No abstract

CD45 is a key protein tyrosine phosphatase regulating Src-family protein tyrosine kinases (Src-PTKs) in lymphocytes; precisely how it exerts its effect remains controversial, however. We previously demonstrated that CD45 negatively regulates Lyn in the WEHI-231 B-cell line. Here we show that negative regulation by CD45 is physiologically significant in B cells and that some CD45 is constitutively associated with glycolipid-enriched mi- IntroductionUpon B-cell receptor (BCR) ligation, signals are transmitted downstream through multiple pathways, ultimately leading to cell activation, proliferation, death, or anergy. [1][2][3][4] Initiation of such immune responses is driven primarily by activation of Src-family protein tyrosine kinases (Src-PTKs) whose activity is regulated, in part, by phosphorylation of 2 tyrosine residues: the autophosphorylation site, located in the activation loop of the catalytic domain, and the COOH-terminal negative regulatory site. 5 Phosphorylation of the former is a prerequisite for Src-PTK activation. Phosphorylation of the latter by COOH-terminal Src kinase (Csk) results in the intramolecular binding of the phosphotyrosine (PY) in the COOHterminal tail to the Src homology 2 (SH2) domain, yielding a closed conformation in which Src-PTK activity is attenuated. Thus, phosphorylation of the respective regulatory tyrosine residues has opposing effects on Src-PTK activity.CD45, a receptor-type protein tyrosine phosphatase (PTP) exclusively expressed in nucleated hematopoietic cells, is known to be a major regulator of Src-PTK activation and of lymphocyte fate, 6-8 though its precise role in the regulation of Src-PTKs remains controversial. [9][10][11][12][13] One conventional model holds that when Csk phosphorylates the negative regulatory COOH-terminal tyrosine residue, leading to the aforementioned closed and inactive conformation, the dominant role of CD45 is to dephosphorylate this site, setting the stage for antigen receptor signaling to activate Src-PTKs by autophosphorylation. 14,15 This scenario is based on the findings that the COOH-terminal tyrosine residues of Lck and Fyn are hyperphosphorylated and T-cell receptor (TCR) signaling is attenuated in CD45-deficient T-cell lines and in thymocytes from Ptprc-targeted mice. [16][17][18][19][20] Moreover, similar results have also been obtained in B cells. [21][22][23] However, CD45 is also shown to dephosphorylate both of Src-PTK's regulatory tyrosine residues in both T cells 24-26 and B cells, 27,28 thereby inactivating it.We previously showed that CD45 exerts a negative regulatory effect on BCR-induced Ca 2ϩ mobilization, activation of c-Jun NH 2 -terminal kinase (JNK) and p38, and growth arrest in immature WEHI-231 cells, 29,30 but exerts a positive regulatory effect on these processes in mature BAL-17 cells. 30,31 We further showed that, in WEHI-231 cells, CD45 constitutively inhibits Lyn by dephosphorylating both the COOH-terminal and autophosphorylation sites, but that BCR ligation induces phosphorylation of the 2 sites. 27, 2...

Nitrogen dioxide is a highly toxic reactive nitrogen species (RNS) recently discovered as an inflammatory oxidant with great potential to damage tissues. We demonstrate here that cell death by RNS was caused by c-Jun N-terminal kinase (JNK). Activation of JNK by RNS was density dependent and caused mitochondrial depolarization and nuclear condensation.

Eosinophilic influx is characteristic of numerous inflammatory conditions. Eosinophil peroxidase (EPO) is a major enzyme present in eosinophils and upon degranulation, becomes released into the airways of asthmatics. As a result of its cationic nature and its ability to catalyze the formation of highly toxic oxidants, EPO has significant potential to induce cellular injury. The focus of the present study was to determine the cell-signaling events important in EPO-induced death of lung epithelial cells. In the presence of hydrogen peroxide and nitrite (NO2-; hereafter called EPO with substrates), EPO catalyzes the formation of nitrogen dioxide. EPO with substrates induced rapid and sustained activation of c-Jun-NH2-terminal kinase (JNK) and led to cell death, as was evidenced by enhanced mitochondrial depolarization, cytochrome c release, cleavage of caspases 9 and 3, poly-adenosine 5'-diphosphate ribosylation of proteins, the formation of single-stranded DNA, and membrane permeability. Moreover, EPO with substrates caused Rho-associated coiled coil-containing kinase-1-dependent dynamic membrane blebbing. Inhibition of JNK activity in cells expressing a dominant-negative JNK-1 construct (JNK-APF) prevented mitochondrial membrane depolarization and substantially decreased the number of cells blebbing compared with vector controls. The cellular responses to EPO with substrates were independent of whether NO2-, bromide, or thiocyanide was used as substrates. Our findings demonstrate that catalytically active EPO is capable of causing significant damage to lung epithelial cells in vitro and that this involves the activation of JNK.

Anticancer agents effect tumor cell killing both in vivo and in vitro through the induction of apoptosis. Endonuclease-mediated internucleosomal DNA fragmentation, the most widely used biochemical marker of apoptosis, has been shown to play a central role in apoptosis in many experimental systems. In the present investigation, we report that activation of endonuclease(s) leading to oligonucleosomal DNA fragmentation is common and an essential event in apoptosis, induced by different anticancer drugs, adriamycin, etoposide and cisplatin. The endonuclease inhibitors aurintricarboxylic acid and zinc ion prevented apoptotic cell death in human monocytic leukemic cell line U937, as documented by DNA fragmentation, morphological and nuclear alterations, and cell viability assay. Additional studies suggest endonuclease(s)-mediated DNA fragmentation may not play a central role in apoptosis in the same cell line in response to other inducers such as heat shock and cells may undergo cell death showing all morphological features of apoptosis even in the absence of DNA fragmentation.