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The heterogeneous nuclear ribonucleoprotein (hnRNP) K, a component of the hnRNP particles, appears to be involved in several steps of regulation of gene expression. To gain insight into mechanisms of K protein action, we performed two-hybrid screens using full-length hnRNP K as a bait. Several novel protein partners were identified, including Y-box-binding protein (YB-1), splicing factors 9G8 and SRp20, DNA-methyltransferase, hnRNP L, and hnRNP U. In vitro binding studies and co-immunoprecipitation from cellular extracts provided evidence for direct interaction between hnRNP K and YB-1. Two distinct domains in YB-1 were responsible for binding to K protein. Each protein was able to transactivate transcription from a polypyrimidine-rich promoter; however, this effect was reduced when K and YB-1 proteins were coexpressed suggesting a functional interaction between these two proteins.

The heterogeneous nuclear ribonucleoprotein K protein recruits a diversity of molecular partners and may act as a docking platform involved in such processes as transcription, RNA processing, and translation. We show that K protein is tyrosine-phosphorylated in vitro by Src and Lck. Treatment with H(2)O(2)/Na(3)VO(4), which induces oxidative stress, stimulated tyrosine phosphorylation of K protein in cultured cells and in intact livers. Tyrosine phosphorylation increased binding of Lck and the proto-oncoprotein Vav to K protein in vitro. Oxidative stress increased the association of K protein with Lck and Vav, suggesting that tyrosine phosphorylation regulates the ability of K protein to recruit these effectors in vivo. Translation-based assay showed that K protein is constitutively bound to many mRNAs in vivo. Native immunoprecipitated K protein-mRNA complexes were disrupted by tyrosine phosphorylation, suggesting that the in vivo binding of K protein to mRNA may be responsive to the extracellular signals that activate tyrosine kinases. This study shows that tyrosine phosphorylation of K protein regulates K protein-protein and K protein-RNA interactions. These data are consistent with a model in which functional interaction of K protein is responsive to changes in the extracellular environment. Acting as a docking platform, K protein may bridge signal transduction pathways to sites of nucleic acid-dependent process such as transcription, RNA processing, and translation.

The heterogeneous nuclear ribonucleoprotein (hnRNP) K protein recruits a diversity of molecular partners that are involved in signal transduction, transcription, RNA processing, and translation. K protein is phosphorylated in vivo and in vitro by inducible kinase(s) and contains several potential sites for protein kinase C (PKC) phosphorylation. In this study we show that K protein is phosphorylated in vitro by PKCdelta and by other PKCs. Deletion analysis and site-directed mutagenesis revealed that Ser302 is a major K protein site phosphorylated by PKCdelta in vitro. This residue is located in the middle of a short amino acid fragment that divides the two clusters of SH3-binding domains. Mutation of Ser302 decreased the level of phosphorylation of exogenously expressed K protein in phorbol 12-myristate 13-acetate-treated COS cells, suggesting that Ser302 is also a site for PKC-mediated phosphorylation in vivo. In vitro, PKCdelta binds K protein via the highly interactive KI domain, an interaction that is blocked by poly(C) RNA. Mutation of Ser302 did not alter the K protein-PKCdelta interaction in vitro, suggesting that phosphorylation of this residue alone is not sufficient to alter this interaction. Instead, binding of PKCdelta to K protein in vitro and in vivo was greatly increased by K protein phosphorylation on tyrosine residues. The ability of PKCdelta to bind and phosphorylate K protein may serve not only to alter the activity of K protein itself, but K protein may also bridge PKCdelta to other K protein molecular partners and thus facilitate molecular cross-talk. The regulated nature of the PKCdelta-K protein interaction may serve to meet cellular needs at sites of active transcription, RNA processing and translation in response to changing extracellular environment.

A microelectrometric titration method is described to measure picomole amounts of Cl- present in solutions at micromolar concentrations. This method was used to measure total intracellular chloride concentration ([Cl-]i) in leukocytes. Through the use of submicroliter samples, [Cl-] can be measured in the range of 5-500 microM. There is no measurable interference from other ions normally present in the cell and no intracellular ion is falsely measured as Cl-. [Cl-]i determined by the conventional coulometric titration and the new microelectrometric titration method was the same. Among the commonly used substituting anions, thiocyanate was the only one falsely measured as Cl- and should be avoided in experiments using this method. Because only picomole amounts of Cl- are required, measurements of intracellular pH and total concentrations of other intracellular ions can be done in the same lysate of as few as 5 X 10(5) cells. This feature should make it possible to study the time course of changes in [Cl-]i together with measurements of other intracellular ions following various physiological or experimental maneuvers.

We have previously shown that in a murine pre-B lymphocyte cell line, 70Z/3, interleukin-1-induced IgM expression (differentiation) was associated with Na+/H+-mediated cytoplasmic alkalinization. Because interferon-gamma also induces 70Z/3 differentiation, in this study we examined the effects of recombinant murine interferon-gamma (rIFN-gamma) on cell pH. We found that rIFN-gamma (50 units/ml) induced a sustained increase in cell pH, averaging 0.09 pH units above the base line at 30 min and 0.08 at 4 h. Because rIFN-gamma also induced increases in cell Na+ concentration, the data suggests stimulation of Na+/H+ exchange across the cell membrane. Amiloride inhibited the rIFN-gamma-mediated pH rise by only 31% at 30 min, but at 4 h the inhibition was more complete (89%). In contrast to pHi, amiloride totally blocked the Na+ rise at both 30 min and 4 h. This indicates that at the earlier time point the pH rise had a Na+/H+ exchange-dependent and -independent component while at the later time most of the pH rise was due to the Na+/H+ exchanger. The presence of a Na+ independent amiloride-insensitive component was further confirmed by the 0.05 pH rise induced by rIFN-gamma in Na+-free media. Failure to block the Na+-independent rIFN-gamma-mediated pHi rise by anion exchange inhibitors or removal of Ca2+ indicate that this component was not mediated by Cl-/OH- or Ca2+/H+ exchange. The nature of the Na+-independent cell alkalinization process or its role in rIFN-gamma-mediated 70Z/3 differentiation remains to be determined. Because amiloride did not have an effect on rIFN-gamma-induced surface Ig expression or the rIFN-gamma-mediated increase in new kappa light chain-specific mRNA the results indicate that rIFN-gamma-stimulated Na+/H+ is not required for 70Z/3 differentiation.

We have previously shown that recombinant murine interferon-gamma, rIFN-gamma, and recombinant human interleukin-1 alpha, rIL-1 alpha, induce differentiation of murine pre-B-like cell line 70Z/3, a finding associated with stimulation of Na+/H+ exchange across the plasma membrane. The present study was designed to test whether the enhanced Na+/H+ exchange is mediated by Ca2+/phospholipid-dependent protein kinase C. The results show that two structurally different peptides, rIFN-gamma and rIL-1 alpha, induce identical patterns of transient translocation of protein kinase C from the cytosol to the membranes. The increase in membrane-associated protein kinase C activity was first detected 20 min after exposure to the lymphokines. This activity peaked at 30 min and was back to baseline by 2 h. At each time point, the increase in membrane-associated protein kinase C activity corresponded to a decrease in the activity of protein kinase C in the cytoplasmic fraction. The total cellular activity (cytosol + membrane) remained the same. Two series of experiments were carried out to test the role of protein kinase C in mediating the lymphokine-stimulated Na+/H+ exchange. In the first, the effects of rIFN-gamma and rIL-1 alpha on cytoplasmic pH were measured in the presence of a protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, H-7. In the second, rIFN-gamma- and rIL-1 alpha-induced cytoplasmic alkalinization was determined in cells containing decreased protein kinase C activity. Under both experimental conditions, lymphokine-induced cytoplasmic alkalinization was not attenuated. These results indicate that, although both rIFN-gamma and rIL-1 alpha cause association of protein kinase C with membranes, activation of protein kinase C is not required for rIFN-gamma or rIL-1 alpha to stimulate Na+/H+ exchange across the plasma membrane.

Treatment of mesangial cells with either phorbol 12-myristate 13-acetate (PMA) or interleukin-1beta induces an increase in laminin B2 chain mRNA levels. In other systems, activation of gene expression by these agents is transcriptionally mediated. To identify transcription factors that control expression of laminin B2 chain gene, we employed a strategy consisting of a computer-based analysis of murine and human gene promoter sequences and gel shift assays. Although overall the laminin B2 chain promoters from the two species have low sequence similarity, the mouse promoter contained sequences that were also contained in one motif, 5'-CCCCGCCCACCTCGCGCGC-3', designated bcn-1, from the human promoter. Treatment of mesangial cells with either PMA or interleukin-1beta induced a transient increase in nuclear DNA binding activity, designated BCN-1, recognized the bcn-1 motif in a gel shift assay. A single nucleotide replacement in the bcn-1 motif abolished DNA binding, indicating that bcn-1.BCN-1 complex formation is highly specific. In transient transfections, the ability of PMA to induce the laminin B2 chain promoter was abolished by mutating the bcn-1 motif. These results suggest that the bcn-1 element and its cognate inducible BCN-1 protein regulate laminin B2 chain gene transcription.

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) K protein is comprised of multiple modular domains that serve to engage a diverse group of molecular partners including DNA, RNA, the product of the proto-oncogene vav, and tyrosine and serine/threonine kinases. To identify additional K protein molecular partners and to further understand its function, we used a fragment of K protein as a bait in the yeast two-hybrid screen. The deduced primary structure of one of the positive clones revealed a novel zinc finger protein, hereby denoted as Zik1. In addition to the nine contiguous zinc fingers in the C terminus, Zik1 contains a KRAB-A domain thought to be involved in transcriptional repression. Zik1 and K protein bound in vitro and co-immunoprecipitated from cell extracts indicating that in vivo their interaction is direct. Expression of Gal4 DNA-binding domain-Zik1 fusion protein repressed a gene promoter bearing Gal4-binding elements, indicating that from cognate DNA elements Zik1 is a transcriptional repressor. The known diverse nature of K protein molecular interactions and now the identification of a K protein partner that is a transcriptional repressor lends support to the notion that K protein is a remarkably versatile molecule that may be acting as a docking platform to facilitate communication among molecules involved in signal transduction and gene expression.

We have previously shown that interleukin-la (IL-1α) and interferon-γ (IFN-γ) induce surface IgM expression, stimulate Na+/H+exchange, and activate protein kinase C in the murine pre-B lymphocyte cell line, 70Z/3. Because the two structurally different lymphokines induce similar effects, in this study we set out to compare the properties of the IL-1 and IFN-γ surface receptors. In contrast to their similar cellular effects, we found that IL-1α and IFN-γ receptors have different properties. 70Z/3 have high (100 sites/cell) and low (900 sites/cell) affinity IL-1 receptors with dissociation constants (KD) 6 × 10−11and 10−9M, respectively. In contrast, IFN-γ receptors are of one class with a KDof 3 × 10−10Mand are at a higher number, 8000 sites/cell. After binding to their receptors both IL-1α and IFN-γ are internalized and intracellularly degraded, but the rate of internalization of IFN-γ is greater than IL-1α. The effective median concentrations (EC50) of IL-1α- or IFN-γ-induced surface IgM expression are similar (4–5 × 10−12M). However, at this concentration 10-fold more of IFN-γ than IL-1α molecules are bound per cell.

The heterogeneous nuclear ribonucleoprotein K protein represents a novel class of proteins that may act as docking platforms that orchestrate cross-talk among molecules involved in signal transduction and gene expression. Using a fragment of K protein as bait in the yeast two-hybrid screen, we isolated a cDNA that encodes a protein whose primary structure has extensive similarity to the Drosophila melanogaster extra sex combs (esc) gene product, Esc, a putative silencer of homeotic genes. The cDNA that we isolated is identical to the cDNA of the recently positionally cloned mouse embryonic ectoderm development gene, eed. Like Esc, Eed contains six WD-40 repeats in the C-terminal half of the protein and is thought to repress homeotic gene expression during mouse embryogenesis. Eed binds to K protein through a domain in its N terminus, but interestingly, this domain is not found in the Drosophila Esc. Gal4-Eed fusion protein represses transcription of a reporter gene driven by a promoter that contains Gal4-binding DNA elements. Eed also represses transcription when recruited to a target promoter by Gal4-K protein. Point mutations within the eed gene that are responsible for severe embryonic development abnormalities abolished the transcriptional repressor activity of Eed. Results of this study suggest that Eed-restricted homeotic gene expression during embryogenesis reflects the action of Eed as a transcriptional repressor. The Eed-mediated transcriptional effects are likely to reflect the interaction of Eed with multiple molecular partners, including K protein.

Previous studies have shown that binding of interleukin 1 (IL-1) to its receptor and intracellular processing of the IL-1/IL-1 receptor complex appear to be different in B- and T-lymphocyte cell lines. In this study we used a B-lymphoid cell line, 70Z/3, and T-lymphoid cell line, EL-4 6.1 C10, to explore further the differences that exist between IL-1 receptors on cells of B and T lineage. We show that a monoclonal antibody against the IL-1 receptor on EL-4 cells does not bind to the IL-1 receptor on 70Z/3 cells. This finding suggests that there are structural differences in the extracellular domains of the IL-1 receptors on the two cell lines. Furthermore, affinity crosslinking showed that the molecular mass of the IL-1 receptor on EL-4 is 87 kDa, whereas that of 70Z/3 is significantly lower (66 kDa). Activation of phospholipid/Ca2+-dependent protein kinase, protein kinase C, by phorbol 12-myristate 13-acetate (PMA) greatly reduced the number of IL-1 binding sites on 70Z/3. But, in sharp contrast, PMA had no effect on surface IL-1 receptor expression on EL-4 cells despite having an equally potent effect in activating protein kinase C. The different effects of protein kinase C suggest that the cytoplasmic domains of the IL-1 receptors in 70Z/3 and EL-4 may also be different. Lastly, a probe containing the entire coding region of the murine T-cell IL-1 receptor hybridized under high stringency conditions with mRNA from EL-4 cells but not with mRNA from 70Z/3 cells. Taken together, the observations made in this study suggest that major structural differences exist between the IL-1 receptors on B and T lymphocytes.

The heterogeneous ribonucleoprotein particle (hnRNP) K protein interacts with multiple molecular partners including DNA, RNA, serine/threonine, and tyrosine kinases and the product of the proto-oncogene, Vav. The K protein is phosphorylated in vivo and in vitro on serine/threonine residues by an interleukin 1 (IL-1)-responsive kinase with which it forms a complex. In this study we set out to map the K protein domains that bind kinases. We demonstrate that the K protein contains a cluster of at least three SH3-binding sites (P1, PPGRGGRPMPPSRR, amino acids 265-278; P2, PRRGPPPPPPGRG, 285-297; and P3, RARNLPLPPPPPPRGG, 303-318) and that each one of these sites is capable of selectively engaging c-Src and Vav SH3 domains but not SH3 domains of Abl, p85 phosphatidylinositol 3-kinase, Grb-2, and Csk. We demonstrate that the K protein domain that recruits and is phosphorylated in an RNA-dependent manner by the IL-1-responsive kinase, designated KPK for K protein kinase, is contained within the 338-425-amino acid stretch and thus is contiguous but does not include the cluster of the SH3-binding sites. K protein and KPK co-immunoprecipitate from cell extracts with either c-Src or Vav, suggesting that K protein-KPK-c-Src and K protein-KPK-Vav complexes exist in vivo. Furthermore, in the context of K protein, c-Src can reactivate KPK in vitro. The succession of kinase-binding sites contained within the K protein that allow it to form multienzyme complexes and facilitate kinase cross-talk suggest that K protein may serve as a docking platform that promotes molecular interactions occurring during signal transduction.

To examine the role of protein kinase C (PKC) on the acid secretory activity of isolated rat parietal cells, histamine-and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated [14C]aminopyrine accumulation was determined in the presence of agents that redistribute PKC activity to plasma membranes. Phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetylglycerol (OAG), and phospholipase C inhibited, in a dose-dependent fashion, histamine- and DBcAMP-stimulated [14C]aminopyrine accumulation. Because PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) reversed the effect, the results suggest that inhibition of histamine- or DBcAMP-stimulated [14C]aminopyrine accumulation induced by PMA, OAG, or phospholipase C was caused by increased activity of PKC in plasma membrane. To determine where in the cascade of events PKC inhibits acid secretion, histamine-, cholera toxin-, and forskolin-stimulated [14C]aminopyrine accumulation was measured with or without PMA. Because the percent of inhibition by PMA of [14C]aminopyrine accumulation was similar with the three secretagogues, the results suggest that PKC inhibits acid secretion at a point beyond adenosine 3',5'-cyclic monophosphate (cAMP) production. This was supported by the fact that PMA had no effect on histamine-stimulated production of cAMP and by the finding that activation of PKC had the same effect on histamine- or DBcAMP-stimulated [14C]aminopyrine accumulation. Histamine and DBcAMP inhibited PKC activity, suggesting a reciprocal interaction between PKC and histamine-triggered signal transduction pathway.

Interleukin-1 a polypeptide hormone produced by activated macrophages is a mixture of at least two proteins, interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta). We have previously shown that macrophage-derived interleukin-1 induced new kappa light chain synthesis for surface IgM expression in a murine pre-B like cell line 70Z/3, a finding associated with an early amiloride-sensitive rise in the total intracellular sodium concentration. Because IL-1 alpha and IL-1 beta are structurally quite different, in this study their effect on 70Z/3 was examined separately. The results show that both human rIL-1 alpha and rIL-1 beta induce the differentiation of 70Z/3, but a higher concentration of rIL-1 beta compared to rIL-1 alpha is needed for a maximal response. At saturating concentrations, both rIL-1 alpha and rIL-1 beta induce a simultaneous rise in intracellular pH and sodium concentration. Because rIL-1 mediated intracellular alkalinization and sodium rise are amiloride sensitive, they likely occur through stimulation of the Na+/H+ exchanger across the cell membrane. Inhibition of the Na+/H+ antiport with an amiloride analog did not have an effect on rIL-1 induced surface IgM expression or the rIL-1-mediated increase in kappa light chain specific mRNA level. Therefore, these results indicate that an increase in pHi or [Na]i is not required for IL-1 induced 70Z/3 differentiation.