MAPK/ERK signaling in activated T cells inhibits CD95/Fas-mediated apoptosis downstream of DISC assembly

When T cells are activated, the expression of the CD95 ligand is elevated, with the purpose of inducing apoptosis in target cells and to later eliminate the activated T cells. We have shown previously that mitogen-activated protein kinase (MAPK or ERK) signaling suppresses CD95-mediated apoptosis in different cellular systems. In this study we examined whether MAPK signaling controls the persistence and CD95-mediated termination of an immune response in activated T cells. Our results show that activation of Jurkat T cells through the T cell receptor immediately suppresses CD95-mediated apoptosis, and that this suppression is mediated by MAPK activation. During the phase of elevated MAPK activity, the activation of caspase-8 and Bid is inhibited, whereas the assembly of a functional death-inducing signaling complex (DISC) is not affected. These results explain the resistance to CD95 responses observed during the early phase of T cell activation and suggest that MAPK-activation deflects DISC signaling from activating caspase-8 and Bid. The physiological relevance of the results was confirmed in activated primary peripheral T cells, in which inhibition of MAPK signaling markedly sensitized the cells to CD95-mediated apoptosis.     

Identification of a novel protein complex containing annexin VI, Fyn, Pyk2, and the p120(GAP) C2 domain

p120(GAP) (RasGAP) has been proposed to function as both an inhibitor and effector of Ras. Previously we have shown that RasGAP contains a C2 domain which mediates both Ca(2+)-dependent membrane association and protein-protein interactions. Specifically, three proteins have been isolated in a complex with the C2 domain of RasGAP; these are the Ca(2+)-dependent lipid binding protein annexin VI (p70) and two previously unidentified proteins, p55 and p120. Here we provide evidence that p55 is the Src family kinase Fyn and p120 is the focal adhesion kinase family member Pyk2. In addition, in vitro binding assays indicate that Fyn, but not Pyk2 binds directly to annexin VI. Finally, co-immunoprecipitation studies in Rat-1 fibroblasts confirm that Fyn, Pyk2, annexin VI and RasGAP can form a protein complex in mammalian cells.     

Double C2 protein. A review

Concerted effort has led to the identification of dozens of synaptic proteins and has thereby opened the door for the characterisation of the molecular mechanisms underlying regulated exocytosis. Calcium is known to play a number of roles in regulated exocytosis, acting as the trigger for fast synaptic transmission and also acting at some of the steps preceding vesicle fusion. Investigators have therefore focussed considerable attention on possible calcium sensors. What many of the candidate proteins have in common is a C2 domain, one of the four conserved domains originally described in protein kinase C. Such domains have been shown to bind calcium and phospholipid in a large number of intracellular proteins. Synaptotagmin, a C2-domain protein, is a very strong candidate for the protein involved in triggering fast calcium-dependent vesicle fusion. Recent attention has also concerned the other calcium sensors, which may play roles in the 'priming' or transport of vesicles. This review concerns one of these tentative calcium-binding proteins, double C2 or DOC2. DOC2 was originally isolated from nervous tissue but subsequently has been found to be more widely expressed. DOC2 is a vesicular protein that may be involved in the early stages of preparing vesicles for exocytosis.     

Cell surface-bound collagenase-1 and focal substrate degradation stimulate the rear release of motile vascular smooth muscle cells

To migrate in the vessel wall, smooth muscle cells (SMCs) must contend with abundant type I collagen. We investigated the mechanisms used by human SMCs to efficiently migrate on type I collagen, following stimulation with fibroblast growth factor-2 (FGF-2). FGF-2-stimulated migration was inhibited by a hydroxamic acid inhibitor of matrix metalloproteinases and by a neutralizing anti-collagenase-1 antibody. Moreover, migration speed of SMCs plated on mutant collagenase-resistant type I collagen was not increased by FGF-2. Time-lapse video analysis of unstimulated SMCs migrating on collagen revealed discrete phases of leading edge membrane extension and rear retraction, the latter often after rupture of an elongated tail. FGF-2 stimulation yielded a more synchronous, gliding motion with a collagenase-1-mediated decrease in tail ripping. Surface labeling of SMCs with biotin followed by immunoprecipitation revealed that a proportion of active collagenase-1, expressed in response to FGF-2, was bound to the plasma membrane. Pericellular collagen substrate cleavage was verified by immunostaining for neoepitopes generated by collagenase-1 action and was localized to discrete zones beneath the cell tail and the leading edge. These results identify a novel mechanism by which SMC migration on collagen is enhanced, whereby rear release from the substrate is orchestrated by the localized actions of membrane-bound collagenase-1.     

Human immunodeficiency virus Env-independent infection of human CD4(-) cells

CD4(-) epithelial cells covering mucosal surfaces serve as the primary barrier to prevent human immunodeficiency virus type 1 (HIV-1) infection. We used HIV-1 vectors carrying the enhanced green fluorescent protein gene as a reporter gene to demonstrate that HIV-1 can infect some CD4(-) human epithelial cell lines with low but significant efficiencies. Importantly, HIV-1 infection of these cell lines is independent of HIV-1 envelope proteins. The Env-independent infection of CD4(-) cells by HIV-1 suggests an alternative pathway for HIV-1 transmission. Even on virions bearing Env, a neutralizing antibody directed against gp120 is incapable of neutralizing the infection of these cells, thus raising potential implications for HIV-1 vaccine development.     

Integrase-lexA fusion proteins incorporated into human immunodeficiency virus type 1 that contains a catalytically inactive integrase gene are functional to mediate integration

Purified fusion proteins made up of a retroviral integrase and a sequence-specific DNA-binding protein have been tested in in vitro assays for their ability to direct integration into specific target sites. To determine whether these fusion proteins can be incorporated into human immunodeficiency virus type 1 (HIV-1) and are functional to mediate integration, we used an in trans approach to deliver various integrase-LexA proteins to an integrase-defective virus containing an integrase mutation at aspartate residue 64. Integrase-LexA, integrase-LexA DNA-binding domain, or N- or C-terminally truncated integrase-LexA proteins were fused to the HIV-1 accessory protein, Vpr. Coexpression of the Vpr fusion proteins and an integrase-defective HIV-1 molecular clone by a producer cell line resulted in efficient incorporation of the fusion protein into the integrase-mutated virus. In addition, each of these viruses was infectious and capable of performing integration, as determined by two independent cellular assays that measure reporter gene expression. With the exception of the N-terminally truncated integrase fused to LexA, which was at about 1%, all of the fusion proteins restored integration to a similar level, at 17 to 24% of that of the wild-type virus. The low level observed with the N-terminally truncated integrase fused to LexA is consistent with previous results implying that the N terminus of integrase is involved in multiple steps of the retroviral life cycle. These data indicate that the integrase-fusion proteins retain catalytic function in the integrase-mutated viruses and demonstrate the feasibility of incorporating integrase fusion proteins into HIV-1 for the development of site-directed retroviral vectors.     

Role of capsaicin sensory nerves and EGF in the healing of gastric ulcer in rats

Accumulating evidence indicates that capsaicin sensitive afferent fibers play a pivotal role not only in gastroprotection but also in ulcer healing. Denervation of capsaicin sensitive afferent fibers exerts an adverse action on these effects. However, whether such an action is mediated through a depression on epidermal growth factor (EGF) is undefined. In this study, the effects of denervation of sensory neurons with capsaicin (100 mg/kg, s.c.) on acetic acid-induced chronic gastric ulcers and their relationship with the EGF expression in salivary glands, serum and gastric mucosa were investigated. Capsaicin significantly increased ulcer size, decreased gastric mucosal cell proliferation at the ulcer margin, angiogenesis in the granulation tissue and also gastric mucus content. Ulcer induction by itself dramatically elevated EGF levels in salivary glands and serum on day 1 and 4, and also in the gastric mucosa on day 4. However, capsaicin completely abolished these effects. It is concluded that stimulation of EGF expression in salivary glands and serum may be one of the mechanisms by which capsaicin sensitive nerves contribute to the gastroprotective and ulcer healing actions in the stomach.