Rinehart J , Vázquez N , Kahle KT , Hodson CA , Ring AM , Gulcicek EE , Louvi A , Bobadilla NA , Gamba G , Lifton RP .

2P30DA018343 NIDA NIH HHS , Howard Hughes Medical Institute , P30 DA018343 NIDA NIH HHS , K01 DK089006 NIDDK NIH HHS

	FIGURE 1. Analysis of WNK2 mRNA expression in mouse brain and kidney as compared with other WNK kinases. WNK2 localization (blue) by in situ hybridization shows a high level of expression in mouse brain both in embryonic development (E14.5) and in the adult (top two panels). A representative coronal section of the E14.5 brain (top left) is compared with sections of the adult brain (top right). Whole sections are shown in the inset on top of representative magnified region of the cortex. WNK2 is highly expressed in post-mitotic neurons in the cortical plate (CP) and intermediate zone (IZ) at E14.5. WNK2 is expressed in all cortical layers in the adult cortex (ctx) (top right). WNK2 expression in the adult cerebellum (bottom left). A whole section is shown in the inset on top of a magnified region of the cerebellum. Purkinje cells (pjk), the granular cell layer (gr), and the white matter (wm) are indicated. WNK2, WNK1, and WNK4 mRNA expression in the mouse kidney (bottom right). A whole sagittal section is shown in the insets below images of a magnified region of the cortex (ctx) for each WNK kinase indicated.
	FIGURE 2. Characterization of WNK2-expressing cells in mouse brain. SMI-32 co-localization (brown) highlights WNK2 expression in pyramidal neurons (pyr) in the developing neocortex (top panel). GFAP-expressing astrocytes do not express Wnk2 (middle). The arrowhead marks an astrocyte (ast). WNK2 mRNA is expressed in the Purkinje cells (pc) in the cerebellum, labeled with calbindin (bottom). The molecular layer (mol) and granular layer (gr) border the Purkinje cell layer in the cerebellum.
	FIGURE 3. Modulation of KCC2, KCC4, and NKCC1 activity by WNK2. Xenopus laevis oocytes were injected with (A) water or 10 ng/oocyte of KCC2 cRNA, (B) water or 10 ng/oocyte of KCC2 or KCC4 cRNA, and (C) water, or 10 ng/oocyte of NKCC1 cRNA. In all figures, cotransporters cRNA were injected alone or together with 10 ng/oocyte of WNK2 or WNK3 cRNA, as stated. Four days later, 86Rb+ influx experiments were performed in isotonic (A and C) or hypotonic conditions (B) in the presence (open bars) or absence (closed bars) of extracellular chloride (A and B) or in the absence (open bars) or presence (closed bars) of 100 μm bumetanide (C). *, significantly different from the uptake observed in corresponding control. Each figure depicts combined data of at least three different experiments.
	FIGURE 4. Effect of WNK2 and WNK3 on the protein level and surface expression of NKCC1 and KCC4. Western blot analysis of the total and biotinylated fraction of proteins extracted from oocytes injected with NKCC1 (A) or KCC4 (B) cRNA in the absence or presence of WNK2 or WNK3 cRNA. C and D depict the results of the functional expression performed the same day using oocytes from the same batch for NKCC1 or KCC4, respectively, expressed as bumetanide-sensitive (C) or chloride-dependent (D) 86Rb+ uptake.
	FIGURE 5. Effect of kinase-dead (DA) WNK2 upon NKCC1 and KCCs cotransporters activity. A, 86Rb+ uptake in oocytes injected with water, NKCC1 cRNA alone or together with WNK2-DA or WNK3-DA cRNA. Uptake was performed in isotonic conditions in the absence (open bars) or presence (closed bars) of 100 μm bumetanide. *, significantly different from the uptake observed in NKCC1 cRNA group. B, oocytes were injected with water, KCC2 cRNA alone or together with kinase-dead WNK2 or WNK3, as stated. C, oocytes were injected with water, KCC2, or KCC4 cRNA alone or together with kinase-dead WNK2 or WNK3, as stated. 86Rb+ Uptake was performed in in the presence (open bars) or absence (closed bars) of extracellular Cl−, in hypotonic (B) or isotonic (C) conditions. *, significantly different from the uptake observed in corresponding control. D, representative Western blot analysis of protein homogenates extracted from oocytes injected with wild type or kinase-dead HA-WNK2 cRNA, together with NKCC1 or KCC2 cRNA, as stated. Western blots were then performed using anti-HA monoclonal antibodies. Similar results were observed in the absence of NKCC1 cRNA injections.
	FIGURE 6. MS/MS analysis of WNK2 phosphorylation. A, phosphorylation sites are shown in the context of full-length human WNK2. The conserved kinase domain and two coiled-coiled regions are labeled. Phosphorylation sites identified in human WNK2 expressed in HEK cells are numbered on the bottom of the diagram. Sites unique to this study are in black, and sites in common with other studies are in red. Phosphorylation sites identified in native mouse WNK2 are numbered according to their position in the mouse sequence and mapped onto the corresponding position in human WNK2. Phosphorylation at Ser-1770 (mouse) and Ser-1862 (human) are homologous. B, SDS-PAGE analyisis of native WNK2 immunoprecipitates prepared from mouse brain for MS/MS. An example gel is shown with the results of the MS/MS analysis for each protein band shown to the left. Phosphorylation sites from full-length WNK2 and SPAK identified by MS/MS are listed (for additional peptide information see supplemental Tables S1 and S2. A brief Coomassie-staining method was used to reveal the proteins bands to the naked eye. Bands that were unique to brain IP and not visible in a control IP from mouse kidney (not shown) were cut and subjected to MS analysis. C, table of WNK2 phosphopeptides identified in MS/MS analysis of purified human and mouse WNK2. Mascot searches followed by manual MS/MS spectra interpretation places the possible phosphorylation site within each peptide. Unambiguous assignment of phosphorylation sites are indicated in underlined, red capital S, while ambiguous assignments have the second most likely site as a lowercase s. Kinase motifs corresponding to each phosphorylation site were identified with NetworKIN and NetPhorest analysis. Abbreviations are for Rho-associated protein kinase 1 (DMPK), cAMP-dependent protein kinase (PKA), casein kinase II (CKII), PKC protein kinase CΔ (PKC), p21-activated kinase (PAK), p38 mitogen-activated protein kinase (p38MAPK), and cyclin-dependent kinases (CDK2, 3, and 5).
	FIGURE 7. Relationship between GABA excitatory and inhibitory effect and the expression of WNK2, WNK3, NKCC1, and KCC2 in the CNS from embryonic to postnatal life.

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