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DIRECT PKC-DEPENDENT PHOSPHORYLATION REGULATES THE CELL SURFACE STABILITY AND ACTIVITY OF THE POTASSIUM CHLORIDE COTRANSPORTER,

KCC2 Henry H. C. Lee1, Joshua A. Walker1, Jeffery R. Williams2, Richard J. Goodier2, John A.

Payne2 and Stephen J. Moss1,3. 1Department of Neuroscience, School of Medicine, University of Pennsylvania, PA 19104, USA;

2University of California Davis, University California, Department of Physiology & Membrane Biology, One Shields Ave, Davis CA 95616-8644, USA; 3Department of Pharmacology, University

College London, WC1E 6BT, UK. Address correspondence to: Stephen J. Moss: Department of Neuroscience, University of Pennsylvania, 215 Stemmler Hall, 3450 Hamilton Walk, Philadelphia, PA 19104. Tel: 215-898-3420; E-mail: sjmoss@mail.med.upenn.edu

The potassium chloride co-transporter KCC2 plays a major role in the maintenance of transmembrane chloride potential in mature neurons; thus KCC2 activity is critical for hyperpolarizing-membrane currents generated upon the activation of γ-aminobutyric acid type A (GABAA) and glycine (Gly) receptors that underlie fast synaptic inhibition in the adult CNS. However, to date an understanding of the cellular mechanism that neurons use to modulate the functional expression of KCC2 remains rudimentary. Using E. coli expression coupled with in vitro kinase assays we first established that protein kinase C (PKC) can directly phosphorylate Serine 940 (S940) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that S940 is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of S940 increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of S940 increased the rate of ion transport by KCC2. It was further evident that phosphorylation of endogenous KCC2 in cultured hippocampal neurons is regulated by PKC-dependent activity. Moreover, in keeping with our recombinant studies, enhancing PKC-dependent phosphorylation increased the targeting of KCC2 to the neuronal cell surface. Our studies thus suggest that PKC-dependent phosphorylation of KCC2 may play a central role in modulating both the functional expression of this critical transporter in the brain and the strength of synaptic inhibition.

Cation-chloride cotransporters (CCC) regulate Cl- homeostasis in cells and the generation of transmembrane chloride gradients (1). Adult mammalian neurons maintain low intracellular Cl- concentrations, which arise principally from the activity of the potassium chloride cotransporter-2 (KCC2). The maintenance of such low levels of intracellular Cl- ions is responsible for hyperpolarizing Cl- currents upon activation of GABAA and Gly receptors which are responsible for fast synaptic inhibition in the adult CNS (2-5). Molecular studies have demonstrated that KCC2 is a member of a CCC superfamily and that these transporters are composed of 12-transmembrane domains with N- and C-terminal cytoplasmic domains (2,6,7). KCC2 is expressed exclusively in neurons throughout the adult brain. Developmentally KCC2 is first detected around 10 days in vitro (DIV) in cultured rat neurons, which is coincident with the emergence of hyperpolarizing GABAA receptor mediated Cl- currents (4,8). Gene knock-out of KCC2 has revealed that ablating the expression of this protein results in early postnatal death. Neurons derived from these animals exhibited compromised GABAA receptor mediated synaptic inhibition (9). In pathological conditions such as epilepsy or ischemic brain injury, deficits in the expression of KCC2 are evident together with decreased efficacy of GABAergic inhibition and with the emergence of depolarizing GABAA receptor mediated currents that reflect decreased neuronal Cl- extrusion (10). These changes in functional expression are believed in part to be transcriptional (11,12) but post-translational modification of KCC2 is also likely to be of central importance. Intriguingly the activity of a number of protein kinases including

http://www.jbc.org/cgi/doi/10.1074/jbc.M705053200The latest version is at JBC Papers in Press. Published on August 10, 2007 as Manuscript M705053200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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WNK3, WNK4, brain-type creatine kinase, TrkB receptors, tyrosine kinases and PKC have all been reported to influence KCC2 activity (13). However, it remains to be established whether these varying kinase activities actually directly regulate KCC2 phosphorylation and whether altered levels of phosphorylation modulate the function or membrane trafficking of this key transporter. To further address the role of phosphorylation in regulating KCC2 we have assessed whether this protein is directly phosphorylated and whether this covalent modification alters transporter functional expression. Our studies demonstrated that KCC2 is directly phosphorylated by PKC activity on S940 within the major C-terminal intracellular domain of this protein. PKC-dependent phosphorylation of S940 increased KCC2 cell surface stability and activity by decreasing endocytosis from the plasma membrane. Endogenous KCC2 expressed in hippocampal neurons was also phosphorylated by PKC activity and, in common with our recombinant studies activation of PKC increased the accumulation of KCC2 on the neuronal plasma membrane. Together our studies suggest a critical role for PKC-mediated phosphorylation of S940 in KCC2 in regulating the functional expression of this transporter in the brain.

Experimental Procedures

Antibodies- Monoclonal mouse anti-KCC2 antibody clone N1/12 was purchased from the UC Davis/NINDS/NIMH NeuroMab facility. Polyclonal rabbit anti-KCC2 antibody was purchased from Upstate. Biotinylation and endocytosis assays- Cells were washed 2 times with 1xPBS containing 0.5mM MgCl2 and 1mM CaCl2 (PBS-CM) then incubated with 2mL of 1xPBS-CM containing 1mg/mL Sulfo-NHS-SS-Biotin for 30 min for biotin labeling. After labeling, the biotin reaction was quenched by washing 3 times with 1xPBS-CM containing 50mM glycine and 0.1% bovine serum albumin (14,15). Cells were then lyzed in 10mM NaPO4, 2% Triton X-100, 0.5% deoxycholate, 10mM Na pyrophosphate, 25mM NaF, 1mM Na orthovanadate, 100µM PMSF, 10µg aprotinin, leupeptin, pepstatin, 5mM EDTA, EGTA, and 100mM and biotinylated proteins were purified on

immobilized avidin eluted in SDS-PAGE sample buffer. KCC2 levels were then measured by immunoblotting. To measure endocytosis cells were labeled on ice as described above and incubated at 37oC for varying time periods. Remaining cell surface biotin was then cleaved by exposure to 2mM reduced glutathione for 10 min on ice (14). Cells were lyzed and biotinylated proteins were purified as detailed above. Data were then correct for the efficiency of GST cleavage (biotin remaining at 0 time) the proportion of the total cell surface population of KCC2 internalized over time was then calculated. Expression and purification of KCC2 fusion proteins- The respective nucleotides encoding amino acids 1-102 and 645-1116 of KCC2 (3) were amplified from rat brain cDNA using the following primer pairs: AAGTCGACCATGCTCAACAACCTGACGGACTGCGAG/AAGCGGCCGCTCAGGAGTAGATGGTGATGACCTCTCGGC and CAAGGATCCGATCCGAGGCCTGTCTCTCAGTGCAGC/CAACTCGAGTCAGGAGTAGATGGTGATGACCTCTCG respectively. They were then cloned into pTrcHis2C (Invitrogen) to yield His-N/KCC2 and His-C/KCC2. After DNA sequencing fusion proteins were expressed in E. coli strain BL21. Exponentially growing cultures were treated with 100µM IPTG for 3 h and bacterial pellets were lyzed using 6M guanidine hydrochloride (pH7.8) and sonication. The fusion protein was then bound to ProBondTM resin (Invitrogen) in 8M urea (pH 7.8) for 30 min and washed with decreasing pH of buffer. Elution of fusion protein was then carried out by washing resin with a buffer of pH 4.0. The eluted protein was dialyzed extensively in PBS and stored at -800C before use. Expression of KCC2 in HEK-293 cells- Wild-type and mutant KCC2 cDNAs were cloned into the mammalian expression vector PRK5, which utilizes the human cytomegalovirus promoter for transgene expression (16). Cells were transfected using electroporation with a total of 10 µg of DNA and utilized 24-48 h after transfection (17). Immunoblotting- Cells were lyzed by lysis buffer containing 10mM Na2HPO4, 2% Triton X-100, 0.5% deoxycholate, 10mM Na pyrophosphate, 25mM NaF, 1mM Na orthovanadate, 100µM PMSF, 10µg aprotinin, leupeptin, pepstatin, 5mM

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EDTA, EGTA, and 100mM NaCl. Insoluble proteins were removed by centrifugation at 13.2k rpm for 10min. 50µg of protein was loaded into a 6% acrylamide gel and resolved by electrophoresis by 160V for 1 h. The resolved proteins were then electro-transferred by 50mA onto nitrocellulose membrane over 16 h. The protein blots were blocked by 5% skim milk for 1 h. KCC2 protein was recognized by monoclonal anti-KCC2 antibody (from UC Davis) at 1µg/mL concentration diluted in 5% skim milk. HRP-conjugated donkey anti-mouse secondary antibody was used to recognize the anti-KCC2 antibody at a concentration of 0.2µg/mL. Chemiluminescence was generated by a VisigloTM HRP plus substrate kit from Amresco and detected by an LAS-3000 image reader from Fujifilm. Quantification of the chemiluminescence signal was carried out by Multi Gauge v3.0 from Fujifilm. Immunofluorescence- HEK-293 cells transfected with KCC2 plasmids or 4-week-old hippocampal neuron cultures were grown on 1 cm diameter glass coverslips coated with 1mg/mL poly-L-lysine. Cells were fixed with 4% paraformaldehyde in PBS for 15 min, washed 3 times with 1xPBS and blocked with 1xPBS containing 5% skim milk and 0.2% Triton X-100 for 10 min. The cells were incubated with monoclonal anti-KCC2 antibody for 2 h, washed 5 times with 1xPBS, incubated with TRITC-conjugated polyclonal anti-mouse antibody in blocking buffer for 1 h, washed 5 times with 1xPBS and mounted on glass slides with 3µL Vectashield® mounting medium. The prepared slides were imaged with a confocal microscope after 24 h. Acquisition of confocal images was carried out with a Laser Sharp 2000 software from Bio-Rad. Quantification of fluorescence images was carried out using MetaMorph software from Universal Imaging Corporation. In vitro kinase assay- 0.5µg fusion protein was incubated with 10µCi of 32P-γ-ATP and 1-50 ng purified PKC or PKA from Calbiochem in a buffer containing 20mM HEPES (pH 7.5), 10mM MgCl2 and 50µM ATP for 10 min at 30oC and terminated by the addition of 2xSDS-PAGE sample buffer (18,19). The reaction mixture was then analyzed by SDS-PAGE. A phosphor-imager was used to quantify the incorporation of 32P into the proteins.

Neuronal cultures- In brief, rat embryos at E18 were removed and decapitated into 1xHanks’ Balanced Salt Solution (HBSS) from Gibco on ice. Brain tissues from the embryos were removed and transferred into fresh HBSS on ice and hippocampal regions were further dissected out. Dissected hippocampi were placed in 0.25% trypsin at 37oC for 15 min with gentle shaking. Hippocampi were washed by HBSS 2 times for 5 min and passed through Pasteur pipettes 10 times to dissociate. Non-dissociated debris was allowed to settle to the bottom for 10 min. Dissociated hippocampal neurons were counted by hemocytometer and plated on 60mm culture dish at a density of 1 million/dish in attachment medium containing 10% FBS, 1mM sodium pyruvate, 25mM glucose in MEM (Gibco). 4 h after plating attachment medium was replaced with warm maintenance medium containing 2% B-27 neural supplement, 2% FBS, 0.5mM glutamine in Neurobasal medium (Gibco). Hippocampal cultures were kept in an incubator conditioned at 37oC and 5% CO2. 0.5mL of maintenance medium was added into the culture dish every 3 days to replenish the loss of medium from evaporation. Peptide mapping and phospho-amino acid analysis- Gel-slices were excised from SDS-PAGE gels washed and digested with 0.1mg/ml trypsin and subject to 2-dimensional mapping and visualized by autoradiography. The resulting phospho-peptides were also hydrolyzed using 6N HCl and the resulting phospho-amino acids along with phospho-amino acid standards were then separated by thin layer chromatography and visualized by autoradiography as detailed previously (18,19). Site-directed mutagenesis- Mutation of KCC2 was carried out by PCR amplification of the whole plasmid using primer pairs that harbor the desired mutation site as follows: S728A: GAGGCTATCCGGCGCCTGATGGAGGC and CTCTGCCCGCTGAGCCTGAGG T787A: AGGAACTTCATCGAACTCGTCCGGGAAACTAC and CCAAGCCTGATGATCCTCCTTCTGTCGCCAGTTGC S940A: GAATCTCGGGGCGCTATTCGGAGGAAGA and ATCTGTGATGCTCTGGATCTCCCGTTCC

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S1034A: GAAAACTTGAACCAGTCCAACGTGCG and CCACTCCGGCTTCATAGCGAAGAAGTCCTTG. All mutations were verified by DNA sequencing. Whole-cell metabolic labeling and immunoprecipitation- HEK-293 cells or 28-35 Div hippocampal neurons in 60 mm dishes were labeled with 0.5-1.0 mCi/ml 32P-orthophosphoric acid for 4 h in phosphate-free media or with 200 mCi/ml 35S methionine (16). Cell were then lyzed in lysis buffer containing 10mM NaPO4, 5mM EDTA and EGTA, 100mM NaCl, 10mM Na pyrophosphate, 25mM NaF, 2% Triton X-100, 0.5% deoxycholate, 1mM Na orthovanadate, 100µM PMSF, 10µg aprotinin, leupeptin and pepstatin (16). The supernatant was collected after centrifugation. 2µg antibody and 40µL of protein A-sepharose (1:1 slurry) were then added to the supernatant and incubated for 2 h at 4oC with constant agitation. The protein A-sepharose was then washed extensively in lysis buffer supplemented with 500 mM NaCl and analyzed by SDS-PAGE.

RESULTS

In vitro analysis of KCC2 phosphorylation by PKC. Molecular studies have demonstrated that the consensus site for phosphorylation by a number of classical 2nd messenger-dependent protein kinases including both PKC and cAMP-dependent protein kinase (PKA) are evident within both the major intracellular domains of KCC2 (3). To commence our studies we expressed the major N-terminal (amino acids 1-102; His-N/KCC2) and C-terminal (amino acids 645-1116; His-C/KCC2) intracellular cytoplasmic domains of KCC2 as His-tagged fusion proteins in E. coli (Fig. 1A). Purified fusion proteins were then subject to in vitro kinase assays. This revealed that the His-C/KCC2 fusion protein was phosphorylated by purified PKC to a final stoichiometry of approximately 0.6 mol/mol while His-N/KCC2 was not phosphorylated under similar conditions (Fig. 1B). In contrast neither fusion protein was phosphorylated by purified PKA, but the cytoplasmic tail of the GABABR2 subunit, a previously identified of substrate of this enzyme (20), was phosphorylated using the same conditions (Fig. 1B). Peptide mapping and

phospho-amino acid analysis revealed that His-C/KCC2 was primarily phosphorylated on serine residues within 2 major phospho-peptides labeled A and B respectively (Fig. 1C and D).

To further analyze KCC2 phosphorylation site-directed mutagenesis was utilized to convert candidate serine residues within His-C/KCC2 to alanines. Based on the consensus for PKC phosphorylation of R/K-X(1-4)S/T-X(1-3)R/K where X = any amino acid (21,22) mutant fusion proteins were produced in which S728, S940 and S1034 were individually and sequentially mutated to alanines. The phosphorylation of the resulting purified proteins by PKC was then compared to that seen for His-C/KCC2. While mutation of S728 and S1034 did not significantly alter phosphorylation, mutation of S940 reduced phosphorylation to 32.5 + 2.5% of control. Moreover mutation of S940 in combination with either S728 or S1034 had very similar effects on PKC-dependent phosphorylation compared to mutation of S940 alone (Fig. 1E). The phosphorylation of the 32P-His-CS940A/KCC2 was further analyzed using peptide mapping and phospho-amino acid analysis. Significantly this revealed that mutation of S940 ablated peptide A seen on PKC-dependent phosphorylation of His-C/KCC2 (Fig. 1D) and that the remaining sites of phosphorylation for this kinase in His-CS940A/KCC2 are serine residues (Fig. 1C). Together these results suggest that the major site for PKC phosphorylation within His-C/KCC2 is S940. S940 is a major site for PKC-dependent phosphorylation of KCC2 when expressed in HEK-293 cells. To analyze the relevance of our in vitro observations we transiently expressed KCC2 in HEK-293 cells. KCC2 expression was first analyzed by immunoprecipitation with anti-KCC2 antibodies after metabolic labeling with 35S-methionine. This resulted in the isolation of 2 bands with approximate molecular masses of 125 and 130 kDa from cells expressing wild-type KCC2 but not from cells expressing empty vector. Similar bands were also immunoprecipitated from cells expressing a mutant form of KCC2 in which S940 (KCC2S940A) was mutated to an alanine (Fig. 2A). The phosphorylation of KCC2 was examined by immunoprecipitation after metabolic labeling with 32P-orthophosphoric acid. A major band of 130 kDa was evident from cells expressing wild-

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type KCC2 that was not seen in those expressing vector alone, demonstrating basal phosphorylation of this protein in HEK-293 cells. Activation of PKC with phorbol di-butyrate (PDBU) for 10 min produced a significant increase (p<0.01) in KCC2 phosphorylation of 195.7 + 5.6% of that evident under basal conditions (Fig. 2B). Phosphorylation of KCC2S940A was analyzed using similar methodology and this mutant construct exhibited robust levels of basal phosphorylation. However in contrast to wild-type KCC2, PDBU did not significantly enhance the phosphorylation of KCC2S940A (Fig. 2B). These results are consistent with our in vitro experiments and strongly suggest that the major site for PKC phosphorylation within the C-terminal intracellular domain of this transporter is S940. Phosphorylation of S940 enhances KCC2 cell surface expression levels. To address the functional consequences of KCC2 phosphorylation we first compared the proportion of KCC2 and KCC2S940A expressed on the cell surface of HEK-293 cells using biotinylation (14,15). This revealed that 22.9 + 4.5% of wild-type KCC2 was present on the cell surface at steady state, and this could be significantly increased to 40.6 + 5.4% upon activation of PKC over a 10 min time period, but the total levels of KCC2 expression were not altered under the same conditions (Fig. 2C). In contrast to this 38.7 + 4.6 % of KCC2S940A was present on the plasma membrane, a level significantly higher (p<0.01) than that of wild-type KCC2 (22.9 + 4.5%; Fig. 2C). However, PDBU treatment did not significantly increase the cell surface expression level of KCC2S940A (Fig. 2C). To confirm the results of our biotinylation assays we assessed the effects of activating PKC on cell surface expression levels of KCC2 using immunohistochemistry. To do so cells expressing KCC2 or KCC2S940A were treated with PDBU, permeabilized and stained with anti-KCC2 antibodies. Confocal images were then recorded from these cells and the pixel intensity across the entire cell was measured in the presence and absence of PBDU treatment (Fig. 3A). The relative level of membrane expression was determined by calculating the ratio of fluorescence signal associated with the cell periphery and the cytoplasm. Notably treatment of cells expressing wild-type KCC2 with PBDU significantly increased the ratio fluorescence associated with

the cell periphery by 182 + 7.2% of control untreated cells (Fig. 3B and C). Similar experiments were performed on cells expressing KCC2S940A; however, in these cells PDBU did not significantly alter the distribution of KCC2 immunoreactivity (Fig. 3A-C). Together these biochemical and imaging experiments indicate that phosphorylation of KCC2 on S940 increases transporter cell surface expression levels. PKC-dependent phosphorylation of S940 decreases KCC2 endocytosis. To further evaluate the mechanism underlying PKC-dependent modulation of KCC2 cell surface stability, the possible role of this enzyme in regulating transporter endocytosis was evaluated. To do so transfected HEK-293 cells were labeled with NHS-SS-Biotin and incubated at 37oC for up to 20 min in the presence of leupeptin to prevent lysozomal degradation of any internalized protein (14). After cleavage of remaining cell surface NHS-SS-Biotin with reduced glutathione, cells were lyzed and internalized biotinylated proteins were isolated on avidin and immunoblotted with KCC2 antibodies. After controlling for the efficiency of cleavage (remaining biotin at 0 min) this approach revealed that the entire cell surface population of KCC2 was endocytosed within 10 min under basal conditions. This process was linear over the initial 5 min period of the assay (Fig. 4A). Under control conditions 80.7 + 8.2 % of the total cell surface population of KCC2 was internalized within 5 min, while in the presence of PDBU internalization was significantly reduced (p<0.01) to 30.5 + 4.5% (Fig. 4B). We used the KCC2S940A mutant to assess the role of S940 in mediating the effects of PKC on KCC2 endocytosis. Compared to wild-type KCC2 the endocytosis of this mutant appeared to be significantly decreased over a time course of 20 min in the presence or absence of PKC activation (Fig. 4A). To quantify these results we compared the level of endocytosis of KCC2 and KCC2S940A after 5 min incubation at 37ºC in the presence and absence of PDBU. Significantly lower levels (p<0.01) of KCC2S940A endocytosis were evident compared to wild-type KCC2 under these conditions (15.6 + 3.2 vs 80.7 + 8.2% respectively; Fig. 4B). However, in contrast to KCC2 PDBU treatment did not significantly alter the endocytosis of KCC2S940A (Fig. 4B). These results strongly suggest that the phosphorylation of

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S940 acts to modulate cell surface stability of KCC2 by slowing its endocytosis. The activity of KCC2 is increased by PKC activation in cultured cells. To measure the functional effects of PKC activity on KCC2 function, expressing cells were treated with bumetanide and ouabain to inhibit endogenous Na+-K+-2Cl- co-transporter and Na+-K+-ATPase, respectively. KCC2 activity was measured by basal furosemide-sensitive 86Rb+ influx. In cells expressing KCC2, significant levels of 86Rb+ accumulation were evident over a 3-5 min time course that were not seen with cells expressing empty vector (Fig. 5). Influx was significantly enhanced (p<0.01) by a 15 min pre-incubation with N-ethylmaleimide (NEM), an accepted activator of KCC2 (2) (Fig. 5). Pretreatment of cells with phorbol 12-myristate-13-acetate (PMA) produced a large and highly significant increase (p<0.01) in KCC2 activity as measured by furosemide-sensitive 86Rb+ influx (Fig. 5). To test the role that direct phosphorylation of KCC2 plays in this modulation, the effects of PMA on a number of KCC2 mutants were analyzed. Significantly, the ability of PKC to modulate the activity of KCC2 was blocked by mutation of S940, but this treatment did not alter the ability of NEM to stimulate transporter activity (Fig. 5). In contrast mutation of S728 or S1034 in KCC2 was without effect on either PKC, or NEM-dependent stimulation of KCC2 activity but interestingly mutation of S728 appeared to increase constitutive activity of KCC2 (Fig. 5). Together this series of experiments revealed that activation of KCC2 by PKC was dependent upon S940, the major site of phosphorylation for this kinase within this protein. PKC activity modulates the phosphorylation and cell surface stability of endogenous KCC2 in cultured neurons. To examine the relevance of our recombinant studies, we examined the phosphorylation of KCC2 in hippocampal neurons. To initiate these experiments, we first examined the expression of KCC2 in these cells via immunoblotting. A major band of approximately 130kDa was seen with anti-KCC2 antibodies that was first detected at 2 weeks and reached maximal expression levels between 4-5 weeks in culture (Fig. 6A) consistent with other studies on the developmental expression of KCC2 in culture (4,23-25). To examine phosphorylation 4-5 week cultures were labeled with 32P-

orthophosphoric acid and subject to immunoprecipitation with KCC2 antibody. Under control conditions a major phospho-protein of 130 kDa was seen immunoprecipitating with KCC2 antibodies but not with control IgG, demonstrating that KCC2 is basally phosphorylated in hippocampal neurons (Fig. 6B). Treatment of neurons with PDBU produced a highly significant increase (p<0.01) in KCC2 phosphorylation (p<0.01) to 295.5 + 22.3 % of control, an effect reduced by co-application of the PKC inhibitor calphostin (Cal). In addition, inhibition of PKC alone significantly reduced (p<0.01) the basal level of KCC2 phosphorylation to 15.5 + 3.5 % of control (Fig. 6B). KCC2 phosphorylation in this system was further evaluated using peptide mapping and phospho-amino acid analysis. Under basal conditions KCC2 was principally phosphorylated on serine and threonine residues and that activation of PKC also induced tyrosine phosphorylation (Fig. 6C). Intriguingly peptide map analysis of 32P-KCC2 from neurons after activation of PKC revealed the presence of 2 phospho-peptides (A and B) which showed very similar migration to those evident on mapping PKC-phosphorylation 32P-His-C/KCC2 together with a neutral peptide, C (Fig. 1D). Together these results suggest that S940 is likely to represent a site of PKC phosphorylation in neuronal KCC2. We also examined the effects of PKC activation on the cell surface stability of KCC2 using biotinylation. This revealed that activation of PKC with PDBU produced a highly significant increase (p<0.01) in the proportion of KCC2 expressed on the neuronal cell surface (CS) to 295.6 + 9.8% of control, an effect that was blocked by a specific PKC inhibitor (Fig. 6D). To confirm our biotinylation experiments, we measured the effects of PKC activation on the targeting of KCC2 to the plasma membrane using immunohistochemistry followed by confocal microscopy (Fig. 7A). The relative level of membrane expression was determined by measuring the ratio of fluorescence signals associated with the cell surface and the cytoplasm of individual proximal dendrites (Fig. 7B). Treatment of neurons with PDBU significantly increased (p<0.01) the level of KCC2 immunoreactivity at the periphery of dendrites compared to control untreated neurons, an effect that was decreased via inhibition of PKC (Fig.

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7C). Therefore in common with our recombinant studies these observations demonstrated that KCC2 in its native environment is subject to PKC-dependent phosphorylation and that this covalent modification increases the targeting of KCC2 to the neuronal cell surface.

DISCUSSION

The potassium chloride co-transporter KCC2 is the major determinant of Cl- transmembrane gradients in adult neurons. The activity of KCC2 results in low intracellular Cl- concentrations that are responsible for hyperpolarizing responses of GABAA and Gly receptors in fast synaptic inhibition within the CNS (4,8). Deficits in KCC2 activity are of importance in epilepsy and other CNS pathologies (26); therefore comprehending the cellular mechanisms neurons use to regulate the activity of this protein is of significance. Here we have begun to examine molecular sites of phosphorylation for individual protein kinases within KCC2 and the role that these residues play in regulating its functional expression. There are a number of studies that have analyzed the regulation of KCC2 by agents that modify the activity of protein kinases and phosphatases (6, 20-28), however it remains to be demonstrated that KCC2 is actually phosphorylated in neurons. We commenced our analysis by expressing the major N- (residues 1-102) and C-terminal intracellular domains (residues 645-1116) of KCC2 as fusion proteins in E. coli and analyzed their phosphorylation in vitro. This revealed that the C-terminal but not the N-terminal fusion protein was selectively and stochiometrically phosphorylated by PKC. Peptide mapping and phospho-amino acid analysis revealed that this phosphorylation occurred on serine residues within 2 major phospho-peptides. Site-specific mutagenesis of S940 with the C-terminal fusion reduced PKC phosphorylation to approximately 25% of control and ablated one of the major peptides seen on phosphorylation of the wild-type protein. Consistent with our in vitro analysis full-length KCC2 exhibited high levels of basal phosphorylation when expressed in HEK-293 cells as measured via immunoprecipitation after metabolic labeling with 32P-orthophosphoric acid. Moreover, phosphorylation of KCC2 was robustly increased upon activation of PKC, an

effect that was ablated by mutation of S940. Therefore our combined in vitro and in vivo analysis demonstrated that KCC2 is a substrate of PKC and that the major site of phosphorylation within the major intracellular domain of this transporter is S940. In addition to our recombinant experiments we directly analyzed the phosphorylation of KCC2 in hippocampal neurons using immunoprecipitation. This revealed that under basal conditions in 28-35 DIV hippocampal neurons KCC2 was basally phosphorylated on serine and threonine residues. Activation of PKC produced a dramatic increase in the stoichiometry of its phosphorylation to approximately 400% of that seen under basal conditions. Enhanced phosphorylation upon the activation of PKC occurred on serine, threonine, and tyrosine residues. Consistent with our result in hippocampal neurons KCC2 is phosphorylated on serine/threonine residues in cortical neurons (see accompanying manuscript). In addition oxidative stress and/or high metabolic activity have recently been reported to induce phosphorylation of KCC2 on tyrosine residues (27). To directly compare our recombinant and neuronal studies of KCC2 phosphorylation we used peptide mapping. This revealed that KCC2 in hippocampal neurons was phosphorylated within three major tryptic peptides after activation of PKC. Significantly two of these peptides were also evident on tryptic peptide mapping of the C-terminus of KCC2 after PKC phosphorylation in vitro. These results together with our recombinant studies suggest that S940 is phosphorylated in neuronal KCC2 molecules upon the activation of PKC. The effects of PKC activity on KCC2 functional expression were evaluated using both biochemical and functional approaches. As measured by biotinylation activation of PKC increased the cell surface expression levels of KCC2 by approximately 200% relative to control over a 10 min time period. This effect of PKC on KCC2 steady state cell surface expression levels was critically dependent on S940 as mutation of this residue abrogated the effect of activating PKC upon transporter cell surface stability. Likewise immunohistochemistry revealed that activation of PKC increased the targeting of PKC to the periphery of HEK-293 cells, an effect that was also dependent upon S940. To analyze the mechanism underlying the effects of S940

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phosphorylation on cell surface stability the effects of PKC activity on KCC2 transporter endocytosis were measured. Under basal conditions the entire cell surface population of KCC2 was internalized over a time course of 10 min. Activation of PKC dramatically slowed the endocytosis of KCC2 compared to control, an effect that was critically dependent on S940, consistent with the higher steady state levels expression of this mutant. Previous experiments using Xenopus oocytes have illustrated that the activation of PKC decreases cell surface expression levels and activity of KCC2 (6). The varying effects of PMA may result form differing trafficking itineraries between oocytes and HEK293 cells or the varying incubation temperatures; 180C and 370C respectively. However, and in keeping with our results the effects of PMA in oocytes was dependent on S940, suggesting a critical role for this residue in regulating KCC2 functional expression via PKC activity. As S940 is not conserved in other CCCs (3), these results suggest a unique role for this residue in regulating KCC2 function. The effects of PKC activity on the cell surface stability of KCC2 on the neuronal plasma membrane were examined. Using both biotinylation and imaging it was evident that activation of PKC in hippocampal neurons that had been cultured for 4-5 weeks produced a large and highly significant increase in the cell surface

expression levels of KCC2 within 10 min. Whether these effects are mediated via altered endocytosis remains to be ascertained; however, it is interesting to note that studies in hippocampal neurons have previously shown that under basal conditions 50% of cell surface KCC2 is degraded within 20 min. Enhancing PKC-dependent phosphorylation may thus provide a rapid and dynamic mechanism to enhance the cell surface and activity of KCC2. In keeping with this potential mechanism Fiumelli et al., have (38), have demonstrated that that ECl shifts caused by changing [Ca2+]i are that are dependent on PKC-activity. However if these effects are mediated by changes in the stochiometry of KCC2 phosphorylation has not been demonstrated. Our studies have identified that KCC2 is directly phosphorylated by PKC on S940 within the cytoplasmic C-terminal domain of this critical transporter and that phosphorylation of this residue acts to increase KCC2 functional expression by slowing its endocytosis; therefore cell signaling molecules that activate PKC signaling pathways may have profound effects on neuronal Cl- homeostasis by regulating the phosphorylation and functional expression of KCC2. Given the critical role KCC2 plays in regulating Cl- homeostasis this phospho-dependent functional modulation may have significant consequences for the efficacy of synaptic inhibition mediated by GABAA

and Gly receptors.

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FOOTNOTES * We thank Margie Maronski from the Dichter laboratory for preparation of cultured hippocampal neurons and Yolande Haydon for manuscript prepartion. This work was supported in part NIH/NINDS grants; NS-036296 (J.A.P), NS-047478 and NS-048045 (S. J. M.). H.H.C.L. was the recipient of a pre-doctoral fellowship from the Epilepsy Foundation of America. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be marked ‘advertisement’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Abbreviations used include: GABAA, γ-aminobutyric acid type A; ATP, adenosine triphosphate; BSA, bovine serum albumin; CCC, cation-chloride cotransporter; Cal, Calphostin; CNS, central nervous system; DMEM, Dulbecco’s Modified Eagle’s Medium; EGR4, early growth response 4; EGTA, ethylene glycol tetraacetic acid; EDTA, ethylenediamine tetraacetic acid; Gly, Glycine; HEK, human embryonic kidney; KCC2, K+-Cl- cotransporter 2; HEPES, N-(2-hydroxyethyl)-piperazine-N’-2-ethanesulfonic acid; NEM, N-ethylmaleimide; NRSE, neuronal restrictive silencing element; PDBu, phorbol 12, 13-dibutyrate, PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; S940, serine 940; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TRITC, tetramethyl rhodamine iso-thiocyanate; WT, wild-type.

FIGURE LEGENDS Fig. 1. S940 is the major site of PKC phosphorylation within KCC2. A. The major intracellular N- and C-terminals domains of KCC2 were cloned into pTrcHis2C vector to generate His-tagged fusion proteins, His-N/KCC2 and His-C/KCC2 respectively. B. His-N/KCC2 and His-C/KCC2 fusion proteins and the intracellular domain of the GABABR2 subunit (CR2) fused to GST were subject to in vitro kinase assays in the presence of 32P-γ−ATP followed by SDS-PAGE. Phosphorylation was then visualized using a

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phospho-imager (upper panel represents 32P), while the lower panel shows coomassie (Com) staining of the same gel. C. 32P-labeled fusion proteins were subject to phospho-amino acid analysis followed by autoradiography. The migration of phospho-serine (pS), threonine (pT) and tyrosine (pY) standards is indicated. D. 32P-labeled fusion proteins and 32P-KCC2 immunopurified from neurons were digested with trypsin and the resulting peptides were applied to TLC plates, subjected to electrophoresis followed by ascending chromatography as indicated. The small arrows indicate the origins and the letters the major phospho-peptides A-C respectively. E. WT and mutant fusion proteins were phosphorylated in vitro by purified PKC in the presence of 32P-γATP and subject to SDS-PAGE. Phosphorylation was then visualized using a phospho-imager (upper panel represents 32P), while the lower panel shows coomassie (Com) staining of the same gel. The level of phosphorylation for each mutant was then compared to that seen for control WT, which was assigned a value of 100% [*=significantly different from control (p<0.01; student’s t-test, n=3)]. Fig. 2. PKC-dependent phosphorylation of S940 regulates the cell surface stability of KCC2 in HEK-293 cells. A. Cells expressing KCC2 constructs or vector (VT) alone were labeled with 200 µCi/ml 35S-methionine for 4 h before immunoprecipitation with anti-KCC2 antibody followed by SDS-PAGE and visualization using a phospho-imager. B. Cells expressing KCC2 expression constructs or VT were labeled with 0.5mCi/ml 32P orthophosphoric acid for 4 h and a further 10 min in the presence (+) and absence (-) of 100 nM PDBU. KCC2 was immunoprecipitated and subject to SDS-PAGE and visualized via autoradiography (upper panel). The level of phosphorylation for each construct was then normalized to that seen under basal conditions (-PDBU =100%). C. Cells expressing KCC2 constructs were treated with (+) and without (-) 100 nM PDBU and then subject to biotinylation. Purified cell surface (CS) and total fractions (T) were then immunoblotted with KCC2 antibodies as illustrated in the upper panel. The proportion of KCC2 expressed on the cell surface was then determined for each construct and data were normalized to those seen under basal conditions (-PDBU=100%) [*=significantly different from control (p<0.01; student’s t-test, n=3-4]. Fig. 3. The subcellular distribution of KCC2 is regulated by S940 phosphorylation. A. HEK-293 cells expressing KCC2 and KCC2S940A were treated with (+) or without (-) 100 nM PDBU for 10 min. Cells were fixed, permeabilized and stained with anti-KCC2 antibodies and a secondary antibody conjugated to FITC. Confocal images were then acquired from the top to the bottom of expressing cells and the midline image was used for further analysis. In the enlargements the region used for quantification are indicate by the lines and the letters A and B represent the points at which data acquisition commenced and terminated respectfully. B. The pixel intensity per unit length (0.1 micron) was then calculated along this line for PDBU (■) and control cells (C) C. The ratio of plasma membrane to cytosol (PM:cytosol) was calculated from the line scans and values obtained under control conditions (-PDBU) were assigned a value of 100% for each construct [*=significantly different from control (p<0.01; student’s t-test, n=10 cells from 3 differing transfections]. Fig. 4. Phosphorylation of S940 regulates KCC2 endocytosis. A. Transfected HEK-293 cells expressing KCC2 or KCC2S940A were treated with NHS-SS-Biotin and then incubated for varying time periods in the absence (-) and presence (+) of 100 nM PDBU at 37ºC. After cleavage of remaining cell surface NHS-SS-Biotin by incubation with 2 mM reduced GST, remaining internalized biotinylated proteins were purified on avidin and immunoblotted with anti-KCC2 antibody (upper panel). The percentage of cell surface KCC2 that was endocytosed over time was determined for cells expressing KCC2 or KCC2S904A in the presence (■) or absence (■) of PDBU B. Endocytosis of KCC2 and KCC2S940A was analyzed over a 5 min time course in the absence (-) and presence (+) of 100 nM PDBU using biotinylation and immunoblotting (upper panel). Data were then compared to the level of endocytosis seen for KCC2 in the absence of PDBU [*=significantly different from control (p<0.01; student’s t-test, n=3].

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Fig. 5. PKC-dependent phosphorylation of S940 modulates KCC2 activity. K+-Cl- cotransport activity was monitored 48 h after transfection by measuring the furosemide-sensitive 86Rb+ influx in cells expressing a range of KCC2 constructs or VT. Influx of 86Rb+ was linear for 10 min in all KCC2 constructs and 3-5 min influxes were routinely performed. HEK-293 cells were pretreated with 2µM bumetanide prior to the influx assay to inhibit endogenous Na+-K+-2Cl- cotransport activity and 100µM ouabain to inhibit endogenous Na+-K+-ATPase. The level of 86Rb+ influx under basal conditions (❏) in the presence of 100 nM PMA (■) or 50 µM NEM (■) over a time course of 3-5 min. 86Rb+ accumulation was then compared to that under basal conditions. [*=significantly different from control (p<0.01; student’s t-test, n= 3]. Fig. 6. Activation of PKC increases both the phosphorylation and cell surface expression levels of KCC2 in cultured hippocampal neurons. A. 50µg of total cellular lysate derived from hippocampal neurons maintained in culture for 0-8 weeks was subject to immunoblotting using anti-KCC2 antibody. B. 4-5 week cultured hippocampal neurons were radiolabeled with 0.75mCi/ml 32P orthophosphate for 4 h before treatment with 100nM PDBU and/or 10µM Cal for 10 min. Cell lysates were immunoprecipitated with either control IgG or KCC2 antibody and precipitated material was subject to SDS-PAGE and visualized using a phospho-imager. KCC2 phosphorylation was compared to the level seen under basal conditions, (-PDBU) which was assigned a value of 100%. C. 32P-labeled KCC2 immunopurified from neurons was subject to phospho-amino acid analysis followed by autoradiography. The migration of phospho-serine (pS), threonine (pT) and tyrosine (pY) standards is indicated. D. Neurons were treated with or without 100nM PDBU or 100nM PDBU + 10µM Cal for 10 min as indicated and subject to biotinylation. CS and T fractions were then immunoblotted with anti-KCC2 antibody as illustrated in the upper panel. The level of cell surface expression was then compared to that evident under basal conditions (100%) [*=significantly different from control (p<0.01; student’s t-test, n= 3]. Fig. 7. The subcellular distribution of KCC2 in hippocampal neurons is modulated by PKC activity. A. 4-week-old cultured hippocampal neurons were treated with (+) or without (-) 100nM PDBU and/or 10µM Cal for 10 min. Neurons were fixed, permeabilized and stained with anti-KCC2 antibody coupled to a rhodamine conjugated secondary. Confocal images were then acquired from the bottom to top of proximal dendrites. In the enlargements the region used for quantification are indicate by the lines and the letters A and B represent the points at which data acquisition commenced and terminated respectfully. B. The pixel intensity per unit length (0.1 micron) was then calculated along for cells treated with PDBU (grey line) and PDBU/Cal (broken black line) and control cells (solid black line). C. The ratio of plasma membrane to cytosol (PM:cytosol) signals was calculated and values were then compared to levels seen under control conditions (-PDBU; 100%) [*=significantly different from control (p<0.01; student’s t-test, n=10 cells from 3 differing transfections].

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Payne and Stephen J. MossHenry H. C. Lee, Joshua A. Walker, Jeffery R. Williams, Richard R. Goodier, John A.

of the potassium chloride cotransporter, KCC2Direct PKC-dependent phosphorylation regulates the cell surface stability and activity

published online August 10, 2007J. Biol. Chem. 

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