Over-expression of the p110beta but not p110alpha isoform of PI 3-kinase inhibits motility in breast cancer cells

Phosphoinositide 3-kinase (PI 3-kinase) activity is required for growth factor-induced cytoskeletal regulation and cell migration. We previously found that in MTLn3 rat adenocarcinoma cells, EGF-stimulated induction of actin barbed ends and lamellipod extension specifically requires the p85/p110alpha isoform of PI 3-kinase. To further characterize signaling by distinct PI 3-kinase isoforms, we have developed MTLn3 cells that transiently or stably overexpress either p110alpha or p110beta. Transient overexpression of p110beta inhibited EGF-stimulated lamellipod extension, whereas p110alpha-transfected cells showed normal EGF-stimulated lamellipod extension. Similar results were obtained by overexpression of kinase-dead p110beta, suggesting that effects on cytoskeletal signaling were due to competition with p85/p110alpha complexes. Stable overexpression of p110alpha appeared to be toxic, based on the difficulty in obtaining stable overexpressing clones. In contrast, cells expressing a 2-fold increase in p110beta were readily obtainable. Interestingly, cells stably expressing p110beta showed a marked inhibition of EGF-stimulated lamellipod extension. Using computer-assisted analysis of time-lapse images, we found that overexpression of p110beta caused a nearly complete inhibition of motility. Cells overexpressing p110beta showed normal activation of Akt and Erk, suggesting that overall PI 3-kinase signaling was intact. A chimeric p110 molecule containing the p85-binding and Ras-binding domains of p110alpha and the C2, helical, and kinase domains of p110beta, was catalytically active yet also inhibited EGF-stimulated lamellipod extension. These data highlight the differential signaling by distinct p110 isoforms. Identification of effectors that are differently regulated by p110alpha versus p110beta will be important for understanding cell migration and its role in metastasis.     

Differential subcellular localization of hZip1 in adherent and non-adherent cells.

Two human divalent cation transporters of the ZIP family, hZip1 and hZip2, homologous to Irt1 (Arabidopsis thaliana), the first identified member, have been described. They were shown by transfection into K562 cells to be localized at the plasma membrane and to mediate zinc uptake. Here we report a differential subcellular localization of hZip1 according to cell type. By transient expressions of EGFP-hZip1, FLAG-tagged or native hZip1, we observed that hZip1 has a vesicular localization in COS-7 cells or in several epithelial cell lines, corresponding partially to the endoplasmic reticulum. Using anti-hZip1 antibodies, we confirmed the intracellular localization of the endogenous protein in PC-3, a prostate cancer cell line.     

Selective regulation of neurite extension and synapse formation by the beta but not the alpha isoform of CaMKII

Neurite extension and branching are important neuronal plasticity mechanisms that can lead to the addition of synaptic contacts in developing neurons and changes in the number of synapses in mature neurons. Here we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates movement, extension, and branching of filopodia and fine dendrites as well as the number of synapses in hippocampal neurons. Only CaMKIIbeta, which peaks in expression early in development, but not CaMKIIalpha, has this morphogenic activity. A small insert in CaMKIIbeta, which is absent in CaMKIIalpha, confers regulated F-actin localization to the enzyme and enables selective upregulation of dendritic motility. These results show that the two main neuronal CaMKII isoforms have markedly different roles in neuronal plasticity, with CaMKIIalpha regulating synaptic strength and CaMKIIbeta controlling the dendritic morphology and number of synapses.     

Retinoic acid alters subcellular compartmentalization of ATP pools in 3T3 cells but not in HeLa cells

From the Chinese hamster ovary (CHO) cell, genetic variants (MonR-31 and MonR-32) relatively resistant to monensin, an ionophoric antibiotic, have been isolated. Growth of both MonR-31 and MonR-32 clones required higher doses of serum than CHO. Addition of insulin to media containing a low dose of serum restored full colony formation, but growth of MonR-31 or MonR-32 cells required more insulin than CHO cells. Specific binding of [125I]insulin was observed in these cell lines. The two MonR clones bound about one-half or less the [125I]insulin bound by CHO cells. Scatchard analysis for [125I]insulin binding at 4 degrees C and 37 degrees C showed altered number of binding sites, but not insulin affinity: The number of binding sites in the MonR cell was about a half or less that of the parental CHO cell. Down-regulation of insulin receptor was assayed when both CHO and MonR cells were incubated with 1 microgram/ml insulin. A 50-60% decrease in levels of insulin surface binding capacities was observed in CHO after exposure to insulin, whereas there was no decrease in MonR cell. The cellular uptake of 2-[3H]deoxyglucose into CHO cells was significantly enhanced in the presence of insulin, but only slight, if any, increase was observed in MonR cells.     

Differential isoform expression and interaction with the P32 regulatory protein controls the subcellular localization of the splicing factor U2AF26

The U2 auxiliary factor (U2AF) is an integral part of the spliceosome that is important for the recognition of the 3' splice site. U2AF consists of a large and a small subunit, the prototypes of which are U2AF65 and U2AF35. Recent evidence suggests that several homologs of both U2AF subunits exist that are able to regulate alternative splicing. Here we have investigated the expression, intracellular localization, and nucleo-cytoplasmic shuttling of one homolog of the small U2AF subunit, U2AF26, and a splice variant lacking exon 7, U2AF26DeltaE7. In contrast to the nuclear U2AF26, which displays active nucleo-cytoplasmic shuttling, U2AF26DeltaE7 is localized in the cytoplasm. Our studies reveal a nuclear localization sequence in the C-terminal exons 7 and 8 of U2AF26 that differs from the known nuclear localization sequence in U2AF35. In addition, we could identify P32 as a protein that is able to interact with U2AF26 through this domain, and we demonstrate that this interaction is required for the nuclear translocation of U2AF26. Our results suggest the existence of two distinct nuclear import pathways for U2AF26 and U2AF35 that could independently control their intracellular distribution and availability to the splicing machinery. Such a mechanism could work in addition to the differential expression of U2AF homologs to contribute to the regulation of alternative splicing.     

Fibulin-5 binds human smooth-muscle cells through alpha5beta1 and alpha4beta1 integrins, but does not support receptor activation

Fibulin-5, an extracellular matrix glycoprotein expressed in elastin-rich tissues, regulates vascular cell behaviour and elastic fibre deposition. Recombinant full-length human fibulin-5 supported primary human aortic SMC (smooth-muscle cell) attachment through alpha5beta1 and alpha4beta1 integrins. Cells on fibulin-5 spread poorly and displayed prominent membrane ruffles but no stress fibres or focal adhesions, unlike cells on fibronectin that also binds these integrins. Cell migration and proliferation were significantly lower on fibulin-5 than on fibronectin. Treatment of cells on fibulin-5 with a beta1 integrin-activating antibody induced stress fibres, increased attachment, migration and proliferation, and stimulated signalling of epidermal growth factor receptor and platelet-derived growth factor receptors alpha and beta. Fibulin-5 also modulated fibronectin-mediated cell spreading and morphology. We have thus identified the beta1 integrins on primary SMCs that fibulin-5 interacts with, and have shown that failure of fibulin-5 to activate these receptors limits cell spreading, migration and proliferation.     

Regulation of subcellular localization of alpha1-adrenoceptor subtypes

Alpha1-adrenergic receptors (AR) are members of the superfamily of G protein-coupled receptors (GPCRs) which mediate the effects of the sympathetic nervous system. Alpha1-AR comprise a heterogeneous family of three distinct isoforms of alpha1A, alpha1B and alpha1D; however, very little is known about their difference in physiological role or regulation. We have recently observed a subtype-specific differences in subcellular localization of alpha1-ARs; thus, alpha1A-AR predominantly localize intracellularly, while alpha1B-AR on the cell surface. To examine the molecular mechanism for the subtype-specific differences in subcellular localization, we conducted a search for novel proteins that interact with the alpha1B-AR, specifically focusing on the carboxyl-terminal cytoplasmic domain. Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R interacts with alpha1B-AR in vitro and in vivo through the specific site, and that in cells which co-express alpha1B-AR and gC1q-R, the subcellular localization of alpha1B-AR is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as well as the function of alpha1B-ARs.     

Specific subcellular localization of siRNAs delivered by lipoplex in MCF-7 breast cancer cells

In order to better understand the mechanism of delivery of siRNAs by lipid-based vectors, we investigated the subcellular distribution of siRNAs directed against cyclin D1 delivered by the DLS system in the breast cancer cell line MCF-7. Cells were treated with cyclopentenone or 17beta-estradiol to modulate the level of expression of cyclin D1 mRNA. We qualitatively observed that siRNA localized to specific cytoplasmic compartments in the periphery of the nucleus in granular-like structures that do not correspond to early endosomal vesicles. In cells treated with either cyclopentenone or 17beta-estradiol cellular distribution of siRNAs was not affected but variations in the amount of siRNAs present in cells were found. We suggest these variations might be associated with the effects of cyclopentenone and 17beta-estradiol in cyclin D1 gene expression. Low cytotoxicity and highly cellular uptake of lipoplexes was observed in the presence of serum indicating that the DLS system could be a useful tool for siRNA vectorization in vitro and in vivo.     

Protein subcellular localization profiling of breast cancer cells by dissociable antibody microarray staining

We have developed dissociable antibody microarray (DAMA) staining technology that provides a new approach to the global analysis of protein subcellular localization (SCL) in fixed cells. We have developed and optimized this technology for protein SCL profiling, generated ChipView, a program for management and analysis of molecular image database, and utilized the technique to identify proteins with unique SCL in breast cancer cell lines. We compared the SCL profiles of 325 proteins among nine different breast cell lines, and have identified one protein, Cyclin B1, with distinctively different SCLs between normal and cancer cell lines. With classic individual immunostaining, Cyclin B1 was confirmed to localize to the cytoplasm of seven breast cancer cell lines and in both cytoplasm and nuclei of two normal breast cell lines, and to have higher expression levels in the cancer cell lines tested.     

Differential subcellular targeting and activity-dependent subcellular localization of diacylglycerol kinase isozymes in transfected cells

Diacylglycerol kinase (DGK) plays a pivotal role in cellular signal transduction through regulating levels of the second messenger diacylglycerol (DG). Previous studies have revealed that DGK is composed of a family of isozymes that show remarkable heterogeneity in terms of molecular structure, functional domains, tissue and cellular gene expression. Recently, it has been shown that DG is produced in various subcellular compartments including the plasma membrane, internal membranes, cytoskeleton, and nucleus. However, it remains unclear how DG is regulated at distinct subcellular sites. To address this point, we have used an epitope-tag expression system in cultured cells and investigated the subcellular localization of DGK isozymes under the same experimental conditions. We show here that DGK isozymes are targeted differentially to unique subcellular sites in transfected COS7 cells, including the cytoplasm, actin stress fibers, Golgi complex, endoplasmic reticulum, and nucleus. It is also shown that among the isozymes overexpression of DGKbeta causes fragmentation of actin stress fibers while a kinase-dead mutant of DGKbeta abolishes its colocalization with actin stress fibers. These data strongly suggest that each isozyme may be responsible for the metabolism of DG that is produced upon stimulation at a different and specific subcellular site and that DGKbeta activity might have effects on the reorganization of actin stress fibers in transfected COS7 cells.     

TAB1beta (transforming growth factor-beta-activated protein kinase 1-binding protein 1beta ), a novel splicing variant of TAB1 that interacts with p38alpha but not TAK1

The mitogen-activated protein kinases (MAPKs) play an important role in a variety of biological processes. Activation of MAPKs is mediated by phosphorylation on specific regulatory tyrosine and threonine sites. We have recently found that activation of p38alpha MAPK can be carried out not only by its upstream MAPK kinases (MKKs) but also by p38alpha autophosphorylation. p38alpha autoactivation requires an interaction of p38alpha with TAB1 (transforming growth factor-beta-activated protein kinase 1-binding protein 1). The autoactivation mechanism of p38alpha has been found to be important in cellular responses to a number of physiologically relevant stimuli. Here, we report the characterization of a splicing variant of TAB1, TAB1beta. TAB1 and TAB1beta share the first 10 exons. The 11th and 12th exons of TAB1 were spliced out in TAB1beta, and an extra exon, termed exon beta, downstream of exons 11 and 12 in the genome was used as the last exon in TAB1beta. The mRNA of TAB1beta was expressed in all cell lines examined. The TAB1beta mRNA encodes a protein with an identical sequence to TAB1 except the C-terminal 69 amino acids were replaced with an unrelated 27-amino acid sequence. Similar to TAB1, TAB1beta interacts with p38alpha but not other MAPKs and stimulates p38alpha autoactivation. Different from TAB1, TAB1beta does not bind or activate TAK1. Inhibition of TAB1beta expression with RNA interference in MDA231 breast cancer cells resulted in the reduction of basal activity of p38alpha and invasiveness of MDA231 cells, suggesting that TauAlphaBeta1beta is involved in regulating p38alpha activity in physiological conditions.     

Hypotonic cell swelling induces translocation of the alpha isoform of cytosolic phospholipase A2 but not the gamma isoform in Ehrlich ascites tumor cells.

We demonstrate that two isoforms of the cytosolic phospholipase A2, cPLA2alpha and cPLA2gamma, are present in Ehrlich ascites tumor cells. Both enzymes are almost uniformly distributed throughout the cells under control conditions, as visualized by laser-scanning confocal microscopy. Stimulation by either hypotonic cell swelling or addition of the Ca2+ ionophore A23187 results in translocation of cPLA2alpha, but not cPLA2gamma, to the nucleus, where it forms hot-spot-like clusters. Our group previously showed that release of radioactively labeled arachidonic acid, incorporated into the phospholipids of Ehrlich cells, was immediately and transiently increased on hypotonic cell swelling [Thoroed, S.M., Lauritzen, L., Lambert, I.H., Hansen, H.S. & Hoffmann, E.K. (1997) J. Membr. Biol. 160, 47-58]. We now demonstrate that arachidonic acid is released from the nuclear fraction following hypotonic exposure. Stimulation of Ehrlich cells with A23187 also leads to an increase in arachidonic acid release from the nucleus. However, as hypotonic cell swelling is not accompanied by any detectable increase in intracellular concentration of free cytosolic Ca2+ ([Ca2+]i), stimulus-induced translocation of cPLA2alpha can also occur without elevation of [Ca2+]i. The stimulus-induced translocation of cPLA2alpha appears not to be prevented by inhibition of mitogen-activated protein (MAP) kinase activation, p38 MAP kinase, tyrosine kinases and protein kinase C, hence, phosphorylation is not crucial for the stimulus-induced translocation of cPLA2alpha. Disruption of F-actin did not affect the translocation process, thus, an intact F-actin cytoskeleton does not seem to be required for translocation of cPLA2alpha.     

NDPK-A (but not NDPK-B) and AMPK alpha1 (but not AMPK alpha2) bind the cystic fibrosis transmembrane conductance regulator in epithelial cell membranes

Cystic fibrosis (CF) results from mutations within the cystic fibrosis transmembrane-conductance regulator (CFTR) protein. The AMP-activated protein kinase (AMPK) is a heterotrimer composed of different isoforms of the alphabetagamma subunits, where the alpha1 catalytic subunit binds CFTR. Nucleoside diphosphate kinase (NDPK, NM23/awd) converts nucleoside diphosphates to nucleoside triphosphates but also acts as a protein kinase. We recently showed that AMPK alpha1 binds NDPK-A in lung epithelial cytosol. Here we report that in the plasma membrane of human airway epithelial cells, NDPK-A and AMPK alpha1 associate with the plasma membrane via CFTR. We show that the regulatory domain of CFTR binds NDPK-A whereas AMPK gamma1 or gamma2 bind the first nucleotide binding domain (NBD1) and AMPK alpha1 binds the second (NBD2) of CFTR. We also show that NDPK-A specifically binds AMPK alpha1 and AMPK gamma2 subunits, thereby specifying the isozyme of AMPK heterotrimer that associates with CFTR at the membrane. Thus, the combined data provide novel insight into the subunit composition of the epithelial CFTR/AMPK/NDPK complex, such that: CFTR interacts specifically with AMPK alpha1, gamma2 and NDPK-A and not NDPK-B or AMPK gamma1.