We have investigated the mechanism of mitochondrialnuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Δψ m ) and electron transport-coupled ATP synthesis, which was also accompanied by an elevated steady-state cytosolic Ca 2⍣ level ([Ca 2⍣ ] i ). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor-1 (RyR-1), hence potentiating the Ca 2⍣ release in response to its modulator, caffeine; (ii) enhanced levels of Ca 2⍣ -responsive factors calineurin, calcineurindependent NFATc (cytosolic counterpart of activated T-cell-specific nuclear factor) and c-Jun N-terminal kinase (JNK)-dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF-κB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady-state [Ca 2⍣ ] i were normalized in genetically reverted cells which contain near-normal mtDNA levels. We propose that the mitochondria-to-nucleus stress signaling occurs through cytosolic [Ca 2⍣ ] i changes, which are likely to be due to reduced ATP and Ca 2⍣ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

SLAP-130/Fyb (SLP-76-associated phosphoprotein or Fyn-binding protein; also known as Fyb/Slap) is a hematopoietic-specific adapter, which associates with and modulates function of SH2-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76). T cells from mice lacking SLAP-130/Fyb show markedly impaired proliferation following CD3 engagement. In addition, the T cell receptor (TCR) in SLAP-130/Fyb mutant cells fails to enhance integrin-dependent adhesion. Although TCR-induced actin polymerization is normal, TCR-stimulated clustering of the integrin LFA-1 is defective in SLAP-130/Fyb-deficient cells. These data indicate that SLAP-130/Fyb is important for coupling TCR-mediated actin cytoskeletal rearrangement with activation of integrin function, and for T cells to respond fully to activating signals.

Actomyosin dynamics and T cell receptor signaling are tightly coupled to ensure proper dynamics and function of signaling microclusters within the immunological synapse.

These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

HS1, the leukocyte-specific homolog of cortactin, regulates F-actin in vitro and is phosphorylated in response to TCR ligation, but its role in lymphocyte activation has not been addressed. We demonstrate that HS1-deficient T cells fail to accumulate F-actin at the immune synapse (IS) and, upon TCR ligation, form actin-rich structures that are disordered and unstable. Early TCR activation events are intact in these cells, but Ca2+ influx and IL-2 gene transcription are defective. Importantly, HS1 tyrosine phosphorylation is required for its targeting to the IS and for its function in regulating actin dynamics and IL-2 promoter activity. Phosphorylation also links HS1 to multiple signaling proteins, including Lck, PLCgamma1, and Vav1, and is essential for the stable recruitment of Vav1 to the IS. Taken together, our studies show that HS1 is indispensable for signaling events leading to actin assembly and IL-2 production during T cell activation.

Human immunodeficiency virus type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry. It was recently demonstrated that HIV-1 glycoprotein 120 (gp120) elevated calcium and activated several ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. This study shows that chemokine receptor engagement by both CCR5-dependent (R5) and CXCR4-dependent (X4) gp120 led to rapid phosphorylation of the focal adhesion-related tyrosine kinase Pyk2 in macrophages. Pyk2 phosphorylation was also induced by macrophage inflammatory protein-1␤ (MIP-1␤) and stromal cell-derived factor-1␣, chemokine ligands for CCR5 and CXCR4. Activation was blocked by EGTA and by a potent blocker of calcium release-activated Ca ؉؉ (CRAC) channels, but was insensitive to pertussis toxin (PTX), implicating CRAC-mediated extracellular Ca ؉؉ influx but not G␣ i protein-dependent mechanisms. Coreceptor engagement by gp120 and chemokines also activated 2 members of the mitogen-activated protein kinase (MAPK) superfamily, c-Jun amino-terminal kinase/stress-activated protein kinase and p38 MAPK. Furthermore, gp120-stimulated macrophages secreted the chemokines monocyte chemotactic protein-1 and MIP-1␤ in a manner that was dependent on MAPK activation. Thus, the gp120 signaling cascade in macrophages includes coreceptor binding, PTX-insensitive signal transduction, ionic signaling including Ca ؉؉ influx, and activation of Pyk2 and MAPK pathways, and leads to secretion of inflammatory mediators. HIV-1 Env signaling through these pathways may contribute to dysregulation of uninfected macrophage functions, new target cell recruitment, or modulation of macrophage infection. IntroductionHuman immunodeficiency virus type 1 (HIV-1) infection is initiated by the formation of a trimolecular complex on the cell surface, consisting of the HIV-1 envelope (Env) glycoprotein 120 (gp120), the principal cellular receptor CD4, and a chemokine receptor that functions as a coreceptor (reviewed by Berger 1 ). Conformational changes induced in gp120 on binding to CD4 increase its affinity for the coreceptor, leading to fusion between the viral and cellular membranes. The chemokine receptors that serve as HIV coreceptors are members of the 7 transmembrane G protein-coupled receptor (GPCR) family. CCR5 and CXCR4 play a dominant role in HIV-1 entry into primary cells, and are the principal coreceptors used by macrophage (M)-tropic (R5) and T-cell line (T)-tropic (X4) isolates, respectively.Given their normal cell signaling function, the interaction of gp120 with the chemokine receptors may, in addition to facilitating viral entry, also result in activation of cellular responses that could modulate the activation status of the cells and affect postentry stages of HIV replication (reviewed by Popik and Pitha 2 ). Although chemokine receptors and their ligands play central roles in both HIV infection and in immune regulation, how signaling pathways mediated by CCR5 and CXCR4 contribute to the immunopathogenesis...

The highly homologous proteins ezrin, radixin, and moesin link proteins to the actin cytoskeleton. The two family members expressed in T cells, ezrin and moesin, are implicated in promoting T cell activation and polarity. To elucidate the contributions of ezrin and moesin, we conducted a systematic analysis of their function during T cell activation. In response to TCR engagement, ezrin and moesin were phosphorylated in parallel at the regulatory threonine, and both proteins ultimately localized to the distal pole complex (DPC). However, ezrin exhibited unique behaviors, including tyrosine phosphorylation and transient localization to the immunological synapse before movement to the DPC. To ask whether these differences reflect unique requirements for ezrin vs moesin in T cell signaling, we generated mice with conditional deletion of ezrin in mature T cells. Ezrin−/− T cells exhibited normal immunological synapse organization based upon localization of protein kinase C-θ, talin, and phospho-ZAP70. DPC localization of CD43 and RhoGDP dissociation inhibitor, as well as the novel DPC protein Src homology region 2 domain-containing phosphatase-1, was also unaffected. However, recruitment of three novel DPC proteins, ezrin binding protein of 50 kDa, Csk binding protein, and the p85 subunit of PI3K was partially perturbed. Biochemical analysis of ezrin−/− T cells or T cells suppressed for moesin using small interfering RNA showed intact early TCR signaling, but diminished levels of IL-2. The defects in IL-2 production were more pronounced in T cells deficient for both ezrin and moesin. These cells also exhibited diminished phospholipase C-γ1 phosphorylation and calcium flux. We conclude that despite their unique movement and phosphorylation patterns, ezrin and moesin function together to promote T cell activation.

HIV type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry into target cells. Here we show that the HIV-1 envelope gp120 (Env) activates multiple ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. Env from both CCR5-dependent JRFL (R5) and CXCR4-dependent IIIB (X4) HIV-1 opened calcium-activated potassium (K Ca), chloride, and calcium-permeant nonselective cation channels in macrophages. These signals were mediated by CCR5 and CXCR4 because macrophages lacking CCR5 failed to respond to JRFL and an inhibitor of CXCR4 blocked ion current activation by IIIB. MIP-1␤ and SDF-1␣, chemokine ligands for CCR5 and CXCR4, respectively, also activated K Ca and Cl ؊ currents in macrophages, but nonselective cation channel activation was unique to gp120. Intracellular Ca 2؉ levels were also elevated by gp120. The patterns of activation mediated by CCR5 and CXCR4 were qualitatively similar but quantitatively distinct, as R5 Env activated the K Ca current more frequently, elicited Cl ؊ currents that were Ϸ2-fold greater in amplitude, and elevated intracellular Ca ؉2 to higher peak and steady-state levels. Env from R5 and X4 primary isolates evoked similar current responses as the corresponding prototype strains. Thus, the interaction of HIV-1 gp120 with CCR5 or CXCR4 evokes complex and distinct signaling responses in primary macrophages, and gp120-evoked signals differ from those activated by the coreceptors' chemokine ligands. Intracellular signaling responses of macrophages to HIV-1 may modulate postentry steps of infection and cell functions apart from infection.