Regulation of antigen receptor signal transduction by protein tyrosine kinases

The purpose of this study was to assess the value of electron beam computed tomography in the detection of cardiac calcifications in coronaries and valves of dialysis patients and to determine the rate at which calcification progresses. Forty-nine chronic hemodialysis patients aged 28 to 74 years were compared with 102 non-dialysis patients aged 32 to 73 years with documented or suspected coronary artery disease, all of whom underwent coronary angiography. We used high-resolution electron beam computed tomography scanning to make 30 axial slices with a distance of 3 mm between each slice. The number of calcifications, the surface area, and the average and highest density values were measured. We calculated a quantitative coronary artery calcium score and assessed calcification of mitral and aortic valves. In dialysis patients, the measurements were repeated after 12 months. The coronary artery calcium score was from 2.5-fold to fivefold higher in the dialysis patients than in the non-dialysis patients. Hypertensive dialysis patients had higher calcium scores than non-hypertensive dialysis patients (P < 0.05). A stepwise, multiple regression analysis confirmed the importance of age and hypertension. No correlation between calcium, phosphate, or parathyroid hormone values and the coronary calcium score was identified; however, the calcium score was inversely correlated with bone mass in the dialysis patients (r = 0.47, P < 0.05). The mitral valve was calcified in 59% of dialysis patients, while the aortic valve was calcified in 55%. The coronary artery calcium score was correlated with aortic valvular, but not mitral valvular calcification. A repeat examination of the dialysis patients at an interval of 1 year showed a disturbing tendency for progression. Our data under-score the frequency and severity of coronary and valvular calcifications in dialysis patients, and illustrate the rapid progression of this calcification. Finally, they draw attention to hypertension as an important risk factor in this process.     

Regulation of cell-matrix adhesion by receptor tyrosine kinases

Cell-cell and cell-matrix adhesive interactions mediated by integrins play crucial roles in leukocyte migration to inflamed tissues, and also in cell migration during embryogenesis. Much remains to be learned about the molecular mechanisms of regulation of adhesion mediated by integrins. Recently we found that steel factor and c-kit induce adhesion to fibronectin by VLA-5 in mast cells. Activation of adhesiveness is transient, and occurs at concentrations of steel factor 100-fold lower than required for growth stimulation. This suggests that regulation of adhesion is an important biological function of steel factor and c-kit. Other receptor tyrosine kinases such as the PDGF receptor can substitute for c-kit. Signaling through receptor tyrosine kinases may offer a general mechanism for the regulation of integrin avidity.     

Altered efficacy and selectivity of tyrosine kinase inhibitors of the activated states of protein tyrosine kinases

Listeria monocytogenes is a pathogenic bacterium which has been implicated in several foodborne illnesses. This microorganism grows into biofilms attached to the surfaces in food-processing plants, increasing its resistance to antimicrobial agents. The present work was realized to investigate the attachment of L. monocytogenes isolates to glass surfaces and to find a decontamination procedure to remove these bacteria in biofilms. Three-day biofilms were prepared by growing L. monocytogenes isolates from food plant environments on glass surfaces. Sixteen decontamination treatments at different pHs, temperatures, and times of exposure were tested against L. monocytogenes biofilms. The most efficient treatments were those applied at 63 degrees C. Combinations of decontamination treatments applied at 55 degrees C for 30 min provided different results according to the other factors used. In general, L. monocytogenes biofilms were found to be not very susceptible to high osmolarity (10.5% NaCl), and the interaction of sodium chloride and acid did not seem to have important effects in inactivating these bacteria (from a 1.3-to a 1.9-log-CFU/cm2 reduction). The combination of NaOH (pH 10.5; 100 mM) and acetic acid (pH 5.4; 76.7 mM) applied sequentially at 55 degrees C for even 5 min was shown to be the most effective treatment to remove L. monocytogenes from biofilms (at least a 4.5-to 5.0-log-CFU/cm2 decline).     

Regulation of Zap-70 by Src family tyrosine protein kinases in an antigen-specific T-cell line

To further understand the interactions between Zap-70, Src family kinases, and other T-cell proteins, we have examined the regulation of Zap-70 in the antigen-specific T-cell line BI-141. By analyzing derivatives containing an activated version of either p56lck or p59fynT, it was observed that the two Src-related enzymes augmented T-cell receptor (TCR)-mediated tyrosine phosphorylation of Zap-70, as well as its association with components of the antigen receptor complex. Importantly, the accumulation of TCR.Zap-70 complexes quantitatively and temporally correlated with the induction of tyrosine phosphorylation of the CD3 and zeta chains of TCR. Using a CD4-positive variant of BI-141, we also found that the ability of Zap-70 to undergo tyrosine phosphorylation and associate with TCR was enhanced by aggregation of TCR with the CD4 co-receptor. Further studies allowed the identification of two distinct pools of tyrosine-phosphorylated Zap-70 in activated T-cells. While one population was associated with TCR, the other was co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated protein of unknown identity. In addition to supporting the notion that Src-related enzymes regulate the recruitment of Zap-70 in TCR signaling, these data added further complexity to previous models of regulation of Zap-70. Furthermore, they suggested that p120 may be an effector and/or a regulator of Zap-70 in activated T-lymphocytes.     

Regulation of N-acetylglucosaminyltransferase V by protein kinases

When 7721 human hepatocarcinoma cells were treated with 100 nM phorbol-12-myristate-13-acetate (PMA), the activity of N-acetylglucosaminyltransferase V(GnT-V) in the cells varied in accordance with the activity of membranous protein kinase C (PKC), but not with that of cytosolic PKC. Quercetin, a non-specific inhibitor of Ser/Thr protein kinase, and D-sphingosine and staurosporine, two specific inhibitors of PKC, blocked the activation of membranous PKC and GnT-V by PMA. Among the three inhibitors, quercetin was least effective. The inhibitory rates of quercetin and staurosporine toward membranous PKC and GnTV were proportional to the concentrations of the two inhibitors. The activities of GnTV and membranous protein kinase A (PKA) were also induced in parallel by dibutyryl cAMP (db-cAMP) and this induction was blocked by a specific PKA inhibitor. When cell free preparations of 7721 cells and human kidney were treated with alkaline phosphatase (ALP) to remove the phosphate groups, the GnTV activities were decreased. These results suggest that GnTV may be activated by membranous PKC or PKA, indirectly or directly, via phosphorylation of Ser/Thr residues.     

Regulation of fibroblast motility by the protein tyrosine phosphatase PTP-PEST

The protein tyrosine phosphatase PTP-PEST is a cytosolic enzyme that displays a remarkable degree of selectivity for tyrosine-phosphorylated p130(Cas) as a substrate, both in vitro and in intact cells. We have investigated the physiological role of PTP-PEST using Rat1 fibroblast-derived stable cell lines that we have engineered to overexpress PTP-PEST. These cell lines exhibit normal levels of tyrosine phosphorylation of the majority of proteins but have significantly lower levels of tyrosine phosphorylation of p130(Cas) than control cells. Initial cellular events occurring following integrin-mediated attachment to fibronectin (cell attachment and spreading) are essentially unchanged in cells overexpressing PTP-PEST; similarly, the extent and time course of mitogen-activated protein kinase activation in response to integrin engagement is unchanged. In contrast, the reduced phosphorylation state of p130(Cas) is associated with a considerably reduced rate of cell migration and a failure of cells overexpressing PTP-PEST to accomplish the normally observed redistribution of p130(Cas) to the leading edge of migrating cells. Furthermore, cells overexpressing PTP-PEST demonstrate significantly reduced levels of association of p130(Cas) with the Crk adaptor protein. Our results suggest that one physiological role of PTP-PEST is to dephosphorylate p130(Cas), thereby controlling tyrosine phosphorylation-dependent signaling events downstream of p130(Cas) and regulating cell migration.     

Mutated connexin43 proteins inhibit rat glioma cell growth suppression mediated by wild-type connexin43 in a dominant-negative manner

To explore the role of calcitonin gene-related peptide (CGRP) in rat pregnancy, we determined the density of myometrial CGRP-encoded nerve fibre terminals and examined, in an organ bath, the relaxant effect of the peptide on uterine strips near parturition. Comparisons were made with the uterus and aorta of nonpregnant rats. In the myometrium, CGRP immunoreactive nerve fibers were abundant in nonpregnant rats and scarce at the parturient stage. In the aorta there was no variation in the density of CGRP fibres with gestation. In nonpregnant rats only, CGRP relaxed spontaneous and tetrodotoxin (TTX)-sensitive electrically-evoked uterine contractions (EC50 40 nM, Emax 80%). The effect was antagonized by CGRP[8-37] (pKB 6.47) but was not affected by either blockers of nitricoxid synthase or ATP-sensitive potassium channels. CGRP was also able to relax contractions evoked by direct depolarization of the cells (TTX-insensitive contractions) (EC50, 2 nM, Emax 70%). In aorta contracted with arginine vasopressin, CGRP-induced relaxation was the same in nonpregnant and parturient animals. It was antagonized by CGRP [8-371 (pKB 6.90) and was abolished in presence of the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). Amylin neither relaxed the uterus nor the aorta. In pregnant rats, the relaxant effect of CGRP on the uterus was limited on day 21 and was totally absent on day 22 of gestation. We conclude that the primary relaxant effect of CGRP on the uterus occurs at the level of myometrial smooth muscle cells. In the myometrium, gestation decreases CGRP innervation and impairs the relaxant responses to CGRP. Such changes are not observed in vascular tissues like aorta.     

Regulation of DNA-dependent protein kinase by the Lyn tyrosine kinase

The Src-like protein-tyrosine kinase Lyn is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of the catalytic subunits (DNA-PKcs) and Ku DNA-binding components, requires DNA double-stranded breaks for activation. Here we demonstrate that Lyn associates constitutively with DNA-PKcs. The SH3 domain of Lyn interacts directly with DNA-PKcs near a leucine zipper homology domain. We also show that Lyn phosphorylates DNA-PKcs but not Ku in vitro. The interaction between Lyn and DNA-PKcs inhibits DNA-PKcs activity and the ability of DNA-PKcs to form a complex with Ku/DNA. These results support the hypothesis that there are functional interactions between Lyn and DNA-PKcs in the response to DNA damage.     

Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling

TCR stimulation results in the tyrosine phosphorylation of a number of cellular substrates. We have recently identified a 70-kDa protein tyrosine kinase, ZAP-70, which associates with the human TCR zeta-chain after TCR stimulation. We report here the isolation and sequence of a cDNA clone that encodes murine ZAP-70. Murine and human ZAP-70 share 93% amino acid identity and are homologous to the 72-kDa protein tyrosine kinase Syk. Syk has been implicated in the signal transduction pathways of the B cell membrane Ig and high affinity IgE receptors, Fc epsilon RI. In addition, we examined the tissue distribution of ZAP-70 and Syk in human and murine thymocyte subsets, B cells, and peripheral T cell subsets. ZAP-70 protein is expressed in all major thymocyte populations, with the level of expression being comparable to that found in both CD4+ and CD8+ peripheral T cells. Although Syk protein is also present in all thymocyte subsets, expression of Syk protein is down-regulated threefold to fourfold in peripheral T cells. In contrast to ZAP-70, expression of Syk is 12- to 15-fold higher in peripheral B cells when compared with peripheral T cells. In addition, whereas T cell stimulation results in down-regulation of Lck, no significant change in ZAP-70 or Syk protein is detected. Finally, we provide evidence that both ZAP-70 and Syk can associate with the TCR after TCR stimulation. With the use of a heterologous expression system, we show that, like ZAP-70, Syk is dependent upon a Src-family protein tyrosine kinase for association with the phosphorylated zeta-chain. Thus, the differential expression of these kinases suggests the possibility of different roles for ZAP-70 and Syk in TCR signaling and thymic development.     

Regulation of T-cell antigen receptor signalling by Syk tyrosine protein kinase

T-cell antigen receptor (TCR) signalling has been shown to involve two classes of tyrosine protein kinases: the Src-related kinases p56(lck) and p59(fyr), and the Zap-70/Syk family kinases. Lck and FynT are postulated to initiate TCR-triggered signal transduction by phosphorylating the CD3 and zeta subunits of the TCR complex. This modification permits the recruitment of Zap-70 and Syk, which are presumed to amplify the TCR-triggered signal, by phosphorylating additional intracellular proteins. While Zap-70 is expressed in all T cells, Syk is present in thymocytes and mature T-cell populations such as intraepithelial gammadelta T cells and naive alphabeta T cells. To better understand the role of Syk in these cells, its impact on the physiology of an antigen-specific T-cell line was tested. Our results showed that compared to Zap-70 alone, Syk was a strong positive regulator of antigen receptor-induced signals in BI-141 cells. Surprisingly, they indicated that, like Src family kinases, Syk augmented TCR-triggered tyrosine phosphorylation of CD3/zeta. Syk, but not Zap-70 alone, could also stimulate tyrosine phosphorylation of a zeta-bearing chimera in transiently transfected Cos-1 cells. Finally, evidence was provided that Syk has the capacity to directly phosphorylate a zeta-derived peptide in vitro. These findings suggested that Syk may have a unique role in T cells, as a consequence of its ability to efficiently phosphorylate multiple components of the TCR signalling cascade. Furthermore, they raised the possibility that Syk can regulate the initiation of TCR signalling, by promoting phosphorylation of the immunoreceptor tyrosine-based activation motifs of the TCR complex.     

Structural changes in lenses of mice lacking the gap junction protein connexin43

PURPOSE: To investigate the role of the gap junction protein connexin43 (Cx43), which is predominantly expressed in lens epithelial cells in the control of lens development and organization. METHODS: Newborn mice in which the Cx43 gene was disrupted by homologous recombination were used. Lenses from Cx43 (-/-) mice and wild-type littermates were processed by using 2% glutaraldehyde fixation for light and transmission electron microscopy and by freezing in liquid nitrogen for light and confocal microscopy of immunofluorescence in cryosections. RESULTS: In wild-type mice, Cx43 was immunolocalized to apical and lateral regions of lens epithelial cells and throughout the cornea, iris, ciliary body, and retina. In the bow, or equatorial, region of the lens, Cx43 disappeared gradually at the margins of the epithelial layer, whereas major intrinsic polypeptide, MP26, and alpha-crystallins were only detected in differentiated fiber cells. Ultrastructural studies revealed that epithelial cells and epithelial fiber cells were connected by large gap junctions. Lens fiber cells were closely apposed to apical boundaries of epithelial cells and apposed to one another along their entire lengths. In Cx43 (-/-) mice, epithelial cells were connected more loosely. The distribution of MP26 and alpha-crystallin in bow region fiber cells in Cx43 (-/-) lenses was not distinguishable from that in the lenses of wild-type mice. Cx46 and Cx50 were also expressed in superficial and cortical fiber cells, with similar distributions in Cx43 (-/-) and wild-type mice. However, organization of appositional membranes between lens fiber cells and between fiber and epithelial cells differed dramatically in the Cx43 (-/-) lens. In contrast to the close apposition of cells in lenses of normal mice, fiber cells in Cx43 (-/-) lenses were largely separated from apical surfaces of epithelial cells, and large vacuolar spaces were apparent between fiber cells, most prominently in deeper cortical regions. CONCLUSIONS: The normal differentiation of lens fiber cells in the bow region in lenses of Cx43 (-/-) mice, evidenced by similar distributions of Cx46, Cx50, MP26, and alpha-crystallin, suggests that the expression of Cx43 is not required for this process. However, these lenses exhibit grossly dilated extracellular spaces and intracellular vacuoles, indicative of early stages of cataract formation. These changes suggest that osmotic balance within the lens is markedly altered in Cx43 (-/-) animals, highlighting the importance of intercellular communication mediated by lens epithelial Cx43 gap junctions in the function of this tissue.     

Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase

Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.     

The Eek receptor, a member of the Eph family of tyrosine protein kinases, can be activated by three different Eph family ligands

The Eph family of receptors, the largest subgroup within the tyrosine protein kinase receptor family, are comprised of at least thirteen members, many of which are predominantly expressed in the developing and adult nervous system. In this study, we have isolated a full-length cDNA, encoding the mouse homologue of a previous partially characterized Eek protein, a member of Eph receptor tyrosine kinase family. In a comparison of the amino acid sequences of various Eph family members, Eek is most similar to Ehk-3/MDK1, Sek/Cek8, Ehk-2, Hek/Mek4/Cek4, and Bsk/Ehk1/Rek7/Cek7, which are predominantly expressed in the nervous system. Additionally, we have used a low-stringency PCR cloning technique to identify ligands, related to B61, that may interact with Eek. Three different GPI-linked ligands, namely Elf-1/Cek7-L, Ehk1-L/Efl-2/Lerk3 and AL-1/RAGS, were isolated from mouse brain. To study the functional interactions between these ligands and the Eek receptors, we have constructed chimeric ligands consisting of the Fc portion of human IgG fused to their carboxyl-terminus. These chimeric ligands bound to, and activated both the Eek receptors and the Eek-TrkB chimeric receptors expressed in NIH3T3 cells. These findings suggest that Eek receptor can be activated by at least three different GPI-linked ligands.     

rlk/TXK encodes two forms of a novel cysteine string tyrosine kinase activated by Src family kinases

Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR.     

Involvement of protein tyrosine kinases in regulation of endothelial cell organization by basement membrane proteins

A complete understanding of the biological mechanisms that take part in the hyperacute vascular rejection is necessary to prevent its development. Nitric Oxide (NO) release by the graft could be one of those mechanisms as cytokine induction of NO synthase in reticuloendothelial cells produces NO with cytotoxic activity. We have performed 20 heterotopic pig-to-dog kidney xenotransplantations in order to determine if cytotoxic NO is released during the hyperacute rejection. Nitrites and cGMP levels were determined in plasma samples taken from the graft artery and vein until vessel thromboses. NO synthase activity was measured in pieces from renal artery and renal tissue. The results show that after discordant xenotransplantation, inducible NO synthase is activated in the transplanted organ, indicating that NO can mediate host tissue damage during the hyperacute rejection.     

Role of the mitogen-activated protein kinases and tyrosine kinases during leukotriene B4-induced eosinophil activation

Exposure of guinea-pig eosinophils to leukotriene B4 (LTB4; 1 microM) resulted in a rapid generation of H2O2 (index of NADPH oxidase activation), stimulated [3H]arachidonic acid (AA) release (index of phospholipase A2 activity), and promoted CD18-dependent homotypic aggregation. Under similar conditions, LTB4 (1 microM) induced a rapid activation of extracellular-regulated kinases-1 and 2 (ERK-1/2) but not c-jun N-terminal kinases 46 and 54 (JNK-46/54) or p38 mitogen-activated protein kinase (p38 MAP kinase). To examine the role of ERK-1/2 in the mechanism of eosinophil activation, a selective inhibitor of MAP kinase kinase-1/2 (MEK-1/2), PD098059, was employed. However, PD 098059 at concentrations that attenuated ERK-1/2 activation had no significant affect on eosinophil activation. In contrast, a role for tyrosine kinases in LTB4-induced eosinophil activation was suggested by studies with the tyrosine kinase inhibitors, herbimycin A and lavendustin A. However, the results of those experiments implied divergent pathways for the control of eosinophil responses because the inhibitors were more effective at attenuating H2O2 generation than [3H]AA release, and had little effect on homotypic aggregation.     

Gap junctional communication and the tyrosine phosphorylation of connexin 43 in interaction between breast cancer and endothelial cells

An antigenic double mutant of rabies virus (challenge virus standard [CVS] strain) was selected by successive use of two neutralizing antiglycoprotein monoclonal antibodies, both specific for antigenic site III. This mutant differed from the original virus strain by two amino acid substitutions in the ectodomain of the glycoprotein. The lysine in position 330 and the arginine in position 333 were replaced by asparagine and methionine, respectively. This double mutant was not pathogenic for adult mice. When injected intramuscularly into the forelimbs of adult mice, this virus could not penetrate the nervous system, either by the motor or by the sensory route, while respective single mutants infected motoneurons in the spinal cord and sensory neurons in the dorsal root ganglia. In vitro experiments showed that the double mutant was able to infect BHK cells, neuroblastoma cells, and freshly prepared embryonic motoneurons, albeit with a lower efficiency than the CVS strain. Upon further incubation at 37 degrees C, the motoneurons became resistant to infection by the mutant while remaining permissive to CVS infection. These results suggest that rabies virus uses different types of receptors: a molecule which is ubiquitously expressed at the surface of continuous cell lines and which is recognized by both CVS and the double mutant and a neuron-specific molecule which is not recognized by the double mutant.