Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis.

Arabidopsis WRKY proteins comprise a family of plant specific zinc-finger-type transcription factors involved in the regulation of gene expression during pathogen defense, wounding, trichome development, and senescence. To understand the regulatory role of the senescence-related WRKY53 factor, we identified target genes of this transcription factor by a pull down assay using genomic DNA and recombinant WRKY53 protein. We isolated a number of candidate target genes including other transcription factors, also of the WRKY family, stress- and defence related genes, and senescence-associated genes (SAGs). WRKY53 protein could bind to these different promoters in vitro and in vivo and it could act either as transcriptional activator or transcriptional repressor depending on the sequences surrounding the W-boxes. Overexpression, RNAi and knock-out lines showed accelerated and delayed senescence phenotypes, respectively, and exhibited altered expression levels of the target genes. WRKY53 can be induced by H2O2 and can regulate its own expression in a negative feed back loop. Our results suggest that WRKY53 acts in a complex transcription factor signalling network regulating senescence specific gene expression and that hydrogen peroxide might be involved in signal transduction.

A src-like kinase activates outwardly rectifying chloride channels in CFTR-defective lymphocytes.

Defective activation of chloride channels is a hallmark of cystic fibrosis (CF). Recently we have described activation of a volume-sensitive, outwardly rectifying chloride conductance (I(OR)) through the src-like tyrosine kinase p56(lck). Here we show that p56(lck) activates I(OR) independently of CFTR. In lymphocytes from healthy donors, chloride channels could be opened by either intracellular cAMP, p56(lck) or osmotic swelling. In CF lymphocytes, p56(lck) and cell swelling but not cAMP could activate chloride channels. Regulation of I(OR) by p56(lck) thus represents an alternative pathway of stimulating membrane chloride conductance that is left intact in cystic fibrosis.

Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids.

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca(2+) influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca(2+) entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca(2+) influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.

The tyrosine kinase p56lck mediates activation of swelling-induced chloride channels in lymphocytes.

Osmotic cell swelling activates Cl- channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl-swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl-swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl-swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl-swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, slow single-channel gating events can be observed revealing a unitary conductance of 25-28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56lck. We conclude that osmotic swelling activates ICl-swell in lymphocytes via the tyrosine kinase p56lck.