gastric irritant-induced apoptosis seems to be mediated by mitochondrial dysfunction. Caspase-3, caspase-8, and caspase-9-like activities were all activated simultaneously by each of these irritants and the activation was concomitantly with cell death and apoptotic DNA fragmentation. Furthermore, pre-treatment of gastric mucosal cells with an inhibitor of caspase-8 suppressed the onset of cell death as well as the stimulation of caspase-3- and caspase-9-like activities caused by each of these gastric irritants. Based on these results, we consider that caspase-8, an initiator caspase, plays an important role in gastric irritant-induced apoptosis.
Keywords: Apoptosis; Gastric mucosal cell; Caspase; Gastric irritant;

Characterization and location of Src-dependent tyrosine phosphorylation in rat brain mitochondria by Mauro Salvi; Anna Maria Brunati; Luciana Bordin; Nicoletta La Rocca; Giulio Clari; Antonio Toninello (181-195).
Analysis of protein phosphorylation in highly purified rat brain mitochondria revealed the presence of several alkali-stable phosphoproteins whose phosphorylation markedly increases upon treatment with peroxovanadate and Mn2+, a property indicating tyrosine phosphorylation. These include three prominent bands, with apparent sizes of 50, 60, and 75 kDa, which are detectable by anti-phosphotyrosine. Tyrosine phosphorylation disappears when mitochondria are treated with PP2, an inhibitor of the Src kinase family, suggesting the presence of members of this family in rat brain mitochondria. Immunoblotting and immunoprecipitation assays of mitochondrial lysates confirmed the presence of Fyn, Src and Lyn kinases, as well as Csk, a protein kinase which negatively controls the activity of the Src kinase family. Results show that tyrosine-phosphorylated proteins are membrane-bound and that they are located on the inner surface of the outer membrane and/or the external surface of the inner membrane. Instead, Src tyrosine kinases are mainly located in the intermembrane space – in particular, as revealed by immunogold experiments for Lyn kinase, in the cristal lumen. Rat brain mitochondria were also found to possess a marked level of tyrosine phosphatase activity, strongly inhibited by peroxovanadate.
Keywords: Mitochondria; Tyrosine kinase; Src kinase family;

Biochemical analysis of a T cell receptor α-like molecule involved in antigen-nonspecific suppression by Morihiko Nakamura; Tokugoro Tsunematsu; Yoshinori Tanigawa (196-202).
Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses a pleiotropic antigen-nonspecific suppressive function. We have shown that 70 kDa MNSF comprises an 8 kDa ubiquitin-like polypeptide (Ubi-L) and 62 kDa T cell receptor (TCR) α-like molecule. Ubi-L binds specifically to its 82 kDa receptor protein on target cells. In the current study, we have further characterized the biochemical nature of the TCRα-like molecule. The 62 kDa protein was separated into two species of 46 kDa and 16 kDa on reverse-phase HPLC. Anti-TCRα monoclonal antibody recognized the 46 kDa, but not the 16 kDa protein. Anti-TCRβ monoclonal antibody failed to recognize these proteins. Ubi-L conjugated to the 46 kDa protein, whereas Ubi-L lacking its C-terminal Gly-Gly did not. Although Ubi-L was labile both to heating at 56°C and to acidification to pH 4, the Ubi-L-46 kDa protein complex was unaffected by these treatments. In addition, the 46 kDa protein elongated the Ubi-L-induced protein tyrosine phosphorylation in a concanavalin A-activated murine T helper type 2 clone, D10 cells. One of the four tryptic peptide sequences derived from the 46 kDa protein was in alignment with a related sequence found in the Jα region of the TCRα, including the highly conserved motif F-G-X-G-T-X-L.
Keywords: Ubiquitin-like protein; Ubiquitin; T cell receptor; Suppressor factor;

To understand the transport mechanism of the bovine heart mitochondrial ADP/ATP carrier at the atomic level, we studied the four-dimensional features of the interaction of various purine nucleotides with the adenine nucleotide binding region (ABR) consisting of Arg151-Asp167 in the second loop facing the matrix side. After three-dimensional modeling of ABR based on the experimental results, its structural changes on interaction with purine nucleotides were examined by molecular dynamics computation at 300 K. ATP/ADP were translocated to a considerable degree from the matrix side to the inner membrane region accompanied by significant backbone conformational changes, whereas neither appreciable translocation nor a significant conformational change was observed with the untransportable nucleotides AMP/GTP. The results suggested that binding of the terminal phosphate group and the adenine ring of ATP/ADP with Arg151 and Lys162, respectively, and subsequent interaction of a phosphate group(s) other than the terminal phosphate with Lys162 triggered the expansion and subsequent contraction of the backbone conformation of ABR, leading to the translocation of ATP/ADP. Based on a simplified molecular dynamic simulation, we propose a dynamic model for the initial recognition process of ATP/ADP with the carrier.
Keywords: Subnanosecond order; Molecular dynamics; Conformational change; Transporter; Mitochondrial ADP/ATP carrier; Bovine heart mitochondria;

Molecular cloning and characterisation of p15CDK-BP, a novel CDK-binding protein by Lee Vogel; Blandine Baratte; Lénaı̈ck Détivaud; Lyamine Azzi; Pierre Leopold; Laurent Meijer (219-231).
The suc1/Cks proteins are well-conserved regulatory components of cyclin-dependent kinases 1 and 2 (CDK1/2). These small molecular mass proteins form a stable complex with CDK1/2 and are essential for normal regulation of CDKs during the cell division cycle and for degradation of p27kip1. Despite the high degree of homology between the nine known CDKs, only CDK1, CDK2 and, to a lesser extent, CDK3 are able to bind to the suc1/Cks proteins. No additional suc1/Cks-related proteins interacting with other CDKs have been reported. We have purified, from starfish oocytes, a 15 kDa protein, p15CDK-BP, which cross-reacts with anti-Cks antibodies (L. Azzi, L. Meijer, A.C. Ostvold, J. Lew, J.H. Wang, J. Biol. Chem. 269 (1994)). Following microsequencing of internal peptides and generation of corresponding oligonucleotides we cloned two cDNAs encoding two closely related proteins, p15A and p15B. The predicted protein sequences display distant but distinct homology with the Suc1/Cks proteins, including the genuine starfish Cks homologue protein, p9CksMg. P15 transcripts are essentially expressed in oocytes. Recombinant p15B or native p15CDK-BP bind a 34 kDa protein cross-reacting with anti-PSTAIRE antibodies, a feature characteristic of CDK-related proteins. In addition p15B interacts tightly with CDK4, CDK6, CDK8 and the yeast CDC28-related kinase Pho85, but not with CDK1, CDK2 or CDK7. P15 does not appear to alter the catalytic activity of the bound kinases.
Keywords: p15CDK-BP; suc1/Cks homologue; Cyclin-dependent kinase;