The human SWI/SNF-B chromatin-remodeling complex is related to yeast Rsc and localizes at kinetochores of mitotic chromosomes

The SWI/SNF family of chromatin-remodeling complexes facilitates gene expression by helping transcription factors gain access to their targets in chromatin. SWI/SNF and Rsc are distinctive members of this family from yeast. They have similar protein components and catalytic activities but differ in biological function. Rsc is required for cell cycle progression through mitosis, whereas SWI/SNF is not. Human complexes of this family have also been identified, which have often been considered related to yeast SWI/SNF. However, all human subunits identified to date are equally similar to components of both SWI/SNF and Rsc, leaving open the possibility that some or all of the human complexes are rather related to Rsc. Here, we present evidence that the previously identified human SWI/SNF-B complex is indeed of the Rsc type. It contains six components conserved in both Rsc and SWI/SNF. Importantly, it has a unique subunit, BAF180, that harbors a distinctive set of structural motifs characteristic of three components of Rsc. Of the two mammalian ATPases known to be related to those in the yeast complexes, human SWI/SNF-B contains only the homolog that functions like Rsc during cell growth. Immunofluorescence studies with a BAF180 antibody revealed that SWI/SNF-B localizes at the kinetochores of chromosomes during mitosis. Our data suggest that SWI/SNF-B and Rsc represent a novel subfamily of chromatin-remodeling complexes conserved from yeast to human, and could participate in cell division at kinetochores of mitotic chromosomes.

In vivo anergized CD4+ T cells express perturbed AP-1 and NF-kappa B transcription factors.

Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell receptor (TCR) variable region beta-chain families and induces strong and rapid production of interleukin 2 (IL-2). In contrast, repeated injections of SEA cause CD4+ T-cell deletion and anergy in the remaining CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. We analyzed expression of AP-1, NF-kappa B, NF-AT, and octamer binding transcription factors, which are known to be involved in the regulation of IL-2 gene promoter activity. Large amounts of AP-1 and NF-kappa B and significant quantities of NF-AT were induced in SEA-activated CD4+ spleen T cells, whereas Oct-1 and Oct-2 DNA binding activity was similar in both resting and activated T cells. In contrast, anergic CD4+ T cells contained severely reduced levels of AP-1 and Fos/Jun-containing NF-AT complexes but expressed significant amounts of NF-kappa B and Oct binding proteins after SEA stimulation. Resolution of the NF-kappa B complex demonstrated predominant expression of p50-p65 heterodimers in activated CD4+ T cells, while anergic cells mainly expressed the transcriptionally inactive p50 homodimer. These alterations of transcription factors are likely to be responsible for repression of IL-2 in anergic T cells.

An inhibitor of p34cdc2/cyclin B that regulates the G2/M transition in Xenopus extracts.

The activity of maturation-promoting factor (MPF), a protein kinase complex composed of p34cdc2 and cyclin B, is undetectable during interphase but rises abruptly at the G2/M transition to induce mitosis. After the synthesis of cyclin B, the suppression of MPF activity before mitosis has been attributed to the phosphorylation of p34cdc2 on sites (threonine-14 and tyrosine-15) that inhibit its catalytic activity. We previously showed that the activity of the mitotic p34cdc2/cyclin B complex is rapidly suppressed when added to interphase Xenopus extracts that lack endogenous cyclin B. Here we show that a mutant of p34cdc2 that cannot be inhibited by phosphorylation (threonine-14-->alanine, tyrosine-15-->phenylalanine) is also susceptible to inactivation, demonstrating that inhibitory mechanisms independent of threonine-14 and tyrosine-15 phosphorylation must exist. We have partially characterized this inhibitory pathway as one involving a reversible binding inhibitor of p34cdc2/cyclin B that is tightly associated with cell membranes. Kinetic analysis suggests that this inhibitor, in conjunction with the kinases that mediate the inhibitory phosphorylations on p34cdc2, maintains the interphase state in Xenopus; it may play an important role in the exact timing of the G2/M transition.

14-3-3 Proteins Act as Negative Regulators of the Mitotic Inducer Cdc25 in Xenopus Egg Extracts

Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2–cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3ε and to a lesser extent with 14-3-3ζ. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G2–M transition.

Positive Regulation of Wee1 by Chk1 and 14-3-3 Proteins

Wee1 inactivates the Cdc2–cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, it has been established previously that Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. We report that Xwee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1.

Cytoplasmic dynein is associated with slow axonal transport.

Neuronal function is dependent on the transport of materials from the cell body to the synapse via anterograde axonal transport. Anterograde axonal transport consists of several components that differ in both rate and protein composition. In fast transport, membranous organelles are moved along microtubules by the motor protein kinesin. The cytoskeleton and the cytomatrix proteins move in the two components of slow transport. While the mechanisms underlying slow transport are unknown, it has been hypothesized that the movement of microtubules in slow transport is generated by sliding. To determine whether dynein, a motor protein that causes microtubule sliding in flagella, may play a role in slow axonal transport, we identified the transport rate components with which cytoplasmic dynein is associated in rat optic nerve. Nearly 80% of the anterogradely moving dynein was associated with slow transport, whereas only approximately 15% of the dynein was associated with the membranous organelles of anterograde fast axonal transport. A segmental analysis of the transport of dynein through contiguous regions of the optic nerve and tract showed that dynein is associated with the microfilaments and other proteins of slow component b. Dynein from this transport component has the capacity to bind microtubules in vitro. These results are consistent with the hypothesis that cytoplasmic dynein generates the movement of microtubules in slow axonal transport. A model is presented to illustrate how dynein attached to the slow component b complex of proteins is appropriately positioned to generate force of the correct polarity to slide microtubules down the axon.

Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation

Antigen presentation by major histocompatibility complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class II-associated invariant chain (Ii). Thus far, only the cysteine protease cathepsin (Cat) S appears essential for complete destruction of Ii. The enzymes involved in degradation of the antigens themselves remain to be identified. Degradation of antigens in vitro and experiments using protease inhibitors have suggested that Cat B and Cat D, two major aspartyl and cysteine proteases, respectively, are involved in antigen degradation. We have analyzed the antigen-presenting properties of cells derived from mice deficient in either Cat B or Cat D. Although the absence of these proteases provoked a modest shift in the efficiency of presentation of some antigenic determinants, the overall capacity of Cat B−/− or Cat D−/− antigen-presenting cells was unaffected. Degradation of Ii proceeded normally in Cat B−/− splenocytes, as it did in Cat D−/− cells. We conclude that neither Cat B nor Cat D are essential for MHC class II-mediated antigen presentation.

Low density lipoprotein receptor-negative mice expressing human apolipoprotein B-100 develop complex atherosclerotic lesions on a chow diet: No accentuation by apolipoprotein(a)

We have generated mice with markedly elevated plasma levels of human low density lipoprotein (LDL) and reduced plasma levels of high density lipoprotein. These mice have no functional LDL receptors [LDLR−/−] and express a human apolipoprotein B-100 (apoB) transgene [Tg(apoB+/+)] with or without an apo(a) transgene [Tg(apoa+/−)]. Twenty animals (10 males and 10 females) of each of the following four genotypes were maintained on a chow diet: (i) LDLR−/−, (ii) LDLR−/−;Tg(apoa+/−), (iii) LDLR−/−;Tg(apoB+/+), and (iv)LDLR−/−;Tg(apoB+/+);Tg(apo+/−). The mice were killed at 6 mo, and the percent area of the aortic intimal surface that stained positive for neutral lipid was quantified. Mean percent areas of lipid staining were not significantly different between the LDLR−/− and LDLR−/−;Tg(apoa+/−) mice (1.0 ± 0.2% vs. 1.4 ± 0.3%). However, the LDLR−/−;Tg(apoB+/+) mice had ≈15-fold greater mean lesion area than the LDLR−/− mice. No significant difference was found in percent lesion area in the LDLR−/−;Tg(apoB+/+) mice whether or not they expressed apo(a) [18.5 ± 2.5%, without lipoprotein(a), Lp(a), vs. 16.0 ± 1.7%, with Lp(a)]. Histochemical analyses of the sections from the proximal aorta of LDLR−/−;Tg(apoB+/+) mice revealed large, complex, lipid-laden atherosclerotic lesions that stained intensely with human apoB-100 antibodies. In mice expressing Lp(a), large amounts of apo(a) protein colocalized with apoB-100 in the lesions. We conclude that LDLR−/−; Tg(apoB+/+) mice exhibit accelerated atherosclerosis on a chow diet and thus provide an excellent animal model in which to study atherosclerosis. We found no evidence that apo(a) increased atherosclerosis in this animal model.

The Yeast Dynactin Complex Is Involved in Partitioning the Mitotic Spindle between Mother and Daughter Cells during Anaphase B

Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability to translocate purified vesicles on microtubules depends on the presence of an accessory complex known as dynactin. We have cloned and characterized a novel gene, NIP100, which encodes the yeast homologue of the vertebrate dynactin complex protein p150glued. Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Δ strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell. Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck. Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase. Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p. Finally, we find that a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle. Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase.

Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation

The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH2) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO2, generating the γ-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684–1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH2) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH2 preincubation. Amino acid analysis of 14C- N-ethyl maleimide-modified human carboxylase revealed 1.8–2.3 reactive residues and a specific activity of 7 × 108 cpm/hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0.2% (Cys-99) or 1% (Cys-450), and increased the Kms for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH2 oxygenation.

Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-κB

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between −5.2 and −6.1 kb in the 5′-flanking hiNOS promoter DNA, which contains multiple nuclear factor κβ (NF-κB) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-α, interleukin-1β, and IFN-γ (TNF-α, IL-1β, and IFN-γ) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at −5.8 kb in the hiNOS promoter identifies a bifunctional NF-κB/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at −5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-κB activates hiNOS promoter–reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-κB function in these cells. These results indicate that both Stat 1 and NF-κB are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.

Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.

Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 125I-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B12 but no measurable affinity of the vitamin complex for the homologous alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor related protein (LRP). 125I-TC-B12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of 125I-TC-B12. The TC-B12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B12 homeostasis and fetal vitamin B12 supply.

Evidence for a preformed transducer complex organized by the B cell antigen receptor.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer, which functions as signaling subunit of the receptor. Stimulation of the BCR activates protein tyrosine kinases (PTKs) that phosphorylate a number of substrate proteins, including the Ig-alpha/Ig-beta heterodimer of the BCR itself. How the PTKs become activated after BCR engagement is not known at present. Here, we show that BCR-negative J558L cells treated with the protein tyrosine phosphatase inhibitor pervanadate/H2O2 display only a weak substrate phosphorylation. However, in BCR-positive transfectants of J558L, treatment with pervanadate/H2O2 induces a strong phosphorylation of several substrate proteins. Treatment with pervanadate/H2O2 does not result in receptor crosslinking, yet the pattern of protein phosphorylation is similar to that observed after BCR stimulation by antigen. The response requires cellular integrity because tyrosine phosphorylation of most substrates is not visible in cell lysates. Cells that express a BCR containing an Ig-alpha subunit with a mutated immunoreceptor tyrosine-based activation motif display a delayed response. The data suggest that, once expressed on the surface, the BCR organizes protein tyrosine phosphatases, PTKs, and their substrates into a transducer complex that can be activated by pervanadate/H202 in the absence of BCR crosslinking. Assembly of this preformed complex seems to be a prerequisite for BCR-mediated signal transduction.

Ultraviolet-B photodestruction of a light-harvesting complex.

Cyanobacteria are important contributors to global photosynthesis in both marine and terrestrial environments. Quantitative data are presented on UV-B-induced damage to the major cyanobacterial photosynthetic light harvesting complex, the phycobilisome, and to each of its constituent phycobiliproteins. The photodestruction quantum yield, phi295 nm, for the phycobiliproteins is high (approximately 10(-3), as compared with approximately 10(-7) for visible light). Energy transfer on a picosecond time scale does not compete with photodestruction. Photodamage to phycobilisomes in vitro and in living cells is amplified by causing dissociation and loss of function of the complex. In photosynthetic organisms, UV-B damage to light-harvesting complexes may significantly exceed that to DNA.