Offringa, R. | Search bioRxiv

bioRxiv2019

Phosphoproteomics of CD2 signaling reveals an AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells

The in-depth analysis of costimulatory signaling enhancing the activity of cytotoxic T cells (CTLs) represents a major approach towards immunotherapy development. Here we report that CD2 costimulation plays a critical role in killing by freshly isolated human CTLs, which represent a challenging but valuable study model to gain insight into CTL biology. We show that CD2 triggering critically aids signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules towards the microtubule-organizing center (MTOC). To gain insight into the underlying elusive mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeleton organization, autophagy and metabolism. Strikingly, signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) represents a functionally critical node of the CD2 network which regulates granule polarization towards the MTOC in CTLs. Granule trafficking is driven by active AMPK enriched on adjacent lysosomes, illustrating a novel signaling cross-talk between vesicular compartments in CTLs. Our results thus establish CD2 signaling as key for regulating cytotoxic killing and granule polarization in freshly isolated CTLs and strengthens the rationale to choose CD2 and AMPK as therapeutic targets to boost CTL activity.

immunology↗

bioRxiv2019

PDK1 has a pleiotropic PINOID-independent role in Arabidopsis development

The 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) is a conserved and important master regulator of AGC kinases in eukaryotic organisms. pdk1 loss-of-function causes a lethal phenotype in animals and yeast. In contrast, only very mild phenotypic defects have been reported for the pdk1 loss-of-function mutant of the model plant Arabidopsis thaliana (Arabidopsis). The Arabidopsis genome contains two PDK1 genes, hereafter called PDK1 and PDK2. Here we show that the previously reported Arabidopsis pdk1 T-DNA insertion alleles are not true loss-of-function mutants. By using CRISPR/Cas9 technology, we created true loss-of-function pdk1 alleles, and pdk1 pdk2 double mutants carrying these alleles showed multiple growth and development defect, including fused cotyledons, a short primary root, dwarf stature, late flowering, and reduced seed production caused by defects in male fertility. Surprisingly, pdk1 pdk2 mutants did not phenocopy pid mutants, and together with the observations that PDK1 overexpression does not phenocopy the effect of PID overexpression, and that pdk1 pdk2 loss-of-function does not change PID subcellular localization, we conclude that PDK1 is not essential for PID membrane localization or functionality in planta. Nonetheless, most pdk1 pdk2 phenotypes could be correlated with impaired auxin transport. PDK1 is highly expressed in vascular tissues and YFP:PDK1 is relatively abundant at the basal/rootward side of root stele cells, where it colocalizes with PIN auxin efflux carriers, and the AGC1 kinases PAX and D6PK/D6PKLs. Our genetic and phenotypic analysis suggests that PDK1 is likely to control auxin transport as master regulator of these AGC1 kinases in Arabidopsis.

plant biology↗