Telomerase activity is induced in human peripheral B lymphocytes by the stimulation to antigen receptor

To understand the molecular events for the proliferation of B cells, we studied the induction of telomerase activity in vitro after stimulation to B-cell antigen receptor (BCR) on human peripheral B cells. Although unstimulated purified B cells of tonsils and peripheral blood from healthy volunteers do not express detectable telomerase activity, anti-IgM beads induce telomerase activity in these B cells. Soluble anti-IgM antibody (Ab) alone does not induce telomerase activity, but the second signal, given by either one of the cytokines of interleukin-2 (IL-2), IL-4, and IL-13 or by anti-CD40 monoclonal Ab (MoAb), is effective as the costimulation for the induction of the activity. Stimulation with anti-IgM Ab and anti-CD40 MoAb induces telomerase activity in most mature B cells of the tonsils and peripheral blood. The stimuli to both IgM and IgD receptors similarly induce the activity. Induction of telomerase activity is accompanied with the proliferation of B cells, but is not absolutely correlated with the extent of B-cell growth. Phorbol dibutylate (PDB) plus calcium (Ca) ionophore (PDB/Ca), which replace the activation through BCR and the costimulatory molecules, also induce telomerase activity. Moreover, it is suggested that phosphoinositide (PI) 3-kinase plays a role for the induction of telomerase activity in B cells stimulated with anti-IgM Ab and anti-CD40 MoAb. These results suggest that telomerase activity is induced in the B-cell activation of the antigen specific immune response.     

Biochemistry of antigen receptor signaling in mature and developing B lymphocytes

The function of the surface antigen receptor of B cells (BCR) has been extensively studied with respect to the activation of mature B lymphocytes. B cells at other points of development (e.g., pre-B cells, immature B cells, and germinal center B cells) also express forms of the BCR, and for cells at these developmental timepoints, signaling through the BCR in some cases may lead to outcomes other than B-cell activation. Understanding the molecular events that are initiated by BCR crosslinking would enhance our understanding of the regulation and functional results of BCR signaling throughout B-cell development. In this article we review the current understanding of BCR signal transduction from initiation of the signal through changes in expression of genes that regulate the activation state. Costimulatory and modulatory molecules are considered with regard to their ability to affect the sensitivity or outcome of the BCR signal. Finally, we discuss how BCR signal transduction and the results of BCR signaling may differ at distinct stages in B-cell development.     

The human type 1 inositol 1,4,5-trisphosphate receptor from T lymphocytes. Structure, localization, and tyrosine phosphorylation

Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular calcium release channels involved in diverse signaling pathways. An IP3R is thought to play a role in mobilizing calcium required for activation of T lymphocytes. The IP3R is a tetrameric structure comprised of four approximately 300-kDa subunits encoded by a approximately 10-kilobase mRNA. In the present study we determined the structure of the human type 1 IP3R expressed in T lymphocytes (Jurkats). The IP3R in human T cells had a predicted molecular mass of 308 kDa and was most similar to the non-neuronal form of the rodent type 1 IP3R. Two putative tyrosine phosphorylation sites were identified, one near the amino terminus and one near the putative channel pore at the carboxyl terminus. During T cell activation the IP3R was tyrosine phosphorylated. A site-specific anti-IP3R antibody was used to localize the carboxyl terminus of the IP3R to the cytoplasm in T cells.     

B cell antigen receptor desensitization: disruption of receptor coupling to tyrosine kinase activation

Antigen binding to the B cell receptor (BCR) induces receptor desensitization, a condition characterized by cellular unresponsiveness to subsequent Ag stimulation despite the continued ability to bind Ag. To better understand the molecular mechanism of this unresponsiveness, we have used complementary lymphoma (K46 mu) and Ig transgenic (3-83 mu delta) mouse models to study regulation of BCR signaling. Our findings in the lymphoma model show that an initial Ag encounter renders receptors unresponsive to subsequent Ag challenge, as measured by their inability to mobilize Ca2+ and to mediate phosphorylation of receptor-proximal kinases, including Lyn, Blk, and Syk. Most importantly, the Ig alpha and Ig beta components of desensitized receptors are not phosphorylated, and receptor-associated kinases are not activated upon Ag challenge. The molecular defect does not appear to result from Lyn inactivation, sequestration, or repression, since Lyn from desensitized cell lysates is activated in vitro by synthetic doubly phosphorylated immunoreceptor tyrosine-based activation motif peptides. A similar deficit in Ag-induced receptor phosphorylation was observed in desensitized B cells from 3-83 mu delta transgenic mice. These studies indicate that Ag receptor desensitization reflects an inability to initiate activation of receptor-associated kinases that normally phosphorylate receptor Ig alphabeta subunits, leading to signal propagation.     

Syk, but not Lyn, recruitment to B cell antigen receptor and activation following stimulation of CD45- B cells

B cell Ag receptor (BCR) signaling occurs via tyrosine phosphorylation of CD79a and CD79b ITAMs, leading to recruitment and activation of Lyn and Syk tyrosine kinases and subsequent downstream events. CD45 expression is required for BCR triggering of certain of these downstream events, such as calcium mobilization and p21ras activation. However, the site in the BCR signaling cascade at which CD45 impinges is poorly defined. To address this question, we have studied CD45 function in the CD45-deficient (CD45-) and CD45-reconstituted (CD45+) J558L mu m3 plasmacytoma. In both CD45+ and CD45- cells, Ag stimulation led to CD79a and CD79b tyrosine phosphorylation as well as Syk tyrosine phosphorylation, recruitment to the receptors, and activation. In contrast to CD45+ cells, Lyn exhibited high basal tyrosine phosphorylation in the CD45- cells and was not further phosphorylated upon Ag stimulation. Mapping studies indicated that the observed constitutive phosphorylation of Lyn reflects phosphorylation of its C-terminal tyrosine, Y508, at high stoichiometry. Constitutively Y508-phosphorylated Lyn was neither recruited to the BCR nor activated upon Ag stimulation. Moreover, CD79a-ITAM phosphopeptides failed to bind Lyn from the CD45- cells. Thus, Y508 phosphorylation of Lyn occurs in the absence of cellular CD45 expression and appears to render the kinase unable to associate with the phosphorylated receptor complex via its Src homology 2 domain and to participate in signal propagation. Surprisingly, in view of previous findings implicating Src family kinases in ITAM phosphorylation, the data indicate that Ag-induced CD79a and CD79b tyrosine phosphorylation and Syk recruitment and activation can occur in the absence of CD45 expression and, hence, Src-family kinase activation.     

The chemokine monocyte chemotactic protein 1 triggers Janus kinase 2 activation and tyrosine phosphorylation of the CCR2B receptor

Gelsolin is an actin filament-capping protein that has been shown to play a key role in cell migration. Here we have studied the involvement of phosphoinositide 3-kinase (PI 3-kinase) and GTP-binding proteins (G-proteins) in the regulation of gelsolin-actin interactions in neutrophils. Inhibition of PI 3-kinase activity in vivo by wortmannin did not affect the dissociation of actin-gelsolin (1:1) complexes induced by neutrophil stimulation with N-formyl-Met-Leu-Phe. Guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) indirectly promoted the dissociation of actin-gelsolin complexes in a cell-free system using neutrophil cytosol, and this effect was blocked by the GDP dissociation inhibitor for Rho (Rho-GDI). The GTPgammaS-loaded ialpha2 and the beta1gamma2 subunits of heterotrimeric G-proteins (Gialpha2 and Gbeta1gamma2) also triggered actin-gelsolin dissociation in a Rho-GDI-sensitive manner. GTP-loaded activated Rac, but not activated Rho, induced the dissociation of cytosolic actin-gelsolin complexes. The guanine nucleotide exchange on Rac was increased by addition of GTPgammaS-loaded Gialpha2 or Gbeta1gamma2 to neutrophil cytosol. These findings suggest that activation of Rac by G-protein-coupled receptors in neutrophils triggers uncapping of actin filaments, independently of PI 3-kinase.     

Induction of the antigen receptor expression on B lymphocytes results in rapid competence for signaling of SLP-65 and Syk

The binding of antigen to the B cell antigen receptor (BCR) results in the activation of protein tyrosine kinases (PTKs) such as Lyn and Syk, and the phosphorylation of several substrate proteins including HS1 and SLP-65. How these signaling elements are connected to the BCR is not well understood. Using an expression vector for a tamoxifen-regulated Cre recombinase, we have developed a method that allows the inducible expression of the BCR. Disruption of the VH leader reading frame of the immunoglobulin heavy chain by two loxP sites is overcome by Cre-mediated DNA recombination and results in the cell surface expression of the BCR starting 4 h after exposure of transfected B cells to tamoxifen. This method can, in principle, be employed for the inducible expression of any secreted or type I transmembrane protein. By monitoring the activation of signaling elements in pervanadate-stimulated B cells expressing different levels of the BCR, we show here that phosphorylation of SLP-65 and Syk, but not of Lyn, is strictly dependent on the expression of the BCR on the cell surface. These data suggest that the BCR reorganizes its signaling molecules as soon as it appears on the cell surface.     

The B cell surface protein CD72 recruits the tyrosine phosphatase SHP-1 upon tyrosine phosphorylation

Activation signals of lymphocytes are negatively regulated by the membrane molecules carrying the immunoreceptor tyrosine-based inhibition motif (ITIM). Upon tyrosine phosphorylation, ITIMs recruit SH2-containing phosphatases such as SHP-1, resulting in down-modulation of cell activation. We showed that the cytoplasmic domain of the CD72 molecule carries an ITIM and is associated in vitro with SHP-1 upon tyrosine phosphorylation. Moreover, cross-linking of B cell Ag receptor (BCR) enhances both tyrosine phosphorylation of CD72 and association of CD72 with SHP-1 in B cell line WEHI-231. These results indicate that CD72 recruits SHP-1 upon tyrosine phosphorylation induced by BCR signaling, suggesting that CD72 is a negative regulator of BCR signaling.     

Low levels of ionic mercury modulate protein tyrosine phosphorylation in lymphocytes

The ability of ionic mercury to induce protein tyrosine phosphorylation in mouse spleen cells and in the mouse WEHI-231 B-cell lymphoma was investigated. We have confirmed previous studies which showed that exposure to high levels (several hundred microM) of mercury lead to very large increases in the level of protein tyrosine phosphorylation in these cell systems. However we have also demonstrated that low levels (in the order of 0.1 to 1.0 microM) of mercury also significantly upregulate protein tyrosine phosphorylation. Mercury induced protein tyrosine phosphorylation is inhibited by the mercury chelator penicillamine and by pretreating treating target cells with the sulfhydryl blocking reagent N-hydroxymaleimide. These results suggest that exposure to low levels of mercury could potentially interfere with lymphocyte signal transduction and so offer a possible explanation as to how mercury exposure could lead to immune cellular dysfunction. On a molecular level, the results suggest that the site(s) of action with respect to mercury dependent induction of protein tyrosine phosphorylation is likely a free disulfide group or groups located on the outer leaflet of the plasma membrane.     

PLD2 complexes with the EGF receptor and undergoes tyrosine phosphorylation at a single site upon agonist stimulation

Mammalian phospholipase D (PLD) activity becomes up-regulated when cells are stimulated by a variety of hormones, growth factors, and other extracellular signals. Two distinct PLDs, PLD1 and PLD2, have been identified. The mechanism through which each PLD is activated, however, is poorly understood. Using transiently transfected human embryonic kidney fibroblasts (HEK293), we demonstrate here that PLD1 activity, and to a lesser extent PLD2 activity, is stimulated in response to epidermal growth factor (EGF). PLD2, but not PLD1, associates with the EGF receptor in a ligand-independent manner and becomes tyrosine-phosphorylated upon EGF receptor activation. Tyrosine 11 (Tyr-11) of PLD2 was identified as the specific phosphorylation site. Mutation of this residue to phenylalanine enhanced basal activity almost 2-fold, but did not alter the magnitude of the EGF-mediated increase in PLD2 activity. In conclusion, we show here for the first time agonist-stimulated activation of both PLD1 and PLD2 in vivo and provide evidence of a distinct type of interaction for each isoform with the EGF receptor. Moreover, our results suggest that agonist-induced tyrosine phosphorylation plays a role in PLD2 regulation.     

TGF-beta inhibits IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 in T lymphocytes

The major yolk protein precursor in mosquito oocytes, vitellogenin (Vg), is internalized by a 205-kDa membrane-bound receptor (VgR). Recently, VgR has been isolated permitting the production of polyclonal anti-VgR antibodies. To elucidate the pathway of VgR internalization and recycling in mosquito oocytes during Vg uptake, we carried out an immunogold electron-microscopic study, labeling both Vg and VgR in ultrathin frozen sections of ovarian tissue. VgR immunolabeling demonstrated that the oocyte plasma membrane was subdivided into microdomains, with VgR being located between and at the lower portions of the oocyte microvilli. During the early stages of internalization, Vg and VgR were observed together in coated pits, coated vesicles, and early endosomes. Fusion of early endosomes created transitional yolk bodies (TYB) in which Vg and VgR became segregated. VgR label was present in the numerous tubular compartments that protruded from the TYBs. These tubular organelles extended to and fused with the plasma membrane, suggesting that they represented the vehicle for VgR recycling. Vg label was not observed in the tubular compartments. Instead, Vg accumulated in the core of the TYB, a region free of VgR label. Mature yolk bodies (MYB) were heavily labeled for Vg, but completely lacked any VgR label, indicating that MYB are storage compartments that do not participate in receptor recycling. Thus, our immunocytochemical data clearly visualize the steps in Vg/VgR internalization, dissociation, sorting, and recycling of the receptor to the plasma membrane.     

Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612

Activation of the endogenous protein kinase Cs in human kidney fibroblast (293) cells was found in the present study to inhibit the subsequent ability of insulin to stimulate the tyrosine phosphorylation of an expressed insulin receptor substrate-1. This inhibition was also observed in an in vitro phosphorylation reaction if the insulin receptor and its substrate were both isolated from cells in which the protein kinase C had been activated. To test whether serine phosphorylation of the insulin receptor substrate-1 was contributing to this process, serine 612 of this molecule was changed to an alanine. The insulin-stimulated tyrosine phosphorylation and the associated phosphatidylinositol 3-kinase activity of the expressed mutant were found to be comparable to those of the expressed wild-type substrate. However, unlike the wild-type protein, activation of protein kinase C did not inhibit the insulin-stimulated tyrosine phosphorylation of the S612A mutant nor its subsequent association with phosphatidylinositol 3-kinase. Tryptic peptide mapping of in vivo labeled IRS-1 and the S612A mutant revealed that PMA stimulates the phosphorylation of a peptide from wild-type IRS-1 that is absent from the tryptic peptide maps of the S612A mutant. Moreover, a synthetic peptide containing this phosphoserine and its nearby tyrosine was found to be phosphorylated by the insulin receptor to a much lower extent than the same peptide without the phosphoserine. Activation of protein kinase C was found to stimulate by 10-fold the ability of a cytosolic kinase to phosphorylate this synthetic peptide as well as the intact insulin receptor substrate-1. Finally, cytosolic extracts from the livers of ob/ob mice showed an 8-fold increase in a kinase activity capable of phosphorylating this synthetic peptide, compared to extracts of livers from lean litter mates. These results indicate that activation of protein kinase C stimulates a kinase which can phosphorylate insulin receptor substrate-1 at serine 612, resulting in an inhibition of insulin signaling in the cell, posing a potential mechanism for insulin resistance in some models of obesity.     

Negative regulation of antigen receptor signaling in lymphocytes

The purpose of this study was to assess the influence of an experimentally induced light premature occlusal contact on tooth sensation. This assessment involved monitoring the electric tooth pain threshold (TPT) at multiple times before and after altering the occlusal contact. This alteration was produced by placing high inlays, which were measured with a custom made measuring device in maximum intercuspation. Data were collected on 10 teeth in 5 subjects, all whom were male and between the ages of 24 and 30 years. The contact area of the high inlays varied from 75 microm to 193 microm. The TPT changes in these teeth ranged from -43 to +21% of their baseline level. Seven of these teeth showed a significant decrease (P < 0.05) in the TPT, one showed an increase and two did not show a significant change. Two of the teeth with a decreased TPT had cold water triggered occlusal pain, and 2 teeth had only occlusal pain. After several inlay adjustments to eliminate interference in maximum intercuspation, all tested teeth returned to their TPT baseline level and all symptoms disappeared. These results suggested that a light premature occlusal contact may change tooth sensation.     

Multiple phosphorylation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B by casein kinase II and p60c-src in vitro

The objectives of this study were to evaluate milk choline as an indicator of choline absorption and to use milk choline to evaluate the efficacy of a rumen-protected choline supplement. In a preliminary 4-wk experiment, two Holstein cows in early lactation were abomasally infused with either 0 or 60 g/d of choline chloride in 2 L of water, which was used as a carrier. Choline infusion increased milk choline secretion from 1.95 to 3.95 g/d during the 2-wk choline infusion period. In Experiment 2, four Holstein cows in early lactation were abomasally infused with 0, 25, 50, and 75 g/d of choline chloride in 2 L of water using a 4 x 4 Latin square design with 1-wk experimental periods. Milk choline secretion was 2.56, 3.62, 3.72, and 3.82 g/d for the respective choline treatments. In Experiment 3, 10 Holstein cows in midlactation were fed either 0 or 50 g/d of choline using an experimental rumen-protected choline supplement during a 2-wk experiment. Milk choline secretion was increased from 2.12 to 2.99 g/d with the supplemental choline. Results of these experiments demonstrated that milk choline is responsive to postruminal choline supply and can be used as a qualitative indicator of choline absorption.