Functional kinomics establishes a critical node of volume-sensitive cation-Cl^- cotransporter regulation in the mammalian brain

This is the final version of the article. Available from the publisher via the DOI in this record.There is another record in ORE for this publication: http://hdl.handle.net/10871/33424Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation – a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl−-sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl− uptake and stimulation of KCC3-mediated Cl− extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought “Cl−/volume-sensitive kinase” of the cation-Cl− cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.We thank the excellent technical support of the MRC-Protein Phosphorylation and Ubiquitylation Unit (PPU) DNA Sequencing Service (coordinated by Nicholas Helps), the MRC-PPU tissue culture team (coordinated by Laura Fin), the Division of Signal Transduction Therapy (DSTT) antibody purification teams (coordinated by Hilary McLauchlan and James Hastie). We are grateful to the MRC PPU Proteomics facility (coordinated by David Campbell, Robert Gourlay and Joby Varghese). We thank for support the Medical Research Council (MC_UU_12016/2; DRA) and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer; DRA). We thank Thomas J. Jentsch (Max-Delbrück-Centrum für Molekulare Medizin) for providing the KCC1/3 double KO mice and his reading of this manuscript. We thank Nathaniel Grey (Harvard) for providing the kinase inhibitor library used in this study (NIH LINCS Program grant U54HL127365). This work was also supported by a Harvard-MIT Neuroscience Grant (to KTK/SJE)

Macroautophagy is essential for killing of intracellular <i>Burkholderia pseudomallei</i> in human neutrophils

<div><p>Neutrophils play a key role in the control of <i>Burkholderia pseudomallei</i>, the pathogen that causes melioidosis. Here, we show that survival of intracellular <i>B. pseudomallei</i> was significantly increased in the presence of 3-methyladenine or lysosomal cathepsin inhibitors. The LC3-flux was increased in <i>B. pseudomallei</i>-infected neutrophils. Concordant with this result, confocal microscopy analyses using anti-LC3 antibodies revealed that <i>B. pseudomallei</i>-containing phagosomes partially overlapped with LC3-positive signal at 3 and 6 h postinfection. Electron microscopic analyses of <i>B. pseudomallei</i>-infected neutrophils at 3 h revealed <i>B. pseudomallei</i>-containing phagosomes that occasionally fused with phagophores or autophagosomes. Following infection with a <i>B. pseudomallei</i> mutant lacking the <i>Burkholderia</i> secretion apparatus Bsa Type III secretion system, neither this characteristic structure nor bacterial escape into the cytosol were observed. These findings indicate that human neutrophils are able to recruit autophagic machinery adjacent to <i>B. pseudomallei</i>-containing phagosomes in a Type III secretion system-dependent manner.</p></div