Summary Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.

Bacterial pathogens interact with host membranes to trigger a wide range of cellular processes during the course of infection. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways involved in vesicle trafficking. Such changes facilitate the entry and replication of the pathogen, and prevent its phagocytosis and degradation. In this Review, we describe the manipulation of host membranes by numerous bacterial effectors that target phosphoinositide metabolism, GTPase signalling and autophagy.

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

Defects in normal autophagic pathways are implicated in numerous human diseases-such as neurodegenerative diseases, cancer, and cardiomyopathy-highlighting the importance of autophagy and its proper regulation. Herein we show that Vibrio parahaemolyticus uses the type III effector VopQ (Vibrio outer protein Q) to alter autophagic flux by manipulating the partitioning of small molecules and ions in the lysosome. This effector binds to the conserved V o domain of the vacuolar-type H + -ATPase and causes deacidification of the lysosomes within minutes of entering the host cell. VopQ forms a gated channel ∼18 Å in diameter that facilitates outward flux of ions across lipid bilayers. The electrostatic interactions of this type 3 secretion system effector with target membranes dictate its preference for host vacuolar-type H + -ATPase-containing membranes, indicating that its pore-forming activity is specific and not promiscuous. As seen with other effectors, VopQ is exploiting a eukaryotic mechanism, in this case manipulating lysosomal homeostasis and autophagic flux through transmembrane permeation.utophagy is a cellular process by which cells degrade and recycle cytoplasmic contents by encapsulating them within a distinctive double bilayer membrane vesicle for delivery to the degradative lysosome (1). Disruption of normal autophagic pathways is implicated in numerous human diseases, stressing the importance of autophagy and its proper regulation (2). Vibrio parahaemolyticus, a Gram-negative marine bacterium and a major cause of gastroenteritis due to the consumption of contaminated raw or undercooked seafood, induces autophagy during infection (3). V. parahaemolyticus harbors two type 3 secretion systems (T3SSs), molecular syringes that enable the translocation of bacterial proteins, known as effectors, into the eukaryotic host (3). The first T3SS (T3SS1) orchestrates a temporally regulated cell death mediated by the induction of autophagy, followed by cell rounding and resulting in lysis of the host cell (4). T3SS1 effector VopQ, also known as VepA (vp1680), is both necessary and sufficient for the rapid induction of autophagy, even in the presence of known chemical inhibitors of autophagy (5).VopQ is a 53-kDa protein with no apparent homology to any proteins outside of the Vibrio species. Vibrio homologs of VopQ have no known function or conserved structural domain. Previous work from our laboratory has shown that VopQ is a cytotoxic effector that accelerates host cell death and is essential in protecting V. parahaemolyticus from phagocytic uptake during infection (5). Based on microbial genetic studies, VopQ was shown to be necessary for the formation of an extensive network of autophagic vesicles in host cells within an hour of V. parahaemolyticus infection (5). Strikingly, recombinant VopQ alone is sufficient to induce this massive accumulation of autophagic vesicles, observed within minutes of microinjection of recombinant VopQ (picomolar concentrations) into eukaryotic cells (5). A recent report shows that Vo...

Highlights d The bacterial effector HopBF1 adopts a minimal protein kinase fold d HopBF1 phosphorylates and inactivates eukaryotic HSP90 d HopBF1 mimics an HSP90 client to achieve specificity d HopBF1 is sufficient to induce disease symptoms in plants