The CD45 tyrosine phosphatase regulates phosphotyrosine homeostasis and its loss reveals a novel pattern of late T cell receptor-induced Ca2+ oscillations.

CD45 is a transmembrane tyrosine phosphatase implicated in T cell antigen receptor (TCR)-mediated activation. In T cell variants expressing progressively lower levels of CD45 (from normal to undetectable), CD45 expression was inversely related to spontaneous tyrosine phosphorylation of multiple proteins, including the TCR zeta chain, and was directly correlated with TCR-driven phosphoinositide hydrolysis. The Ca2+ response in these cells was altered in an unexpected fashion. Unlike wild-type cells, stimulated CD45- cell populations did not manifest an early increase in intracellular Ca2+, but did exhibit a delayed and gradual increase in mean intracellular Ca2+. Computer-aided fluorescence imaging of individual cells revealed that CD45- cells experienced late Ca2+ oscillations that were not blocked by removal of extracellular Ca2+. CD45 revertants had the signaling properties of wild-type cells. Thus, CD45 has a profound influence on both TCR-mediated signaling and phosphotyrosine homeostasis, and its loss reveals a novel role for this tyrosine phosphatase in Ca2+ regulation.

Regulation of TCR signaling by CD45 lacking transmembrane and extracellular domains.

The CD45 protein is a transmembrane tyrosine phosphatase that is required for normal T cell receptor (TCR)-mediated signaling. A chimeric complementary DNA encoding the intracellular enzymatically active portion of murine CD45 preceded by a short amino-terminal sequence from p60c-src was transfected into CD45- T cells. Expression of this chimeric protein corrected most of the TCR signaling abnormalities observed in the absence of CD45, including TCR-mediated enhancement of tyrosine kinase activity and Ca2+ flux. Thus, the enzymatically active intracellular portion of CD45 is sufficient to allow TCR transmembrane signaling.

Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6.

Because ribosome biogenesis plays an essential role in cell proliferation, control mechanisms may have evolved to recognize lesions in this critical anabolic process. To test this possibility, we conditionally deleted the gene encoding 40S ribosomal protein S6 in the liver of adult mice. Unexpectedly, livers from fasted animals deficient in S6 grew in response to nutrients even though biogenesis of 40S ribosomes was abolished. However, liver cells failed to proliferate or induce cyclin E expression after partial hepatectomy, despite formation of active cyclin D-CDK4 complexes. These results imply that abrogation of 40S ribosome biogenesis may induce a checkpoint control that prevents cell cycle progression.

Role of S6 phosphorylation and S6 kinase in cell growth.

This article reviews our current knowledge of the role of ribosomal protein S6 phosphorylation and the S6 kinase (S6K) signaling pathway in the regulation of cell growth and proliferation. Although 40S ribosomal protein S6 phosphorylation was first described 25 years ago, it only recently has been implicated in the translational up-regulation of mRNAs coding for the components of protein synthetic apparatus. These mRNAs contain an oligopyrimidine tract at their 5' transcriptional start site, termed a 5'TOP, which has been shown to be essential for their regulation at the translational level. In parallel, a great deal of information has accumulated concerning the identification of the signaling pathway and the regulatory phosphorylation sites involved in controlling S6K activation. Despite this knowledge we are only beginning to identify the direct upstream elements involved in growth factor-induced kinase activation. Use of the immunosupressant rapamycin, a bacterial macrolide, in conjunction with dominant interfering and activated forms of S6K1 has helped to establish the role of this signaling cascade in the regulation of growth and proliferation. In addition, current studies employing the mouse as well as Drosophila melanogaster have provided new insights into physiological function of S6K in the animal. Deletion of the S6K1 gene in mouse cells led to an animal of reduced size and the identification of the S6K1 homolog, S6K2, whereas loss of dS6K function in Drosophila demonstrated its paramount importance in development and growth control.

Dominant negative variants of the SHP-2 tyrosine phosphatase inhibit prolactin activation of Jak2 (janus kinase 2) and induction of Stat5 (signal transducer and activator of transcription 5)-dependent transcription.

PRL plays a central role in the regulation of milk protein gene expression in mammary epithelial cells and in the growth and differentiation of lymphocytes. It confers its activity through binding to a specific transmembrane, class I hematopoietic receptor. Ligand binding leads to receptor dimerization and activation of the tyrosine kinase Jak (janus kinase) 2, associated with the membrane-proximal, intracellular domain of the receptor. Jak2 phosphorylates and activates Stat5, a member of the Stat (signal transducers and activators of transcription) family. PRL receptor also activates SHP-2, a cytosolic tyrosine phosphatase. We investigated the connection between these two signaling events and derived a dominant negative mutant of SHP-2 comprising the two SH2 domains [SHP-2(SH2)2]. An analogous variant of the SHP-1 phosphatase [SHP-1(SH2)2] was used as a control. The dominant negative mutant of SHP-2 was found to inhibit the induction of tyrosine phosphorylation and DNA-binding activity of m-Stat5a, m-Stat5b, and the carboxyl-terminal deletion variant m-Stat5adelta749, as well as the transactivation potential of m-Stat5a and m-Stat5b. The dominant negative mutant SHP-1(SH2)2 had no effect. The kinase activity of Jak2 is also dependent on a functional SHP-2 phosphatase. We propose that SHP-2 relieves an inhibitory tyrosine phosphorylation event in Jak2 required for Jak2 activity, Stat5 phosphorylation, and transcriptional induction.

Physical and functional interaction of the TPL2 kinase with nucleophosmin.

Tumor Progression Locus 2 (TPL2) is widely recognized as a cytoplasmic mitogen-activated protein 3 kinase with a prominent role in the regulation of inflammatory and oncogenic signal transduction. Herein we report that TPL2 may also operate in the nucleus as a physical and functional partner of nucleophosmin (NPM/B23), a major nucleolar phosphoprotein with diverse cellular activities linked to malignancy. We demonstrate that TPL2 mediates the phosphorylation of a fraction of NPM at threonine 199, an event required for its proteasomal degradation and maintenance of steady-state NPM levels. Upon exposure to ultraviolet C, Tpl2 is required for the translocation of de-phosphorylated NPM from the nucleolus to the nucleoplasm. NPM is an endogenous inhibitor of HDM2:p53 interaction and knockdown of TPL2 was found to result in reduced binding of NPM to HDM2, with concomitant defects in p53 accumulation following genotoxic or ribosomal stress. These findings expand our understanding of the function of TPL2 as a negative regulator of carcinogenesis by defining a nuclear role for this kinase in the topological sequestration of NPM, linking p53 signaling to the generation of threonine 199-phosphorylated NPM.

The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells.

Data on the relationship between ribosome biogenesis and p53 function indicate that the tumour suppressor can be activated by either nucleolar disruption or ribosomal protein defects. However, there is increasing evidence that the induction of p53 does not always require these severe cellular changes, and data are still lacking on a possible role of ribosome biogenesis in the downregulation of p53. Here, we studied the effect of the up- and downregulation of the rRNA transcription rate on p53 induction in mammalian cells. We found that a downregulation of rRNA synthesis, induced by silencing the POLR1A gene coding for the RNA polymerase I catalytic subunit, stabilised p53 without altering the nucleolar integrity in human cancer cells. p53 stabilisation was due to the inactivation of the MDM2-mediated p53 degradation by the binding of ribosomal proteins no longer used for ribosome building. p53 stabilisation did not occur when rRNA synthesis downregulation was associated with a contemporary reduction of protein synthesis. Furthermore, we demonstrated that in three different experimental models characterised by an upregulation of rRNA synthesis, cancer cells treated with insulin or exposed to the insulin-like growth factor 1, rat liver stimulated by cortisol and regenerating rat liver after partial hepatectomy, the p53 protein level was reduced due to a lowered ribosomal protein availability for MDM2 binding. It is worth noting that the upregulation of rRNA synthesis was responsible for a decreased p53-mediated response to cytotoxic stresses. These findings demonstrated that the balance between rRNA and ribosomal protein synthesis controls the function of p53 in mammalian cells, that p53 can be induced without the occurrence of severe changes of the cellular components controlling ribosome biogenesis, and that conditions characterised by an upregulated rRNA synthesis are associated with a reduced p53 response.

Manipulating mammalian genome by gene targeting.

The development of strategies which allow the inactivation of specific murine genes by homologous recombination in embryonic cells has revolutionized biological science in the last 10 years. A large number of mice carrying genetic lesions, generated by gene targeting technology, has tremendously increased our knowledge in many areas of biology, culminating in the identification of mouse models for human genetic disorders. These findings have been recently complemented by "conditional" gene targeting technology, allowing gene inactivation in a defined tissue and at a specific time point during development or adulthood, thereby extending the sophistication and potential of this technology.

Evidence for different mechanisms of growth inhibition of T-cell lymphoma by phorbol esters and concanavalin A.

Stimuli that are mitogenic for mature T-cells induce cell cycle arrest in some T-cell tumors and T-cell hybridomas. The molecular mechanism of this growth inhibition is poorly understood. In this report, we show that in EL4, a murine T-lymphoma cell line, stimulation with concanavalin A or treatment with phorbol 13-myristate 12-acetate (PMA) inhibit growth, due to cell cycle arrest at both the G1 and the G2/M phases. The block at the G1 phase is accompanied by the appearance of a hypophosphorylated form of the retinoblastoma protein (pRb), due to the inhibition of G1 cyclin-Cdk complexes. However, the molecular mechanisms leading to this G1 cell cycle arrest differ between concanavalin A and PMA: concanavalin A inhibits both cyclin E-Cdk2 and cyclin D-Cdk4 complexes, while PMA inhibits only cyclin E-Cdk2. We demonstrate that concanavalin A inhibits cyclin D-Cdk4 activity by decreasing the amount of cyclin D. The inhibition of cyclin E-Cdk2 by both concanavalin A and PMA is due to increased binding of the Cdk inhibitor p21 to this complex. However, while stimulation of the cells with concanavalin A did not result in an evident increase of the total level of p21, treatment of the cells with PMA increased p21 levels significantly. Our results indicate, furthermore, that the G2/M block results from the inhibition of cyclin A- and cyclin B1-associated kinase activities. As for cyclin E-Cdk2, the inhibition of the cyclin A-Cdk2 complex is due to increased binding of the p21 inhibitor.

[The effect of physical exercise on lung diffusing capacity in patients with chronic obstructive lung disease]. / Utjecaj fizickog opterecenja na difuzijski kapacitet pluca u bolesnika s kronicnom opstrukcijskom bolesti pluca.

Diffusing lung capacity (DLCO) was determined at various levels of exercise in three groups of subjects: group with normal spirometry, patients with moderate and severe chronic obstructive pulmonary disease (COPD). Subjects were males with similar anthropometric characteristics and age. Positive correlation between progressive exercise and DLCO was obtained only for the first and second group. Resting and exercise DLCO values were statistically different between the groups. COPD attenuated DLCO at all levels of exercise. However, patients with moderate COPD had DLCO in the normal range, while severe COPD patients had reduction in DLCO during rest and exercise. This test is useful in assessing the working capacity in COPD patients and also as a screening test for exercise-induced hypoxemia.

Activation of STAT proteins and cytokine genes in human Th1 and Th2 cells generated in the absence of IL-12 and IL-4.

We have shown previously that human CD4+ 45RO- T cells could be primed for a Th2 phenotype independent of IL-4 if they were activated by anti-CD28 mAb plus IL-2. If additional TCR signals were provided, the cells differentiated toward Th1 independent of IL-12. Here we show that anti-CD28/IL-2-primed Th2 cells expressed high levels of activated STAT6, but no cytokine mRNA. Moreover, both Th1 and Th2 cells expressed active STAT1 and -3, but not STAT2, -4, and -5. Restimulation of Th1 or Th2 cells via CD3 plus CD28 induced production of IFN-gamma or IL-4, respectively, but did not alter the activation status/DNA binding activity of STATs. Addition of IL-4 (or anti-IL-4 mAb) to restimulated Th2 cells did not modulate STAT6 activation or IL-4 expression, confirming the full commitment. However, Th2 cells remained responsive to IL-12, which repressed STAT6 DNA binding but activated STAT4, and this coincided with a suppression of IL-4/IL-5 and an induction of IFN-gamma. In Th1 cells, IL-12 activated both STAT6 and STAT4, and IL-4 activated STAT6, but in both cases the Th1 phenotype remained. Together the data show that CD28/IL-2-dependent Th2 priming activated STAT6 without inducing IL-4 expression. The primed Th cells resembled memory cells and produced IL-4 upon the first CD3/CD28 costimulus without detectable modulation of STATs. Th2 cells remained responsive to IL-12, which repressed STAT6 DNA binding and activated STAT4, and switched the cells to Th1. The effects of IL-12 may depend on the commitment of the cells, since IL-12 phosphorylated STAT6 in Th1 and dephosphorylated STAT6 in Th2 cells.

Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase.

Immunoprecipitation of Thy-1 from Triton X-100 detergent lysates of surface-iodinated and chemically cross-linked T cells precipitated at least five major and discrete bands. Four of these bands were identified as Thy-1, CD45 (a transmembrane tyrosine phosphatase), a major histocompatibility complex-encoded class I molecule, and beta 2-microglobulin. Similar analyses revealed that CD45 was coprecipitated from lysates of cross-linker-treated cells by antibodies to the T-cell antigen receptor (TCR). The same pattern of coprecipitated bands was observed when digitonin was used to lyse untreated cells. Immunoprecipitation of Thy-1 or the TCR from lysates of cross-linked T cells precipitated CD45 tyrosine phosphatase activity. Calculations based upon the amounts of coprecipitated enzymatic activity or TCR zeta chain indicate that a substantial fraction of Thy-1 and TCR complexes can be cross-linked to CD45. These data support a model in which the dependence of Thy-1 signaling on TCR coexpression is due to their common interaction with a tyrosine phosphatase and provide a possible structural basis for the influence of CD45 on TCR-mediated signaling.

CD45 is required for CD40-induced inhibition of DNA synthesis and regulation of c-Jun NH2-terminal kinase and p38 in BAL-17 B cells.

Stimulation of B cell antigen receptor (BCR) may induce proliferation, differentiation, or apoptosis, depending upon the maturational stage of the cell and the presence or absence of signals transmitted via coreceptors. One such signal is delivered via CD40; for instance, ligation of CD40 rescues B cells from BCR-induced apoptosis. Here we show that, in contrast to WEHI-231 cells, CD40 ligation did not reverse BCR-induced growth inhibition in the BAL-17 mature B cell line and CD40 ligation itself inhibited proliferation. This inhibitory signaling was not observed in CD45-deficient cells. Further analyses demonstrate that transfection of dominant-negative form of SEK1 or treatment with SB203580 strongly reduced CD40-induced inhibition of BAL-17 proliferation, suggesting a requirement for c-Jun NH2-terminal kinase and p38 in CD40-induced inhibition of proliferation. Interestingly, CD40-initiated activation of c-Jun NH2-terminal kinase and p38 was enhanced and sustained in CD45-deficient cells, and these phenotypes were reversed by transfecting CD45 gene. However, CD40-mediated induction of cell surface molecules was not affected in CD45-deficient cells. Taken collectively, these results suggest that CD45 exerts a decisive effect on selective sets of CD40-mediated signaling pathways, dictating B cell fate.