Characterization of 14-3-3sigma Dimerization Determinants: Requirement of Homodimerization for Inhibition of Cell Proliferation

The seven highly conserved 14-3-3 proteins expressed in mammalian cells form a complex pattern of homo- and hetero-dimers, which is poorly characterized. Among the 14-3-3 proteins 14-3-3sigma is unique as it has tumor suppressive properties. Expression of 14-3-3sigma is induced by DNA damage in a p53-dependent manner and mediates a cell cycle arrest. Here we show that the 14-3-3sigma protein exclusively forms homodimers when it is ectopically expressed at high levels, whereas ectopic 14-3-3zeta formed heterodimers with the 5 other 14-3-3 isoforms. The x-ray structure of 14-3-3sigma?revealed 5 residues (Ser⁵, Glu²⁰, Phe²⁵, Q⁵⁵, Glu⁸⁰) as candidate determinants of dimerization specificity. Here we converted these amino-acids to residues present in 14-3-3zeta at the analogous positions. Thereby, Ser⁵, Glu²⁰ and Glu⁸⁰ were identified as key residues responsible for the selective homodimerization of 14-3-3sigma. Conversion of all 5 candidate residues was sufficient to switch the dimerization pattern of 14-3-3sigma to a pattern which is very similar to that of 14-3-3zeta. In contrast to wildtype 14-3-3sigma this 14-3-3sigma variant and 14-3-3zeta were unable to mediate inhibition of cell proliferation. Therefore, homodimerization by 14-3-3sigma is required for its unique functions among the 7 mammalian 14-3-3 proteins. As inactivation of 14-3-3sigma sensitizes to DNA-damaging drugs, substances designed to interfere with 14-3-3sigma dimerization may be used to inactivate 14-3-3sigma function for cancer therapeutic purposes.     

Humanized OKT3 antibodies: successful transfer of immune modulating properties and idiotype expression.

Antibodies that possess the Ag-binding regions of OKT3 within the context of a human framework (Hu-OKT3 Ab) offer distinct advantages for optimizing anti-CD3 mAb therapy. First, manipulation of Ab genes to produce humanized Ab that retain Ag-binding activity may circumvent antigenicity problems. Second, Ab gene engineering provides a means for modifying functional properties, including T cell activation and immune suppression. The purpose of this study was to determine the functional properties of Hu-OKT3 Ab and to compare the functional properties and idiotypes of Hu-OKT3 Ab to those of murine OKT3. Three Hu-OKT3 IgG4 Ab, a chimeric OKT3 antibody (cOKT3-1) (grafted sequences comprising all OKT3 VH and VL regions) and two complementarity determining region (CDR)-grafted antibodies, gOKT3-5 and gOKT3-6 (grafted sequences comprising only OKT3 VH and VL CDR and some framework amino acids, were analyzed. Initial studies demonstrated that the cOKT3 and gOKT3-5 Ab bound selectively to T cells and competitively inhibited OKT3-FITC binding with avidities similar to that of murine OKT3. Binding avidity of the gOKT3-6 Ab was markedly less than that of the other two Hu-OKT3 Ab. Serologic analysis suggested that cOKT3 and gOKT3-5 Ab possess idiotypes (combining sites) similar to murine OKT3. T cell activation potency of all three Hu-OKT3 Ab was assessed by proliferation, induction of activation marker expression (IL-2R and Leu 23), and lymphokine production (TNF-alpha and IFN-gamma). The cOKT3 and gOKT3-5 Ab demonstrated T cell activation potencies similar to murine OKT3 as assessed by each parameter. CD3 coating and modulation by these two Ab was effective but somewhat less potent than that observed with OKT3. Finally, cOKT3 and gOKT3-5 Ab both inhibited CTL activity comparably to murine OKT3. In conclusion, these studies indicate that gOKT3-5 and cOKT3 Ab possess immune modulating properties similar to murine OKT3 and thus offer attractive alternatives to murine OKT3 for in vivo therapy.     

MIP-T3, a novel protein linking tumor necrosis factor receptor-associated factor 3 to the microtubule network

In this study, we report the identification of a novel tumor necrosis factor receptor-associated factor 3 (TRAF3)-interacting protein designated MIP-T3. MIP-T3 is a 83-kDa protein with no significant homology to known mammalian proteins. MIP-T3 mRNA and TRAF3 mRNA are ubiquitously expressed, and TRAF3 is the only TRAF protein to interact with MIP-T3. The MIP-T3-TRAF3 interaction requires the coiled-coil TRAF-N domain of TRAF3. To our knowledge, this is the first case of a TRAF-binding protein that interacts with a single member of the TRAF family specifically through a TRAF-N coiled-coil domain. MIP-T3 binds to Taxol-stabilized microtubules and to tubulin in vitro, and MIP-T3 recruits TRAF3 to microtubules when both proteins are overexpressed in HeLa cells. In a 293 cell line stably expressing CD40, TRAF3 is released from the TRAF3.MIP-T3 complex and recruited to the CD40 receptor upon CD40 ligand stimulation. MIP-T3 may provide a novel mechanism in sequestering TRAF3 to the cytoskeletal network.     

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.     

Action of the C3b-inactivator on the cell-bound C3b.

The action of C3bINA and beta 1H on cell-bound C3b is described in this paper. The alpha-polypeptide of C3b that binds covalently to cell surfaces is cleaved by the C3bINA and beta 1H into two fragments: one of 60,000 (C3b alpha-60) and another of 40,000 (C3b alpha-40) daltons. The beta-chain of C3b is unaffected by the C3bINA and beta 1H. The three polypeptides, C3b alpha-60, C3b alpha-40, and C3 beta, are held together as a single unit by disulfide bonds. This unit, referred to as C3b' is covalently bound to cell surfaces via the C3b alpha-60 polypeptide. The conversion of C3b to C3b' by C3bINA and beta 1H abolishes the ability of the C3b-bearing cells to adhere to human erythrocytes as well as the ability to form, on the cell surface, the B, D, and properdin-dependent amplification C3-convertase. However, the agglutinability of the cells with either anti-C3c or anti-C3d is not affected. Treatment of the C3b'-bearing cells with trypsin releases fragments of C3b' into solution, leaving a polypeptide of 32,000 daltons covalently linked to the membrane. Since the trypsinized cells are agglutinable by anti-C3d but not by anti-C3c, the 32,000 dalton polypeptide appears to correspond antigenically to C3d.     

How to Interpret LC3 Immunoblotting

Microtubule-associated protein light chain 3 (LC3) is now widely used to monitor autophagy. One approach is to detect LC3 conversion (LC3-I to LC3-II) by immunoblot analysis because the amount of LC3-II is clearly correlated with the number of autophagosomes. However, LC3-II itself is degraded by autophagy, making interpretation of the results of LC3 immunoblotting problematic. Furthermore, the amount of LC3 at a certain time point does not indicate autophagic flux, and therefore, it is important to measure the amount of LC3-II delivered to lysosomes by comparing LC3-II levels in the presence and absence of lysosomal protease inhibitors. Another problem with this method is that LC3-II tends to be much more sensitive to be detected by immunoblotting than LC3-I. Accordingly, simple comparison of LC3-I and LC3-II, or summation of LC3-I and LC3-II for ratio determinations, may not be appropriate, and rather, the amount of LC3-II can be compared between samples.     

Significance of 14-3-3 self-dimerization for phosphorylation-dependent target binding

14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other.     

The fission yeast ortholog of eIF3a subunit is not functional inSaccharomyces cerevisiae

The Schizosaccharomyces pombe eIF3a ortholog (SpeIF3a) was shown to be unable to substitute for S. cerevisiae eIF3a (SceIF3a) in its essential function in the initiation of translation. Overproduction of SpeIF3a altered the distribution of SceIF3a but formation of the endogenous eIF3 complex was not affected. SpeIF3a was found to be more tightly bound to S. cerevisiae ribosomes than SceIF3a and other eIF3 subunits (eIF3g, eIF3i, eIF3j). The host cells displayed aberrant morphology and altered chitin deposition. SpeIF3a probably competes with SceIF3a for binding to either ribosomes or yet to be identified substrates.     

Inhibition of DNA synthesis in SV3T3 cultures by isolated 3T3 plasma membranes

3T3 plasma membranes were added to subconfluent cultures of SV3T3 cells in the presence of fusogens. If this protocol results in the introduction into the SV3T3 cell membrane of 3T3 plasma membrane components responsible for density-dependent inhibition of growth, then the SV3T3 cell cultures would be expected to show decreased rates of DNA synthesis as they approach confluence. Results of these experiments indicate that rates of DNA synthesis in SV3T3 cultures so treated were as much as 63% less than in untreated controls. This effect could not be attributed to the fusogens or to the 3T3 plasma membranes alone. This growth-inhibitory effect is specific for 3T3 membranes and is not observed when SV3T3 plasma membranes are fused with SV3T3 cell cultures. These data support the hypothesis that one aspect of the loss of density-dependent inhibition of growth in SV3T3 cells is a deletion or alteration in plasma membrane components and, further, that density- dependent inhibition of growth can be in part restored to SV3T3 cell cultures by fusing the cells with 3T3 plasma membranes.     

Methylation-directed acetylation of histone H3 regulates developmental sensitivity to histone deacetylase inhibition

Hydroxamate-based lysine deacetylase inhibitors (KDACis) are approved for clinical use against certain cancers. However, intrinsic and acquired resistance presents a major problem. Treatment of cells with hydroxamates such as trichostatin A (TSA) leads to rapid preferential acetylation of histone H3 already trimethylated on lysine 4 (H3K4me3), although the importance of this H3K4me3-directed acetylation in the biological consequences of KDACi treatment is not known. We address this utilizing Dictyostelium discoideum strains lacking H3K4me3 due to disruption of the gene encoding the Set1 methyltransferase or mutations in endogenous H3 genes. Loss of H3K4me3 confers resistance to TSA-induced developmental inhibition and delays accumulation of H3K9Ac and H3K14Ac. H3K4me3-directed H3Ac is mediated by Sgf29, a subunit of the SAGA acetyltransferase complex that interacts with H3K4me3 via a tandem tudor domain (TTD). We identify an Sgf29 orthologue in Dictyostelium with a TTD that specifically recognizes the H3K4me3 modification. Disruption of the gene encoding Sgf29 delays accumulation of H3K9Ac and abrogates H3K4me3-directed H3Ac. Either loss or overexpression of Sgf29 confers developmental resistance to TSA. Our results demonstrate that rapid acetylation of H3K4me3 histones regulates developmental sensitivity to TSA. Levels of H3K4me3 or Sgf29 will provide useful biomarkers for sensitivity to this class of chemotherapeutic drug.     

Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.     

The 14-3-3γ isoform binds to and regulates the localization of endoplasmic reticulum (ER) membrane protein TMCC3 for the reticular network of the ER

The reticular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions and undergoes constant remodeling through formation and loss of the three-way junctions. Transmembrane and coiled-coil domain family 3 (TMCC3), an ER membrane protein localizing at three-way junctions, has been shown to positively regulate formation of the reticular ER network. However, elements that negatively regulate TMCC3 localization have not been characterized. In this study, we report that 14-3-3γ, a phospho-serine/phospho-threonine-binding protein involved in various signal transduction pathways, is a negative regulator of TMCC3. We demonstrate that overexpression of 14-3-3γ reduced localization of TMCC3 to three-way junctions and decreased the number of three-way junctions. TMCC3 bound to 14-3-3γ through the N terminus and had deduced 14-3-3 binding motifs. Additionally, we determined that a TMCC3 mutant substituting alanine for serine to be phosphorylated in the binding motif reduced binding to 14-3-3γ. The TMCC3 mutant was more prone than wildtype TMCC3 to localize at three-way junctions in the cells overexpressing 14-3-3γ. Furthermore, the TMCC3 mutant rescued the ER sheet expansion caused by TMCC3 knockdown less than wild-type TMCC3. Taken together, these results indicate that 14-3-3γ binding negatively regulates localization of TMCC3 to the three-way junctions for the proper reticular ER network, implying that the negative regulation of TMCC3 by 14-3-3γ would underlie remodeling of the reticular network of the ER.     

Murine Sarcoma and Leukemia Viruses: Assay Using Clonal Lines of Contact-Inhibited Mouse Cells

A continuous cell line of highly contact-inhibited cells (NIH/3T3) has been developed from NIH Swiss mouse embryo cultures. Its growth properties are similar to those of 3T3 and BALB/3T3. Although 3T3 is relatively insensitive to focus formation by murine sarcoma viruses, cloned lines of both NIH/3T3 and BALB/3T3 have been isolated that are highly sensitive to sarcoma virus focus formation and leukemia virus growth. The sensitivity and specificity are comparable to those found with primary embryo cells. MSV-transformed lines of NIH/3T3 have been obtained.     

Metabolism of alpha-linolenic acid in humans

Alpha-linolenic acid (18:3n-3) is essential in the human diet, probably because it is the substrate for the synthesis of longer-chain, more unsaturated n-3 fatty acids eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) which are required for tissue function. This article reviews the recent literature on 18:3n-3 metabolism in humans, including fatty acid beta-oxidation, recycling of carbon by fatty acid synthesis de novo and conversion to longer-chain polyunsaturated fatty acids (PUFA). In men, stable isotope tracer studies and studies in which volunteers increased their consumption of 18:3n-3 show conversion to 20:5n-3 and 22:5n-3, but limited conversion to 22:6n-3. However, conversion to 18:3n-3 to 20:5n-3 and 22:6n-3 is greater in women compared to men, due possibly to a regulatory effect of oestrogen, while partitioning of 18:3n-3 towards beta-oxidation and carbon recycling was lower than in men. These gender differences may be an important consideration in making dietary recommendations for n-3 PUFA intake.     

Binding, internalization, and degradation of epidermal growth factor by balb 3T3 and BP3T3 cells: relationship to cell density and the stimulation of cell proliferation.

Epidermal growth factor (EGF) stimulates the growth of both benzo[a]pyrene-transformed Balb 3T3 cells (BP3T3) and untransformed Balb 3T3 cells. We describe here the binding, internalization, and degradation of [125I]-EGF by BP3T3 cells and 3T3 cells. Binding of [125I]-EGF reaches a maximum after 45 to 90 minutes incubation at 37 degrees C. In both BP3T3 and 3T3 cells the extent of EGF binding required to stimulate DNA synthesis is density dependent; sparse cultures require a 15-30% occupancy to elicit a maximal response whereas dense cultures require a 70-85% occupancy. At physiological concentrations the total binding of [125I]-EGF to 3T3 cells is higher than to BP3T3 cells, and this difference increases at higher cell densities. The rate of degradation of [125I]-EGF is directly proportional to the total [125I]-EGF binding in each cell type. This supports the hypothesis that one cause of the diminished serum requirement of BP3T3 cells is a reduced rate of utilization of serum growth factors.     

Overexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli and provides enhanced osmotic stress tolerance and ABA sensitivity in Arabidopsis thaliana

Previous studies have shown that the late embryogenesis abundant (LEA) group 3 proteins significantly respond to changes in environmental conditions. However, reports that demonstrate their biological role, especially in Arabidopsis, are notably limited. This study examines the functional roles of the Arabidopsis LEA group 3 proteins AtLEA3-3 and AtLEA3-4 in abiotic stress and ABA treatments. Expression of AtLEA3-3 and AtLEA3-4 is upregulated by ABA, high salinity, and osmotic stress. Results on the ectopic expression of AtLEA3-3 and AtLEA3-4 in E. coli suggest that both proteins play important roles in resistance to cold stress. Overexpression of AtLEA3-3 in Arabidopsis (AtLEA3-3-OE) confers salt and osmotic stress tolerance that is characterized during germination and early seedling establishment. However, AtLEA3-3-OE lines show sensitivity to ABA treatment during early seedling development. These results suggest that accumulation of AtLEA3-3 mRNA and/or proteins may help heterologous ABA re-initiate second dormancy during seedling establishment. Analysis of yellow fluorescent fusion proteins localization shows that AtLEA3-3 and AtLEA3-4 are mainly distributed in the ER and that AtLEA3-3 also localizes in the nucleus, and in response to salt, mannitol, cold, or BFA treatments, the localization of AtLEA3-3 and AtLEA3-4 is altered and becomes more condensed. Protein translocalization may be a positive and effective strategy for responding to abiotic stresses. Taken together, these results suggest that AtLEA3-3 has an important function during seed germination and seedling development of Arabidopsis under abiotic stress conditions.