Calcium is the molecular switch shifting the phytosulfokine receptor 1 (PSKR1) from kinase to guanylate cyclase activity

Conference presentationMany plant responses are mediated by interactions between intracellular calcium and the second messenger cGMP formed by guanylate cyclases (GCs). Previously we identified a novel class of receptor-GCs containing the GC catalytic center embedded within the kinase domain and showed that the recombinant cytoplasmic domain of phytosulfokine receptor AtPSKR1 has both guanylate cyclase and kinase activity in vitro (Kwezi et al. 2011 J Biol Chem 286: 22580-8). We now show that physiological increases in calcium levels enhance GC activity of AtPSKR1 whereas these calcium levels reversibly inhibit kinase activity. In addition PSKR1 kinase activity is reduced in the presence of the GC product cGMP. Recombinant AtPSKR1 can undergo in vitro autophosphorylation and we have confirmed it has 14 phosphorylation sites in its cytoplasmic domain including 8 serine, 3 threonine and 3 tyrosine residues. Three phospho-serine residues at the juxta-membrane position were mutated to either mimic phosphorylation on or off states. Kinase activity was enhanced in the on mutant and suppressed in the off mutant while GC activity was unaffected suggesting calcium acts as a molecular switch of PSKR1- mediated signalling that can be modulated by the phosphorylation state. The challenge now lies in understanding how molecular interactions between the GC and kinase domains are capitalized on in the plant

Plant Individual Nucleotide Resolution Cross-Linking and Immunoprecipitation to Characterize RNA-Protein Complexes.

Köster T, Staiger D. Plant Individual Nucleotide Resolution Cross-Linking and Immunoprecipitation to Characterize RNA-Protein Complexes. Methods in molecular biology (Clifton, N.J.). 2020;2166:255-267.In recent years, it has become increasingly recognized that regulation at the RNA level pervasively shapes the transcriptome in eukaryotic cells. This has fostered an interest in the mode of action of RNA-binding proteins that, via interaction with specific RNA sequence motifs, modulate gene expression. Understanding such posttranscriptional networks controlled by an RNA-binding protein requires a comprehensive identification of its in vivo targets. In metazoans and yeast, methods have been devised to stabilize RNA-protein interactions by UV cross-linking before isolating RNA-protein complexes using antibodies, followed by identification of associated RNAs by next-generation sequencing. These methods are collectively referred to as CLIP-Seq (cross-linking immunoprecipitation-high-throughput sequencing). Here, we present a version of the individual nucleotide resolution cross-linking and immunoprecipitation procedure that is suitable for use in the model plant Arabidopsis thaliana

Proteomics informed by transcriptomics for characterising active transposable elements and genome annotation in Aedes aegypti

This work was funded by Medical Research Council, UK Grant G0801973 to AD, BBSRC grants BB/M020118/1, BB/L018438/1 and BB/K016075/1 to DAM and CB. BBSRC funding BB/M02542X/1 to ADD and DAM, NIH/NIAID R01AI073450 grant to AF-S and a Sir Henry Wellcome fellowship to KM (grant number 096062)