title: Functional kinomics establishes a critical node of volume-sensitive cation-Cl-cotransporter regulation in the mammalian brain.
abstract: Cell 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/Thr1048phosphorylation - 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.
authors: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.|Departments of Neurosurgery and Pediatrics, Yale School of Medicine, New Haven, CT 06511 USA.
authors: Jinwei
authors: J
authors: Zhang
authors: Division of Genetics, Brigham and Women's Hospital, Boston, MA 02115 USA.
authors: Geng
authors: G
authors: Gao
authors: Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors: Gulnaz
authors: G
authors: Begum
authors: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.|Department of Biological Chemistry &Molecular Pharmacology, Harvard Medical School, 250 Longwood Ave, SGM 628, Boston, MA 02115, USA.
authors: Jinhua
authors: J
authors: Wang
authors: Department of Neurosurgery, Massachusetts General Hospital, Boston, MA 02114 USA.
authors: Arjun R
authors: AR
authors: Khanna
authors: Division of Nephrology and Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA -022154 USA.|Department of Medicine, Harvard Medical School, Boston, MA -022154, USA.
authors: Boris E
authors: BE
authors: Shmukler
authors: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
authors: Gerrit M
authors: GM
authors: Daubner
authors: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
authors: Paola
authors: P
authors: de Los Heros
authors: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
authors: Paul
authors: P
authors: Davies
authors: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
authors: Joby
authors: J
authors: Varghese
authors: Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors: Mohammad Iqbal H
authors: MI
authors: Bhuiyan
authors: Departments of Neurosurgery and Pediatrics, Yale School of Medicine, New Haven, CT 06511 USA.|Department of Cardiology, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, Massachusetts 02115, USA.
authors: Jinjing
authors: J
authors: Duan
authors: Department of Cardiology, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, Massachusetts 02115, USA.
authors: Jin
authors: J
authors: Zhang
authors: Departments of Neurosurgery and Pediatrics, Yale School of Medicine, New Haven, CT 06511 USA.
authors: Daniel
authors: D
authors: Duran
authors: Division of Nephrology and Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA -022154 USA.|Department of Medicine, Harvard Medical School, Boston, MA -022154, USA.
authors: Seth L
authors: SL
authors: Alper
authors: Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.|Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA, USA.
authors: Dandan
authors: D
authors: Sun
authors: Department of Genetics, Harvard University Medical School, Howard Hughes Medical Institute, Boston, Massachusetts 02115 USA.
authors: Stephen J
authors: SJ
authors: Elledge
authors: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
authors: Dario R
authors: DR
authors: Alessi
authors: Departments of Pediatrics and Cellular &Molecular Physiology; Interdepartmental Neuroscience Program; and Centers for Mendelian Genomics, Yale School of Medicine, New Haven, CT 06511 USA.
authors: Kristopher T
authors: KT
authors: Kahle
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier; D001921:Brain / Q000378:metabolism*; D048429:Cell Size; D002712:Chlorides / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009997:Osmotic Pressure; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D034622:RNA Interference; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2 / Q000378:metabolism; D027981:Symporters / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D002712:Chlorides; D017346:Protein Serine-Threonine Kinases; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters; C119650:SLC12A6 protein, human; D000096922:K Cl- Cotransporters; C578878:Slc12a2 protein, mouse; C492280:Slc12a6 protein, mouse; C486407:Stk39 protein, mouse; C488586:WNK3 protein, human; C507511:Wnk3 protein, mouse