Tyrosine Phosphorylation Modulates the Activity of TRPV4 in Response to Defined Stimuli*

Src family tyrosine kinases (SFKs) regulate the function of several transient receptor potential (TRP) family members, yet their role in the regulation of the vanilloid subfamily member 4 protein (TRPV4) remains controversial. TRPV4 is a calcium-permeable channel activated by numerous physical and chemical stimuli. Here we show that SFKs mediate tyrosine phosphorylation of TRPV4 in different cell lines. Using mass spectrometric analysis, we identified two novel phosphorylation sites in the cytosolic N- and C-terminal tails of TRPV4. Substitution of either tyrosine with phenylalanine led to a substantial reduction in the overall tyrosine phosphorylation level of TRPV4, suggesting that these two tyrosines constitute major phosphorylation sites. Both mutants efficiently localized to the plasma membrane, indicating that neither tyrosine is required for trafficking of TRPV4 in the secretory pathway. Analysis of the channel function demonstrated a crucial role of the N-terminal tyrosine residue in the activation of TRPV4 by heat, mechanical (shear) stress, hypotonic cell swelling, and phorbol 12-myristate 13-acetate, but not in the activation by synthetic ligand 4α-phorbol 12,13-didecanoate. Furthermore, the response of TRPV4 to phorbol 12-myristate 13-acetate was SFK-dependent. Because the SFK-mediated phosphorylation of the N-terminal tyrosine occurred before TRPV4 activation, tyrosine phosphorylation appears to sensitize rather than activate this channel. Reactive oxygen species, known to mediate inflammatory pain, strongly up-regulated TRPV4 phosphorylation in the presence of SFKs. Our findings indicate that tyrosine phosphorylation of TRPV4 represents an important modulatory mechanism, which may underlie the recently described function of TRPV4 in inflammatory hyperalgesia.

The transient receptor potential (TRP) 3 superfamily consists of Ca 2ϩ -permeable cation channels with a remarkable diversity of activation mechanisms (1). They perform a wide range of physiological functions and are involved in the pathogenesis of several diseases (2). All TRP proteins share the same topology: six transmembrane (TM) segments, a pore-loop situated between TM5 and TM6, and intracellular N-and C-terminal tails (3). Based on sequence similarity, the TRP superfamily can be divided into as many as eight subfamilies, including the vanilloid subfamily (TRPV) (1). The TRPV subfamily contains six mammalian members named TRPV1-6, as well as several invertebrate proteins such as osm-9 from Caenorhabditis elegans.
The polymodal nature of the TRPV4 channel raises the question whether the activating stimuli converge on the same pathway or act independently. Recent studies demonstrate that opening of TRPV4 in response to hypotonic cell swelling involves phospholipase A 2 (PLA 2 )-mediated release of arachidonic acid, which is further metabolized by cytochrome P450 epoxygenase to 5Ј,6Ј-epoxyeicosatrienoic acid (23). Although direct gating of TRPV4 by mechanical force has not been excluded (24), the TRPV4-mediated responses to shear stress and high viscous load appear to depend on PLA 2 activity as well (19,25). In contrast, 4␣PDD stimulates TRPV4 directly by binding to its TM3 and TM4 segments (26). Finally, activation by heat appears to share mechanistic requirements both with 4␣PDD and cell swelling pathways (23,27). Thus, TRPV4 can be gated by at least two independent mechanisms.
In addition to the stimuli described above, activity of TRPV4 can be also regulated by phosphorylation. Treatment with phorbol 12-myristate 13-acetate (PMA) activates TRPV4 in a protein kinase C (PKC)-dependent way (9,28), which is reminiscent of the well studied PKC-mediated phosphorylation and regulation of TRPV1 channels (29). Furthermore, Xu et al. (30) have shown that phosphorylation of TRPV4 on tyrosine 253 by Src family kinases (SFKs) is required for channel activation upon treatment with hypotonic solution. In agreement with this finding, SFKs positively regulate several other TRP channels (31)(32)(33)(34)(35)(36)(37). However, both the involvement of SFKs and the role of tyrosine 253 in the activation of TRPV4 were subsequently contradicted by others (23). Prompted by this controversy, we further explored the function of the SFK-mediated tyrosine phosphorylation of TRPV4. Using mass spectrometry, we unequivocally identified the phosphorylation sites of Src kinase in TRPV4 as tyrosines 110 and 805, and characterized the TRPV4 variants with point mutations at these sites. Our results demonstrate a major role of the Tyr 110 residue in the stimulus-specific modulation of TRPV4 channel function, and contribute to the understanding of the polymodal nature of TRPV4 activation.

EXPERIMENTAL PROCEDURES
Plasmids-TRPV4 with a C-terminal FLAG tag was generated from a pcDNA3-based plasmid containing the mouse Trpv4 cDNA (6). We verified that the addition of the FLAG tag did not inhibit TRPV4 channel function (data not shown). The Trpv4 cDNA was re-cloned into pcDNA6-V5/His vector (Invitrogen) to obtain C-terminal V5/His-tagged constructs. For retrovirus production, Trpv4 cDNA was cloned into pLXSN vector (Clontech). For Ca 2ϩ imaging with HeLa cells, TRPV4 was expressed from the pCAGGS/IRES-GFP vector (6). Y110F and Y805F substitutions were generated by PCR, and short DNA fragments containing these mutations were used to replace corresponding wild-type fragments in target plasmids, to eliminate the possibility of introducing undesired nucleotide changes. The resulting plasmids were verified by sequencing. The T7-tagged fragment of the N-terminal tail of TRPV4 (amino acids 1-321) containing Tyr 110 as the only tyrosine (T7-N-TRPV4 110Y) was prepared by DNA synthesis and cloned into pcDNA3 vector (Invitrogen). V-src was expressed from a plasmid containing Rous sarcoma virus DNA fragment (Schmidt-Ruppin A strain). Human SRC cDNA was cloned into EcoRI and SalI sites of the pIRES-hrGFP-1a vector (Stratagene). Src kinase-dead (KD) mutant was constructed by introducing K297R substitution into the above construct.
Cell Cultures, Transfections, and Retrovirus Production-Human embryonic kidney (HEK) 293T, HeLa, and Madin-Darby canine kidney (MDCK) cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Transient transfections were carried out using the calcium phosphate method or FuGENE 6 reagent (Roche Applied Science). Retroviruses were produced in HEK 293T cells co-trans-fected with appropriate pLXSN-based plasmid and helper plasmids. Transduction of MDCK cells was carried out in the presence of 8 g/ml Polybrene (Sigma). TRPV4-expressing cells were selected with 0.25 mg/ml geneticin (Invitrogen), and analyzed by Western blotting and immunofluorescence.
Enzyme-linked Immunosorbent Assay-The assay was performed as previously described (38). In short, HEK 293T cells transfected by the calcium phosphate method were split into poly-L-lysine-coated 48-well dishes. The cells were incubated with ␣-V5 antibody (1.33 g/ml) in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 20 mM HEPES at 4°C for 45 min, washed three times, and fixed with 3.7% paraformaldehyde in phosphate-buffered saline. Following incubation with alkaline phosphatase-coupled ␣-mouse antibodies (Sigma), the enzymatic reaction was performed using 1 mg/ml p-nitrophenyl phosphate (Sigma), and read at 405 nm in a microplate spectrophotometer. Cells split in parallel were lysed and analyzed by Western blotting. The blots were scanned and the bands were quantified using ImageJ software (National Institutes of Health).
Mass Spectrometry-Purified TRPV4-FLAG proteins were separated by one-dimensional SDS-PAGE using a 6 ϫ 6-cm precast 4 -12% BisTris gel (Invitrogen) under MOPS buffer conditions. After colloidal Coomassie G-250 staining (39) bands corresponding to TRPV4 were excised and processed for mass spectrometric detection as previously described (40). An in-gel proteolytic digest was performed with 12.5 ng/l trypsin (Promega) overnight at 37°C. Peptides were extracted from the gel slices with 15 l of 0.1% trifluoroacetic acid for 30 min.
Mass spectrometric analyses were performed on a linear ion trap coupled online to a nanoLC system (Famos, Switchos, Ultimate, Dionex, Idstein, Germany) comprising a common precolumn concentration setup. For trapping and desalting of peptides from in-gel digests a custom-made 100-m inner diameter ϫ 2-cm length precolumn (Ace C 18 , 5 m particle size, 100 Å pore size, HiChrom Ltd., Berkshire, UK) with 0.1% trifluoroacetic acid as loading buffer was used. Reversed-phase separation was performed on custom-made 75-m inner diam-eter ϫ 150-mm length (Ace C 18 , 3 m particle size, 100 Å pore size, HiChrom Ltd.) main columns. Separations were accomplished at a flow rate of 290 nl/min and gradient slopes of 1% B/min or 0.5% B/min up to 55% buffer B content, followed by a 5-min wash at 95% B. Solvent A was 0.1% formic acid in water and solvent B 0.1% formic acid in 84% acetonitrile. Spectra of eluting peptides were detected using a Qtrap4000 mass spectrometer (Applied Biosystems, Darmstadt, Germany). To focus on the elucidation of phosphotyrosine residues, multiple reaction monitoring (MRM) was used as primary scan event. MRM transitions were calculated using the precursor ion mass of respective in silico phosphorylated TRPV4 peptides as Q1 mass and m/z 216 as Q3 mass corresponding to the phosphotyrosine immonium ion. Q1/Q3 were set to low/unit and each transition had a dwell time of 25-30 ms with a declustering potential of 30. Spray voltage was set to 2300 V using the micro-ionspray 1 source setup (Applied Biosystems). Within each scan cycle the two most intense transitions from the MRM scan were selected for an enhanced resolution scan (250 atomic mass units/s, 2 scans summed) and charge determination. Subsequently, the same ions were fragmented by enhanced product ion scans (4000 atomic mass units/s, 2 scans summed, m/z range 115-1700).
Raw files were processed using Analyst 1.4 software plug-ins (mascot.dll, Matrix Science/Applied Biosystems). All peaks with intensities below 0.1% of the base peak were omitted, whereas data were centroided in the process. The resulting spectra were searched against the Swiss-Prot data base in an automated fashion. Mass deviance was set to 0.4 Da with trypsin specified as protease comprising one missed cleavage site. Carbamidomethylation was set as fixed modification and oxidation of methionine as well as phosphorylation of tyrosine as variable modifications. Spectra with Mascot TM scores Ͼ39 (significance threshold p Ͻ 0.05) were considered for further manual evaluation regarding the range of ion series with a focus on the correct annotation of phosphorylation sites.
Ca 2ϩ Imaging-HeLa, HEK 293T (transfected with FuGENE 6), or MDCK cells were grown on 30-mm round coverslips. The cells were incubated with 2 M fura-2 AM (Biotium) in the presence of pluronic F-127 for 30 min in the perfusion solution. Cells were mounted in a custom-made open chamber on a microscope stage (Zeiss Axiovert 200M with Fluar ϫ40/1.3 oil immersion objective) and perfused with isotonic solution (150 mM NaCl, 6 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 10 mM glucose, 10 mM HEPES, pH 7.4) at 4 -5 ml/min. To examine responses to hypotonic solution (HTS; Fig. 4C and supplemental Fig. S3), cells were first perfused with 15 ml of mannitol isotonic solution (containing 100 mM mannitol and [NaCl] reduced to 100 mM), and stimulated with hypotonic solution ([NaCl] reduced to 100 mM, without mannitol). Secondary responses to HTS, performed as controls in Figs. 6 and supplemental S4, were elicited with a hypotonic solution prepared by mixing isotonic solution and water at a 2:1 ratio. In Ca 2ϩ -free solutions, Ca 2ϩ was replaced by 5 mM EGTA. To examine responses to shear stress, cells were mounted in a custom-made closed chamber and shear stress was applied by increasing flow rate according to the formula, where is shear stress (dyne/cm 2 ); is fluid viscosity (0.69 cP); Q, flow rate (ml/s); b, chamber width (0.2 cm); h, chamber height (0.02 cm). Cells were exposed to the light from a xenon lamp (Hamamatsu) passed through 340-and 380-nm filters. The emitted fluorescence was recorded and analyzed using Metafluor software (Molecular Devices). Background fluorescence was subtracted before calculation of 340/380 ratios. Typically several GFP-positive HeLa or HEK 293T cells and 20 -30 MDCK cells were recorded in one measurement.
Calibration of 340/380 ratios to intracellular resting calcium concentration [Ca 2ϩ ] i in HEK 293T cells was performed as described in Ref. 41, using the following equation, where ␤ is the ratio of fluorescence emission intensity at 380 nm excitation in Ca 2ϩ -depleting and Ca 2ϩ -saturating conditions, R is 340/380 ratio at any time, R min is the minimum ratio in Ca 2ϩ -depleting conditions (5 mM EGTA, 2 M ionomycin), R max is the maximum ratio in Ca 2ϩ -saturating conditions (5 mM Ca 2ϩ , 2 M ionomycin), and K d is the Ca 2ϩ dissociation constant of fura-2 (220 nM).

Src Phosphorylates TRPV4 on Tyrosines 110 and 805-To
investigate Src-mediated phosphorylation of TRPV4, we generated MDCK cell lines stably expressing FLAG-tagged mouse TRPV4 by retroviral gene transfer. TRPV4-FLAG protein purified from these cells contained phosphorylated tyrosine residues, as evidenced by the immunostaining with anti-phosphotyrosine antibodies (Fig. 1A). The phosphotyrosine signal in TRPV4 purified from cells pre-treated with PP2, a specific inhibitor of SFKs, was reduced in a dose-dependent manner, suggesting that Src or related kinases are largely responsible for the basal level of tyrosine phosphorylation of this protein (Fig.  1A). To confirm the involvement of SFKs in the phosphorylation of TRPV4, we transfected HEK 293T cells with plasmids encoding v-src from Rous sarcoma virus together with either FLAGtagged TRPV4 or CD2-associated protein (FLAG-CD2AP). Co-expression of v-src led to a clear increase in the phosphotyrosine level of TRPV4 but not of CD2AP (Fig. 1B).
Subsequently, TRPV4 purified from HEK 293T cells expressing v-src was subjected to mass spectrometry analysis to identify tyrosine phosphorylation sites in this protein. The employed MRM-scanning technique was sufficient to reduce sample complexity and to detect phosphorylation sites. Therefore, no further enrichment technique was required for phosphopeptide detection. This approach identified two phosphorylation sites within the peptide sequences KAPMDSLFDY*GTYR and SEIY*QYYGFSHTVGR, corresponding to Tyr 110 and Tyr 805 in the protein sequence of TRPV4 (Fig.  1C, upper spectra). The spectra allowed an unambiguous assignment of the respective phosphorylation sites by directly annotating sequence ions. Importantly, mass spectrometry analysis of TRPV4-FLAG purified from MDCK cells indicated the same phosphorylation sites (Fig. 1C, lower spectra). Thus, tyrosines 110 and 805 are phosphorylated by endogenous SFKs in TRPV4 at normal cell culture conditions. Applying the outlined experimental conditions, we did not detect phosphorylation at Tyr 253 , a previously reported target site of SKFs during hypotonic cell swelling (30).
We next extended our analysis of TRPV4 phosphorylation in HEK 293T cells with V5/His-tagged TRPV4 variants after replacing either tyrosine by phenylalanine. Tyrosine phosphorylation of wildtype (WT) TRPV4 was induced in the presence of active Src kinase, but not in the presence of kinasedead (KD) mutant of Src (Fig. 1D). The Src-mediated phosphorylation of TRPV4 variants bearing Y110F and Y805F substitutions, singly or in combination, was clearly dimin- Note that TRPV4-FLAG and FLAG-CD2AP have very similar apparent molecular weight. C, TRPV4-FLAG proteins, purified from HEK 293T cells co-expressing v-src (upper spectra) or from MDCK cells (lower spectra) were analyzed by mass spectrometry using targeted MRM scans in conjunction with tandem mass spectrometric sequencing. Phosphorylation of Tyr 110 and Tyr 805 was identified by significant Mascot scores. Asterisks and hashes within the peptide sequences mark the sites of phosphorylation and methionine oxidation, respectively. D, TRPV4 proteins with Y110F and/or Y805F substitutions exhibit reduced levels of Src-induced tyrosine phosphorylation. HEK 293T cells were transfected with plasmids encoding various V5/His-tagged TRPV4 variants (WT, Y110F, Y805F, or Y110F Y805F) along with WT Src or kinase-dead (KD) Src, as indicated. TRPV4 proteins were immunoprecipitated with anti-V5 antibodies and analyzed by Western blotting with antibodies, as indicated. Expression of Src kinase was detected in the lysates with anti-Src antibodies.
ished in comparison to TRPV4 WT, corroborating the mass spectrometry results. Tyrosines 110 and 805 reside in the Nand C-terminal cytosolic tails of TRPV4, respectively. Alignment of mouse TRPV4 with other TRPV proteins indicated that the Tyr 110 residue is evolutionarily conserved; the homologous residue can be found among vertebrate TRPV4 proteins and appears to be present even in the more distantly related osm-9 of C. elegans (supplemental Fig. S1). In contrast, the Tyr 805 residue is conserved only among mammalian TRPV4 proteins. TRPV1-3 do not contain tyrosine residues that correspond to either Tyr 110 or Tyr 805 . In summary, our results demonstrate that Src family kinases phosphorylate TRPV4 on tyrosines 110 and 805 in two different kidney cell lines, suggesting that these modifications play TRPV4-specific functional roles.
TRPV4 Y110F and Y805F Mutants Localize to the Plasma Membrane-TRPV4 channels function at the plasma membrane. To investigate whether mutation of either tyrosine affects trafficking of the channel to the cell surface, we prepared Trpv4 constructs containing Y110F or Y805F substitution in combination with a V5 tag engineered in the first extracellular loop (V5 loop). The abundance of TRPV4-V5 loop proteins at the plasma membrane can be quantified with a modified enzyme-linked immunosorbent assay using alkaline phosphatase-coupled antibodies following incubation of non-permeabilized cells with anti-V5 antibodies, as previously described (38,42). Expression of TRPV4-V5 loop WT, Y110F, or Y805F proteins resulted in a clear increase in the alkaline phosphatase reaction as compared with the vector-transfected cells ( Fig.  2A). Strikingly, the highest alkaline phosphatase activity was always associated with expression of the TRPV4 Y110F mutant ( Fig. 2A, and data not shown). To account for the variability in transfection efficiency, we calculated the ratios of surface expression (quantified by enzyme-linked immunosorbent assay) to total levels of expression (quantified by Western blotting) for each protein. In comparison to the wild-type TRPV4, this ratio was significantly higher for the TRPV4 Y110F mutant and not significantly different for the Y805F variant (Fig. 2B). These results indicated that Y110F substitution increased the relative abundance of TRPV4 at the plasma membrane of HEK 293T cells. Importantly, this experiment also demonstrated that neither mutant is intracellularly retained due to potential folding and/or assembly defects. The expendable role of Tyr 110 in the secretory trafficking of TRPV4 is consistent with our previous finding that TRPV4 mutant lacking amino acids 40 -112 is detectable at the plasma membrane, whereas a mutant with longer truncation (⌬40 -235) is retained in the endoplasmic reticulum (42).
Mutation of Tyr 110 Impairs TRPV4 Channel Function-TRPV4 is a Ca 2ϩ -permeable channel, and its transient expression in cells increases resting intracellular Ca 2ϩ concentrations [Ca 2ϩ ] i due to its spontaneous activity (5,6,8). Transfection of wild-type Trpv4 into HEK 293T results in death of some cells, most likely due to a deregulated Ca 2ϩ balance. Toxicity caused by the expression of TRPV4 Y805F was comparable with that caused by the expression of TRPV4 WT. In contrast, the expression of TRPV4 Y110F did not lead to any detectable cell death (data not shown). To examine whether the toxicity of different TRPV4 variants correlate with cellular basal Ca 2ϩ levels, we measured [Ca 2ϩ ] i in transiently transfected cells using fura-2 ratiometric imaging. Measurement of fura-2 signals revealed no statistically significant difference in [Ca 2ϩ ] i between the cells transfected with empty vector and with the TRPV4 Y110F-encoding plasmid (23.5 Ϯ 0.9 nM, n ϭ 8, and 50 Ϯ 16.1 nM, n ϭ 8, respectively; see also supplemental Fig. S2). In contrast, resting Ca 2ϩ levels in cells expressing TRPV4 WT and Y805F were both significantly higher than vector-transfected cells (137.1 Ϯ 31.8 nM, n ϭ 7, and 253 Ϯ 62.6 nM, n ϭ 8, respectively; see also supplemental Fig. S2).
The results described above suggested that the Tyr 110 residue may be important for the channel function of TRPV4. To investigate this possibility in more detail, we decided to compare responses of the TRPV4 channel and its Y110F variant to known stimuli by measuring Ca 2ϩ transients with fura-2 imaging. For this analysis we used HeLa cells, because these cells respond more robustly to TRPV4 activation than HEK 293T cells. We used Trpv4 wild-type and Y110F cDNAs fused with IRES-GFP sequences to directly identify transfected (GFP-positive) cells. Western blot analysis demonstrated that both TRPV4 variants were expressed at comparable levels with respect to the co-transcribed GFP, justifying the use of GFP signal both as the qualitative marker of transfected cells and the quantitative marker of TRPV4 expression (Fig. 3A). In addition, both TRPV4 variants displayed similar localization in HeLa cells (Fig. 3B). TRPV4 can be activated by an increase in the temperature of bath solution with a threshold of 25-34°C (7,8). In agreement with this, GFP-positive HeLa cells expressing wild-type TRPV4 exhibited a rise in [Ca 2ϩ ] i upon rapid warming of bath solution to 35°C (Fig. 4A). The response to the flow of heated solution was significantly and substantially diminished in GFP-positive HeLa cells expressing TRPV4 Y110F. Because Ca 2ϩ influx in response to the flow of the heated solution may reflect either heat-evoked activation of TRPV4 or, alternatively, temperature-stimulated activation of TRPV4 by mechanical stress imposed by the flow (9, 43), we analyzed Ca 2ϩ responses of Trpv4-transfected HeLa cells to defined fluid shear stress. HeLa FIGURE 2. TRPV4 containing Y110F or Y805F substitutions localizes at the cell surface. A, HEK 293T cells were transfected with plasmids encoding variants of TRPV4 (WT, Y110F, or Y805F) with a V5-tag engineered into the first extracellular loop, or with the empty vector as a control. The amount of the V5 epitope at the cell surface was quantified by enzyme-linked immunosorbent assay, using an alkaline phosphatase enzymatic reaction. Total levels of V5-tagged TRPV4 proteins were controlled by Western blotting. Shown is one representative experiment. B, ratios of surface levels to total levels for Y110F and Y805F mutants as compared with wild-type TRPV4. Shown are mean Ϯ S.E. from n independent experiments (n ϭ 5 for Y110F, n ϭ 4 for Y805F). p value was calculated using the one-sample t test.
cells expressing wild-type TRPV4, subjected to low level of shear stress (0.3 dyne/cm 2 for 10 min at 37°C), exhibited a clear elevation in [Ca 2ϩ ] i upon increasing fluid shear stress to 9 dyne/ cm 2 (Fig. 4B). In contrast, such response was virtually absent in HeLa cells expressing the TRPV4 Y110F mutant. These results suggest that the Tyr 110 residue may be essential for mediating responses of TRPV4 to mechanical stimuli at physiological temperatures. To avoid the influence of mechanical stress and elevated temperatures on TRPV4 activation triggered by other stimuli, we performed the subsequent Ca 2ϩ imaging experiments with HeLa cells at room temperature.
We analyzed responses of Trpv4-transfected HeLa cells to two well established stimuli, HTS and the PKC-non-activating phorbol ester 4␣PDD, which activate TRPV4 through different mechanisms (23). Although HeLa cells expressing TRPV4 Y110F generally responded to HTS, the mean amplitude of Ca 2ϩ transients was significantly smaller and amounted to 35% of that in HeLa cells expressing wild-type TRPV4 (Fig. 4C). In contrast, TRPV4 Y110F mediated responses to 1 M 4␣PDD that were only moderately and not significantly smaller from those mediated by wild-type TRPV4 (Fig. 4D). Finally, we investigated TRPV4 channel function following the treatment with 1 M PMA, a PKC-activating phorbol ester that causes TRPV4 channel opening in a PKC-dependent manner (9,28). In response to PMA, HeLa cells expressing wild-type TRPV4 exhibited a rise in [Ca 2ϩ ] i , with a kinetics slower than treatments with HTS, 4␣PDD, or increasing shear stress, whereas HeLa cells expressing TRPV4 Y110F virtually showed no Ca 2ϩ transients (Fig. 4E). Cytosolic Ca 2ϩ levels in untransfected (GFP-negative) cells or in cells transfected with empty IRES-GFP vector were not clearly affected by any of the described stimuli, indicating absence of endogenous TRPV4 channel in HeLa cells (supplemental Fig. S3, and data not shown). In aggregate, replacement of tyrosine 110 with phenylalanine disrupts normal TRPV4 channel function in response to TRPV4 activating stimuli such as mechanical stress, HTS, and PMA, but not to 4␣PDD.
Subsequently we asked whether induction of TRPV4 tyrosine phosphorylation would facilitate its activation. To this end, we co-expressed TRPV4 and v-src in HeLa cells. We found that the cells transfected with both proteins exhibited a higher rise in [Ca 2ϩ ] i in response to PMA than the cells transfected with Trpv4 alone (supplemental Fig. S4). Thus, up-regulation of the SFK activity and associated TRPV4 phosphorylation appears to lead to an increased Ca 2ϩ influx through TRPV4 channel.
TRPV4-mediated Response to PMA in MDCK Cells Requires the Activity of PKC and SFK-To investigate whether SFK-mediated TRPV4 phosphorylation, occurring in unstimulated MDCK cells (see Fig. 1A), is required for TRPV4 activation, we characterized Ca 2ϩ transients following PMA treatment in MDCK cell lines stably expressing TRPV4-FLAG wild-type (WT), Y110F mutant, or empty pLXSN vector. We confirmed that TRPV4-FLAG WT and Y110F proteins were expressed at comparable levels in these cells (Fig. 5A) and localized at the cell border (Fig. 5B). Furthermore, both TRPV4 proteins were detected in chemically stabilized oligomeric structures, suggesting that the TRPV4 Y110F protein is not defective in the assembly of mature tetrameric channels (supplemental Fig. S5). Because MDCK cells, in contrast to Trpv4-transfected HeLa cells, did not strongly react to an abrupt increase in the temperature of bath solution (data not shown), all experiments were performed at 35°C. Control, wild-type TRPV4 and TRPV4 Y110F cells exhibited an initial rise in [Ca 2ϩ ] i in response to 1 M PMA followed by a further rise in [Ca 2ϩ ] i upon subsequent exposure to HTS (Fig. 6A). Amplitudes of Ca 2ϩ transients upon treatment with PMA were most pronounced in MDCK cells expressing FLAG-tagged wild-type TRPV4, and significantly higher compared with cells expressing TRPV4 Y110F (Fig. 6A). The responses observed in control cells expressing empty vector pLXSN can be explained by the presence of endogenous TRPV4 in MDCK cells (44). The amplitudes of Ca 2ϩ signals following treatments with PMA and HTS in individual cells expressing TRPV4 WT showed a positive correlation (r ϭ 0.46, p Յ 7.86e-14 for n ϭ 236 individual cells taken from 8 independent measurements presented in Fig. 6A), suggesting that both signals are mediated by TRPV4. The responses to PMA and HTS were not evident when the cells were perfused with Ca 2ϩ -free solution (Fig. 6B), and were almost entirely abolished when the cells were pre-treated with ruthenium red (Fig. 6C), an inhibitor of TRPV channels. Together, these results strongly suggest that Ca 2ϩ transients in MDCK cells upon treatments with PMA and HTS are mediated by the plasma membranelocalized TRPV4 channel, a conclusion supported by a recent finding that responses to either stimulus in renal epithelial M-1 cells are dependent on endogenous TRPV4 (45). In addition, these experiments confirmed that the TRPV4 Y110F mutant is defective in response to activation by 1 M PMA.
Phorbol esters can activate TRPV4 in PKC-dependent and independent manners (28). Pretreatment of MDCK cells expressing TRPV4-FLAG WT with BIM I, a broad-spectrum PKC inhibitor, significantly reduced the mean amplitude of Ca 2ϩ transients to 51% in response to 1 M PMA (Fig. 6D). Thus, TRPV4 activation by PMA depends, at least partially, on PKC activity, as shown previously for TRPV4-expressing HEK 293 cells (9,28). Furthermore, we found that responses to PMA in these cells were severely reduced upon pretreatment with the SFK inhibitor PP2 (Fig. 6E). The latter finding indicates that SFKs positively regulate TRPV4 activation by PKC-mediated pathways and is consistent with the severely attenuated responses of the TRPV4 Y110F mutant to PMA.
The Increase in Tyrosine Phosphorylation of TRPV4 upon PMA Treatment Does Not Depend on the Tyr 110 Residue-The phosphotyrosine level of TRPV4 increases upon hypotonic cell swelling (30). This increase was sensitive to pharmacological inhibition of the SFK activity, indicating an involvement of Src or related kinases. Because PKCs may activate Src leading to tyrosine phosphorylation and sensitization of TRPV1 (37), we investigated whether stimulation of MDCK cells with PMA promotes tyrosine phosphorylation of TRPV4. For this purpose, TRPV4-FLAG WT and Y110F mutant proteins were purified from MDCK cells and their phosphorylation status was examined with an anti-phosphotyrosine antibody. The basal level of tyrosine phosphorylation in the Y110F mutant was decreased, although not completely abolished, as compared with the wild-type, demonstrating that Tyr 110 is a major phosphorylation site in TRPV4 (Fig. 7A). Treatment with 1 M PMA led to a modest and transient increase of tyrosine-phosphorylated TRPV4. However, a similar increase was also evident for the TRPV4 Y110F mutant, suggesting that the PMA-induced phosphorylation does not require Tyr 110 . In addition, although inhibition of SFKs with PP2 led to a decrease in resting levels of tyrosine phosphorylation of TRPV4, PP2 did not prevent the PMA-induced increase in tyrosine phosphorylation (Fig. 7B). Therefore, tyrosine kinases other than SFKs may participate in this process. We confirmed that the anti-phosphotyrosine antibody used in these experiments detects phosphorylated Tyr 110 in its native sequence context by analyzing a TRPV4 fragment that contains a tyrosine only at position 110 (Fig. 7C). Of note, Xu et al. (30) who found increased tyrosine phosphorylation in TRPV4 after cell swelling, did not observe any increase of tyrosine phosphorylation after treatment with 100 nM PMA. In summary, the PMA-induced transient increase in TRPV4 tyrosine phosphorylation does not depend on tyrosine 110, and thus phosphorylation at this residue is unlikely to directly gate TRPV4, at least in response to PMA treatment.
Hydrogen Peroxide Strongly Up-regulates Src-mediated Tyrosine Phosphorylation of TRPV4-Because TRPV4 channel is required for the increased pain sensation (hyperalgesia) during inflammation (16 -18) and SFKs promote inflammatory responses (46), we speculated that Src-mediated phosphorylation of TRPV4 may increase in response to inflammatory mediators. While testing various stimuli that activate Src, we found that hydrogen peroxide strongly induced tyrosine phosphorylation of TRPV4 in MDCK cells (Fig. 8A). Simultaneously, we observed increased autophosphorylation of Src within its activation loop, indicating enhanced Src kinase activity (Fig. 8A). Both tyrosine phosphorylation of TRPV4 and autophosphorylation of Src could be blocked by pre-treating the cells with the PP2 inhibitor prior to the application of hydrogen peroxide (Fig. 8A). Our results confirm that Src is activated by hydrogen peroxide in renal epithelial cells (47). Hydrogen peroxide-induced phosphorylation of the TRPV4 Y110F mutant was substantially weaker than the wild-type TRPV4 (Fig. 8B). Thus, hydrogen peroxide, and presumably oxidative stress, leads to a strong up-regulation of Src-dependent phosphorylation of TRPV4 channel, mostly at the Tyr 110 residue.

DISCUSSION
Although the involvement of SFKs in the function of TRP channels is well documented, the functional consequences of SFK-mediated TRPV4 phosphorylation remain controversial. Therefore, we took a novel approach to explore the role of SFKmediated TRPV4 regulation. Using mass spectrometry, we identified Tyr 110 and Tyr 805 as v-src-induced phosphorylation sites in the N-and C-terminal cytosolic tails of TRPV4, respectively. The same TRPV4 sites were phosphorylated in HEK 293T and in renal epithelial MDCK cells, which natively express this channel, suggesting that phosphorylation at these sites may be physiologically important. Although homologous sites are absent in other mammalian TRPV channels, the tyrosine residue corresponding to Tyr 110 is present in osm-9, a functional homolog of TRPV4 in C. elegans. Substitution of tyrosine 110 with phenylalanine in TRPV4 severely altered its Ca 2ϩ channel function. In comparison to wild-type channel, the TRPV4 Y110F mutant exhibited a diminished spontaneous activity, and mediated strongly reduced Ca 2ϩ transients in response to most known stimuli, including the phorbol ester PMA. Importantly, we excluded that the Y110F mutation decreases the abundance of the TRPV4 channel at the plasma membrane by two different immunological methods in various cell types. Thus, replacement of tyrosine 110 with phenylalanine impairs TRPV4 channel function, without affecting its secretory trafficking. These changes were specific for the Y110F mutant because the spontaneous activity of the TRPV4 Y805F variant was comparable with that of the wild-type, although we did not extensively investigate the channel activity of TRPV4 Y805F.
Our data revealed that TRPV4-dependent Ca 2ϩ transients in MDCK cells triggered by PMA were significantly reduced by prior inhibition of SFKs with PP2. Together with the findings discussed above, this indicates that phosphorylation at Tyr 110 regulates the activity of TRPV4, at least in response to PMA. This conclusion is in agreement with several reports showing that SFKs increase phosphorylation and activation potential of various TRP channels (31,(33)(34)(35)37). Moreover, Xu et al. (30) have shown that cell swelling induces SFK-mediated phosphorylation of TRPV4 at tyrosine 253, and proposed that Tyr 253 phosphorylation regulates the channel permeability of TRPV4. However, our data indicate that TRPV4 phosphorylation at Tyr 110 does not occur concomitantly with TRPV4 activation. Thus, we suggest that Tyr 110 phosphorylation modulates rather than activates TRPV4, and provide the following rationale. First, phosphorylation of TRPV4 at Tyr 110 occurs in unstimu- lated cells; second, a modest increase in PMA-induced TRPV4 tyrosine phosphorylation does not depend on Tyr 110 ; third, the reduction of PMA-stimulated Ca 2ϩ transients in MDCK cells requires a prolonged incubation with the SFK-inhibitor PP2 (ϳ45 min in total) as well as prior serum starvation of cells, suggesting that inhibition of SFKs per se is not sufficient to block responses of TRPV4, but entails TRPV4 dephosphorylation. The requirement of Tyr 253 phosphorylation for the activation of TRPV4 by cell swelling has remained controversial. Vriens et al. (23) presented evidence for the involvement of PLA 2 and its metabolites in the cell swelling-induced TRPV4 response. Small Ca 2ϩ transients in cells expressing TRPV4 Y110F upon hypotonic swelling (Fig. 4C) indicate that phosphorylation at this site is not absolutely required for this response but appears to lower the threshold of TRPV4 activation by PLA 2 metabolites.
Phosphorylation of TRPC4 and TRPV1 by SFKs increases their presence at the cell surface, suggesting that SFKs regulate TRP channels by inducing their exocytosis (35,37). Although we cannot completely exclude such a role for Tyr 110 phosphorylation of TRPV4, the following two observations argue against it: first, under steady-state conditions, TRPV4 Y110F was more abundant than wild-type TRPV4 at cell surface of HEK 293T cells; second, if Tyr 110 phosphorylation increases TRPV4 plasma membrane localization, we would expect to see equally impaired responses of the TRPV Y110F mutant to all stimuli. However, the TRPV4 Y110F mutant was strongly defective in responses to shear stress and PMA, and partially defective in response to hypotonic cell swelling, whereas the response to 4␣PDD was not significantly different from that of wildtype TRPV4. These activation properties of TRPV4 Y110F are consistent with recent findings that TRPV4 can be activated through at least two distinct mechanisms, one involving PLA 2 metabolites and the other involving direct binding of 4␣PDD to the TM3 and TM4 of the channel (23,26). We favor the hypothesis that phosphorylation of Tyr 110 triggers a conformational change in TRPV4 and/or affects its interaction with auxiliary proteins, thereby modulating TRPV4 gating in response to certain stimuli. Strikingly, the phenotypes of the TRPV4 Y110F mutant appear to recapitulate the effects of a recently discovered binding partner of TRPV4, PACSIN3 (48). The interaction with PACSIN3 increases TRPV4 apparent levels at the cell surface, inhibits its spontaneous activity, and impairs its responses to heat and hypotonic swelling, but not to 4␣PDD (27,48). PACSIN3 binds to a proline-rich region in TRPV4 that is separated by only 21 amino acids from the Tyr 110 residue. Thus, it is conceivable that binding to PACSIN3 competes with TRPV4 Tyr 110 phosphorylation.
Nociception, the perception of pain, differs from other senses in that its sensitivity increases with time in the continued presence of painful stimulus, a process called hyperalgesia. This sensitization is in part achieved through up-regulation of TRPV1 via diverse mechanisms, including Src-mediated phosphorylation (37). Although TRPV1 appears to be the major player among TRP channels in mediating nociception, other family members including TRPV4 were shown to participate in this process as well (2). Trpv4 knock-out mice are defective in sensing noxious but not low-threshold mechanical stimuli (12,15). In addition, these mice fail to develop mechanical and ther-mal hyperalgesia in inflamed tissues (16 -18). Finally, TRPV4 is required for taxol-induced neuropathic pain in rats, a process that also depends on the integrin/Src pathway (49). These findings indicate that TRPV4 is involved in the sensitization of pain detection under pathological conditions, yet the underlying mechanisms remain unclear. In this study, we found that hydrogen peroxide strongly up-regulates Src-dependent tyrosine phosphorylation of TRPV4, including the Tyr 110 residue. Supported by the established function of reactive oxygen species and SFKs in mediating inflammation and pain (46,50,51), our results suggest that SFK-mediated phosphorylation of Tyr 110 may contribute to the sensitization mechanism of TRPV4 channel in hyperalgesia.
In summary, we identified two sites of Src-mediated phosphorylation in TRPV4. We present evidence that phosphorylation of Tyr 110 is an important mechanism for the modulation of TRPV4 function. Finally, our results demonstrate the importance of the cytosolic N-terminal tail in mediating stimulusspecific responses of TRPV4, reinforcing recent findings by D'Hoedt et al. (27).  A, MDCK cells stably expressing FLAG-tagged TRPV4 WT or empty vector pLXSN were pretreated with 1 M PP2 for 30 min, and then exposed to 2.5 mM H 2 O 2 for 5 or 20 min. Cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibodies, the purified proteins were analyzed by Western blotting (WB) with anti-phosphotyrosine (pY) and anti-FLAG antibodies. Autophosphorylation of endogenous Src was monitored in the cell lysates with anti-p-Src (Tyr 416 ) antibodies. B, MDCK cells stably expressing FLAG-tagged TRPV4 WT, Y110F, or empty vector pLXSN were exposed to 2.5 mM H 2 O 2 for 5 or 20 min, and subsequently lysed. FLAGtagged proteins were immunoprecipitated and analyzed as above. Note, that the basal level of tyrosine phosphorylation of TRPV4 is not detectable on the presented immunoblots, reflecting its strong up-regulation by H 2 O 2 .