A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis.

Phospholipases C are known to be important regulators of cellular processes but may also act as virulence factors of pathogenic microbes. At least three genes in the genome of the human-pathogenic fungus Candida albicans encode phospholipases with conserved phospholipase C (Plc) motifs. None of the deduced protein sequences contain N-terminal signal peptides, suggesting that these phospholipases are not secreted. In contrast to its orthologue in Sacharomyces cerevisiae, CaPLC1 seems to be an essential gene. However, a conditional mutant with reduced transcript levels of CaPLC1 had phenotypes similar to Plc1p-deficient mutants in S. cerevisiae, including reduced growth on media causing increased osmotic stress, on media with a non-glucose carbon source, or at elevated or lower temperatures, suggesting that CaPlc1p, like the Plc1p counterpart in S. cerevisiae, may be involved in multiple cellular processes. Furthermore, phenotypic screening of the heterozygous ΔCaplc1/CaPLC1 mutant showed additional defects in hyphal formation. The loss of CaPLC1 cannot be compensated by two additional PLC genes of C. albicans (CaPLC2 and CaPLC3) encoding two almost identical phospholipases C with no counterpart in S. cerevisiae but containing structural elements found in bacterial phospholipases C. Although the promoter sequences of CaPLC2 and CaPLC3 differed dramatically, the transcriptional pattern of both genes was similar. In contrast to CaPLC1, CaPLC2 and CaPLC3 are not essential. Although Caplc2/3 mutants had reduced abilities to produce hyphae on solid media, these mutants were as virulent as the wild-type in a model of systemic infection. These data suggest that C. albicans contains two different classes of phospholipases C which are involved in cellular processes but which have no specific functions in pathogenicity.

Fas ligand (FasL) is a transmembrane protein that regulates cell death in Fas-bearing cells. FasL-mediated cell death is essential for immune system homeostasis and the elimination of viral or transformed cells. Because of its potent cytotoxic activity, FasL expression at the cell surface is tightly regulated, for example, via processing by ADAM10 and SPPL2a generating soluble FasL and the intracellular fragments APL (ADAM10-processed FasL form) and SPA (SPPL2a-processed APL). In this study, we report that FasL processing by ADAM10 counteracts Fas-mediated cell death and is strictly regulated by membrane localization, interactions and modifications of FasL. According to our observations, FasL processing occurs preferentially within cholesterol and sphingolipid-rich nanodomains (rafts) where efficient Fas–FasL contact occurs, Fas receptor and FasL interaction is also required for efficient FasL processing, and FasL palmitoylation, which occurs within its transmembrane domain, is critical for efficient FasL-mediated killing and FasL processing.

Fas ligand (FasL) not only induces apoptosis in Fas receptor-bearing target cells, it is also able to transmit signals into the FasL-expressing cell via its intracellular domain (ICD). IntroductionFas ligand (FasL, CD95L, CD178, TNSF6) is a 40-kDa glycosylated type II transmembrane protein that belongs to the tumor necrosis factor (TNF) family. Its extracellular portion harbors a C-terminal TNF homology domain that assembles as homotrimers and mediates interactions with receptors. Interestingly, the 77-amino acid (aa)-long N-terminal mouse FasL intracellular domain (ICD) contains several conserved signaling motifs, such as a tandem casein kinase I phosphorylation site (aa 17-21), a proline-rich domain (PRD; aa 45-69) that serves as a docking site for SH3 domain-containing proteins, and a tyrosine phosphorylation site located at aa 7. 1 The FasL/Fas receptor system is renowned as a potent inducer of apoptosis in the receptor-bearing cell and is especially important for numerous immune system functions, including removal of target cells by natural killer and cytotoxic T cells, (self) elimination of effector cells after the proliferative phase of an immune response (activation-induced cell death), and maintenance of immuneprivileged sites. 2,3 Naturally occurring homozygous mutations of the Fas (lpr) and the FasL (gld) genes, which abolish Fas-mediated apoptosis, lead to development of a severe lymphoproliferative disease. 4 Because of its potent proapoptotic potential and important function, expression and activity of FasL is tightly regulated at transcriptional and posttranslational levels and is restricted to a few cell types, such as immune effector cells and cells of immuneprivileged sites (eg, eyes, testis, brain). In contrast, the Fas receptor is expressed in a wide variety of tissues, including the lymphoid tissues, liver, heart, and ovary. 1,5 In addition to its pro-death function, the Fas/FasL system also transduces nonapoptotic signals into the Fas-expressing cells. Among others, Fas is required for cell survival, proliferation and activation of T cells, liver regeneration, and neurogenesis. 6,7 A number of studies have presented evidence for alternative signal transduction via "reverse signaling" into the FasL-bearing cells, although conflicting data have been published regarding the functional implications (for review see Voss et al 1 ). First evidence The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 8, 2018. by guest www.bloodjournal.org From for the existence of retrograde FasL signaling stems from 2 studies that demonstrated either costimulation of murine CD8 ϩ cytotoxic T lymphocyte (CTL) cell lines upon FasL cross-linking 8 or inhibition of activation-induced proliferation in murine CD4 ϩ T cells. 9 In both cases, FasL reverse signaling could...

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