The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation.

The phenotypes of CD19-deficient (CD19-/-) mice, and human CD19-transgenic (hCD19TG) mice that overexpress CD19 indicate that CD19 is a response regulator of B-lymphocyte surface receptor signaling. To further characterize the function of CD19 during B-cell differentiation, humoral immune responses to a T-cell-independent type 1 [trinitrophenyl-lipopolysaccharide (TNP-LPS)], a T-cell-independent type 2 [dinitrophenyl (DNP)-Ficoll], and a T-cell-dependent [DNP-keyhole limpet hemocyanin (KLH)] antigen were assessed in CD19-/- and hCD19TG mice. B cells from CD19-/- mice differentiated and underwent immunoglobulin isotype switching in vitro in response to mitogens and cytokines. In vivo, CD19-/- mice generated humoral responses to TNP-LPS and DNP-KLH that were dramatically lower than those of wild-type littermates. Surprisingly, the humoral response to DNP-Ficoll was significantly greater in CD19-/- mice. In contrast, hCD19TG mice were hyperresponsive to TNP-LPS and DNP-KLH immunization but were hyporesponsive to DNP-Ficoll. These results demonstrate that CD19 is not required for B-cell differentiation and isotype switching but serves as a response regulator which modulates B-cell differentiation. Since humoral responses to both T-cell-dependent and T-cell-independent antigens were similarly affected by alterations in CD19 expression, these differences are most likely to result from intrinsic changes in B-cell function rather than from the selective disruption of B-cell interactions with T cells.

The nuclear matrix protein NMP-1 is the transcription factor YY1.

NMP-1 was initially identified as a nuclear matrix-associated DNA-binding factor that exhibits sequence-specific recognition for the site IV regulatory element of a histone H4 gene. This distal promoter domain is a nuclear matrix interaction site. In the present study, we show that NMP-1 is the multifunctional transcription factor YY1. Gel-shift and Western blot analyses demonstrate that NMP-1 is immunoreactive with YY1 antibody. Furthermore, purified YY1 protein specifically recognizes site IV and reconstitutes the NMP-1 complex. Western blot and gel-shift analyses indicate that YY1 is present within the nuclear matrix. In situ immunofluorescence studies show that a significant fraction of YY1 is localized in the nuclear matrix, principally but not exclusively associated with residual nucleoli. Our results confirm that NMP-1/YY1 is a ubiquitous protein that is present in both human cells and in rat osteosarcoma ROS 17/2.8 cells. The finding that NMP-1 is identical to YY1 suggests that this transcriptional regulator may mediate gene-matrix interactions. Our results are consistent with the concept that the nuclear matrix may functionally compartmentalize the eukaryotic nucleus to support regulation of gene expression.

Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.

Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family.

Philadelphia chromosome-positive leukemias result from the fusion of the BCR and ABL genes, which generates a functional chimeric molecule. The Abr protein is very similar to Bcr but lacks a structural domain which may influence its biological regulatory capabilities. Both Abr and Bcr have a GTPase-activating protein (GAP) domain similar to those found in other proteins that stimulate GTP hydrolysis by members of the Rho family of GTP-binding proteins, as well as a region of homology with the guanine nucleotide dissociation-stimulating domain of the DBL oncogene product. We purified as recombinant fusion proteins the GAP- and Dbl-homology domains of both Abr and Bcr. The Dbl-homology domains of Bcr and Abr were active in stimulating GTP binding to CDC42Hs, RhoA, Rac1, and Rac2 (rank order, CDC42Hs > RhoA > Rac1 = Rac2) but were inactive toward Rap1A and Ha-Ras. Both Bcr and Abr acted as GAPs for Rac1, Rac2, and CDC42Hs but were inactive toward RhoA, Rap1A, and Ha-Ras. Each individual domain bound in a noncompetitive manner to GTP-binding protein substrates. These data suggest the multifunctional Bcr and Abr proteins might interact simultaneously and/or sequentially with members of the Rho family to regulate and coordinate cellular signaling.

Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bc1-2-independent regulator of apoptosis in human B-lineage lymphoma cells.

CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.

Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos.

The dorsoventral axis is established early in Xenopus development and may involve signaling by Wnts, a family of Wnt1-protooncogene-related proteins. The protein kinase shaggy functions in the wingless/Wnt signaling pathway, which operates during Drosophila development. To assess the role of a closely related kinase, glycogen synthase kinase 3 beta (GSK-3 beta), in vertebrate embryogenesis, we cloned a cDNA encoding a Xenopus homolog of GSK-3 beta (XGSK-3 beta). XGSK-3 beta-specific transcripts were detected by Northern analysis in Xenopus eggs and early embryos. Microinjection of the mRNA encoding a catalytically inactive form of rat GSK-3 beta into a ventrovegetal blastomere of eight-cell embryos caused ectopic formation of a secondary body axis containing a complete set of dorsal and anterior structures. Furthermore, in isolated ectodermal explants, the mutant GSK-3 beta mRNA activated the expression of neural tissue markers. Wild-type XGSK-3 beta mRNA suppressed the dorsalizing effects of both the mutated GSK-3 beta and Xenopus dishevelled, a proposed upstream signaling component of the same pathway. These results strongly suggest that XGSK-3 beta functions to inhibit dorsoventral axis formation in the embryo and provide evidence for conservation of the Wnt signaling pathway in Drosophila and vertebrates.

The algT (algU) gene of Pseudomonas aeruginosa, a key regulator involved in alginate biosynthesis, encodes an alternative sigma factor (sigma E).

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is the leading cause of mortality among cystic fibrosis (CF) patients. During the course of sustained infection, the production of an alginate capsule protects the bacteria and allows them to persist in the CF lung. One of the key regulators of alginate synthesis is the algT (algU) gene encoding a putative alternative sigma factor (sigma E). AlgT was hyperproduced and purified from Escherichia coli. The N-terminal sequence of the purified protein matched perfectly with that predicted from the DNA sequence. The purified protein, in the presence of E. coli RNA polymerase core enzyme, was able to initiate transcription of an algT promoter. Deletion of the -35 region of this promoter abolished this activity in vitro as well as in vivo. These data indicate that the algT gene encodes a sigma factor that is autoregulatory.

cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.

Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents.

Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. A combination of channel blockers and analysis of current-voltage relationships were used to dissect the contribution of CFTR and the ORCC to whole cell currents of transfected cells. While CFTR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. These results indicate that CF mutations can affect CFTR functions differently and suggest that severity of pulmonary disease may be more closely associated with the regulatory rather than chloride channel function of CFTR.

The DnaJ chaperone catalytically activates the DnaK chaperone to preferentially bind the sigma 32 heat shock transcriptional regulator.

In Escherichia coli the heat shock response is under the positive control of the sigma 32 transcription factor. Three of the heat shock proteins, DnaK, DnaI, and GrpE, play a central role in the negative autoregulation of this response at the transcriptional level. Recently, we have shown that the DnaK and DnaJ proteins can compete with RNA polymerase for binding to the sigma 32 transcription factor in the presence of ATP, by forming a stable DnaJ-sigma 32-DnaK protein complex. Here, we report that DnaJ protein can catalytically activate DnaK's ATPase activity. In addition, DnaJ can activate DnaK to bind to sigma 32 in an ATP-dependent reaction, forming a stable sigma 32-DnaK complex. Results obtained with two DnaJ mutants, a missense and a truncated version, suggest that the N-terminal portion of DnaJ, which is conserved in all family members, is essential for this activation reaction. The activated form of DnaK binds preferentially to sigma 32 versus the bacteriophage lambda P protein substrate.

A chimeric light-regulated amino acid transport system allows the isolation of blue light regulator (blr) mutants of Neurospora crassa.

We have developed a system for the isolation of Neurospora crassa mutants that shows altered responses to blue light. To this end we have used the light-regulated promoter of the albino-3 gene fused to the neutral amino acid permease gene mtr. The product of the mtr gene is required for the uptake of neutral aliphatic and aromatic amino acids, as well as toxic analogs such as p-flurophenylalanine or 4-methyltryptophan. mtr trp-2-carrying cells were transformed with the al-3 promoter-mtr wild-type gene (al-3p-mtr+) to obtain a strain with a light-regulated tryptophan uptake. This strain is sensitive to p-fluorophenylalanine when grown under illumination and resistant when grown in the dark. UV mutagenesis of the al-3p-mtr(+)-carrying strain allowed us to isolate two mutant strains, BLR-1 and BLR-2 (blue light regulator), that are light-resistant to p-fluorophenylalanine and have lost the ability to grow on tryptophan. These two strains have a pale-orange phenotype and show down-regulation of all the photoregulated genes tested (al-3, al-1, con-8, and con-10). Mutations in the BLR strains are not allelic with white collar 1 or white collar 2, regulatory genes that are also involved in the response to blue light.

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

Activation of mammalian retinoid X receptors by the insect growth regulator methoprene.

We report that methoprene and its derivatives can stimulate gene transcription in vertebrates by acting through the retinoic acid-responsive transcription factors, the retinoid X receptors (RXRs). Methoprene is an insect growth regulator in domestic and agricultural use as a pesticide. At least one metabolite of methoprene, methoprene acid, directly binds to RXR and is a transcriptional activator in both insect and mammalian cells. Unlike the endogenous RXR ligand, 9-cis-retinoic acid, this activity is RXR-specific; the methoprene derivatives do not activate the retinoic acid receptor pathway. Methoprene is a juvenile hormone analog that acts to retain juvenile characteristics during insect growth, preventing metamorphosis into an adult, and it has been shown to have ovicidal properties in some insects. Thus, a pesticide that mimics the action of juvenile hormone in insects can also activate a mammalian retinoid-responsive pathway. This finding provides a basis through which the potential bioactivity of substances exposed to the environment may be reexamined and points the way for discovery of new receptor ligands in both insects and vertebrates.

Oxygen as a key developmental regulator of Rhizobium meliloti N2-fixation gene expression within the alfalfa root nodule.

The symbiotic pattern of expression of Rhizobium meliloti N2-fixation genes is tightly coupled with the histological organization of the alfalfa root nodule and thus is under developmental control. N2-fixation gene expression is induced very sharply at a particular zone of the nodule called interzone II-III that precedes the zone where N2 fixation takes place. We show here that this coupling can be disrupted, hereby resulting in ectopic expression of N2-fixation genes in the prefixing zone II of the nodule. Uncoupling was obtained either by using a R. meliloti strain in which a mutation rendered N2-fixation gene expression constitutive with respect to oxygen in free-living bacterial cultures or by placing nodules induced by a wild-type R. meliloti strain in a microoxic environment. These results implicate oxygen as a key determinant of the symbiotic pattern of N2-fixation gene expression.