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• W2016087379 abstract "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. 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) 3The abbreviations used are: TRP, transient receptor potential; PKC, protein kinase C; GFP, green fluorescent protein; SFK, Src family kinase; PLA2, phospholipase A2; PMA, phorbol 12-myristate 13-acetate; 4αPDD, 4α-phorbol 12,13-didecanoate; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; BIM I, bisindolylmaleimide I hydrochloride; HTS, hypotonic solution; TM, transmembrane; HEK, human embryonic kidney; MDCK, Madin-Darby canine kidney; MOPS, 4-morpholinepropanesulfonic acid; MRM, multiple reaction monitoring; WT, wild type; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; IRES, internal ribosome entry site.3The abbreviations used are: TRP, transient receptor potential; PKC, protein kinase C; GFP, green fluorescent protein; SFK, Src family kinase; PLA2, phospholipase A2; PMA, phorbol 12-myristate 13-acetate; 4αPDD, 4α-phorbol 12,13-didecanoate; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; BIM I, bisindolylmaleimide I hydrochloride; HTS, hypotonic solution; TM, transmembrane; HEK, human embryonic kidney; MDCK, Madin-Darby canine kidney; MOPS, 4-morpholinepropanesulfonic acid; MRM, multiple reaction monitoring; WT, wild type; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; IRES, internal ribosome entry site. superfamily consists of Ca2+-permeable cation channels with a remarkable diversity of activation mechanisms (1Venkatachalam K. Montell C. Annu. Rev. Biochem... 2007; 76: 387-417Google Scholar). They perform a wide range of physiological functions and are involved in the pathogenesis of several diseases (2Nilius B. Owsianik G. Voets T. Peters J.A. Physiol. Rev... 2007; 87: 165-217Google Scholar). 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 (3Ramsey I.S. Delling M. Clapham D.E. Annu. Rev. Physiol... 2006; 68: 619-647Google Scholar). Based on sequence similarity, the TRP superfamily can be divided into as many as eight subfamilies, including the vanilloid subfamily (TRPV) (1Venkatachalam K. Montell C. Annu. Rev. Biochem... 2007; 76: 387-417Google Scholar). The TRPV subfamily contains six mammalian members named TRPV1–6, as well as several invertebrate proteins such as osm-9 from Caenorhabditis elegans. TRPV4 was initially identified as an osm-9-related channel that is activated by hypotonic cell swelling (4Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell.. 2000; 103: 525-535Google Scholar, 5Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol... 2000; 2: 695-702Google Scholar, 6Wissenbach U. Bodding M. Freichel M. Flockerzi V. FEBS Lett... 2000; 485: 127-134Google Scholar). Subsequently, experiments with cultured cells indicated that TRPV4 can also be activated by moderate heat with a threshold of 25–34 °C (7Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci... 2002; 22: 6408-6414Google Scholar, 8Watanabe H. Vriens J. Suh S.H. Benham C.D. Droogmans G. Nilius B. J. Biol. Chem... 2002; 277: 47044-47051Google Scholar), mechanical (shear) stress (9Gao X. Wu L. O'Neil R.G. J. Biol. Chem... 2003; 278: 27129-27137Google Scholar), synthetic ligand 4α-phorbol 12,13-didecanoate (4αPDD) (10Watanabe H. Davis J.B. Smart D. Jerman J.C. Smith G.D. Hayes P. Vriens J. Cairns W. Wissenbach U. Prenen J. Flockerzi V. Droogmans G. Benham C.D. Nilius B. J. Biol. Chem... 2002; 277: 13569-13577Google Scholar), and endogenous compounds such as anandamide, arachidonic acid, and 5′,6′-epoxyeicosatrienoic acid (11Watanabe H. Vriens J. Prenen J. Droogmans G. Voets T. Nilius B. Nature.. 2003; 424: 434-438Google Scholar). Although viable, mice with a genetically disrupted Trpv4 gene are impaired in osmoregulation (12Liedtke W. Friedman J.M. Proc. Natl. Acad. Sci. U. S. A... 2003; 100: 13698-13703Google Scholar, 13Mizuno A. Matsumoto N. Imai M. Suzuki M. Am. J. Physiol... 2003; 285: C96-C101Google Scholar), avoidance of high temperatures (14Lee H. Iida T. Mizuno A. Suzuki M. Caterina M.J. J. Neurosci... 2005; 25: 1304-1310Google Scholar), responses to noxious mechanical stimuli (12Liedtke W. Friedman J.M. Proc. Natl. Acad. Sci. U. S. A... 2003; 100: 13698-13703Google Scholar, 15Suzuki M. Mizuno A. Kodaira K. Imai M. J. Biol. Chem... 2003; 278: 22664-22668Google Scholar), inflammation-induced thermal and mechanical hyperalgesia (16Alessandri-Haber N. Dina O.A. Joseph E.K. Reichling D. Levine J.D. J. Neurosci... 2006; 26: 3864-3874Google Scholar, 17Grant A.D. Cottrell G.S. Amadesi S. Trevisani M. Nicoletti P. Materazzi S. Altier C. Cenac N. Zamponi G.W. Bautista-Cruz F. Lopez C.B. Joseph E.K. Levine J.D. Liedtke W. Vanner S. Vergnolle N. Geppetti P. Bunnett N.W. J. Physiol... 2007; 578: 715-733Google Scholar, 18Todaka H. Taniguchi J. Satoh J. Mizuno A. Suzuki M. J. Biol. Chem... 2004; 279: 35133-35138Google Scholar), as well as in shear stress-induced vasodilation (19Hartmannsgruber V. Heyken W.T. Kacik M. Kaistha A. Grgic I. Harteneck C. Liedtke W. Hoyer J. Kohler R. PLoS ONE.. 2007; 2: e827Google Scholar). TRPV4 protein is strongly expressed in epithelial cells of kidneys (20Tian W. Salanova M. Xu H. Lindsley J.N. Oyama T.T. Anderson S. Bachmann S. Cohen D.M. Am. J. Physiol... 2004; 287: F17-24Google Scholar) and airways (21Sidhaye V.K. Guler A.D. Schweitzer K.S. D'Alessio F. Caterina M.J. King L.S. Proc. Natl. Acad. Sci. U. S. A... 2006; 103: 4747-4752Google Scholar), endothelial cells (22Vriens J. Owsianik G. Fisslthaler B. Suzuki M. Janssens A. Voets T. Morisseau C. Hammock B.D. Fleming I. Busse R. Nilius B. Circ. Res... 2005; 97: 908-915Google Scholar), keratinocytes (7Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci... 2002; 22: 6408-6414Google Scholar), and in sensory neurons (12Liedtke W. Friedman J.M. Proc. Natl. Acad. Sci. U. S. A... 2003; 100: 13698-13703Google Scholar, 15Suzuki M. Mizuno A. Kodaira K. Imai M. J. Biol. Chem... 2003; 278: 22664-22668Google Scholar). This distribution indicates that TRPV4 is well positioned to sense systemic and local environmental changes. 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 A2 (PLA2)-mediated release of arachidonic acid, which is further metabolized by cytochrome P450 epoxygenase to 5′,6′-epoxyeicosatrienoic acid (23Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 396-401Google Scholar). Although direct gating of TRPV4 by mechanical force has not been excluded (24Christensen A.P. Corey D.P. Nat. Rev... 2007; 8: 510-521Google Scholar), the TRPV4-mediated responses to shear stress and high viscous load appear to depend on PLA2 activity as well (19Hartmannsgruber V. Heyken W.T. Kacik M. Kaistha A. Grgic I. Harteneck C. Liedtke W. Hoyer J. Kohler R. PLoS ONE.. 2007; 2: e827Google Scholar, 25Andrade Y.N. Fernandes J. Vazquez E. Fernandez-Fernandez J.M. Arniges M. Sanchez T.M. Villalon M. Valverde M.A. J. Cell Biol... 2005; 168: 869-874Google Scholar). In contrast, 4αPDD stimulates TRPV4 directly by binding to its TM3 and TM4 segments (26Vriens J. Owsianik G. Janssens A. Voets T. Nilius B. J. Biol. Chem... 2007; 282: 12796-12803Google Scholar). Finally, activation by heat appears to share mechanistic requirements both with 4αPDD and cell swelling pathways (23Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 396-401Google Scholar, 27D'Hoedt D. Owsianik G. Prenen J. Cuajungco M.P. Grimm C. Heller S. Voets T. Nilius B. J. Biol. Chem... 2008; 283: 6272-6280Google Scholar). 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 (9Gao X. Wu L. O'Neil R.G. J. Biol. Chem... 2003; 278: 27129-27137Google Scholar, 28Xu F. Satoh E. Iijima T. Br. J. Pharmacol... 2003; 140: 413-421Google Scholar), which is reminiscent of the well studied PKC-mediated phosphorylation and regulation of TRPV1 channels (29Tominaga M. Tominaga T. Pflugers Arch.. 2005; 451: 143-150Google Scholar). Furthermore, Xu et al. (30Xu H. Zhao H. Tian W. Yoshida K. Roullet J.B. Cohen D.M. J. Biol. Chem... 2003; 278: 11520-11527Google Scholar) 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 (31Hisatsune C. Kuroda Y. Nakamura K. Inoue T. Nakamura T. Michikawa T. Mizutani A. Mikoshiba K. J. Biol. Chem... 2004; 279: 18887-18894Google Scholar, 32Jiang X. Newell E.W. Schlichter L.C. J. Biol. Chem... 2003; 278: 42867-42876Google Scholar, 33Jin X. Morsy N. Winston J. Pasricha P.J. Garrett K. Akbarali H.I. Am. J. Physiol... 2004; 287: C558-C563Google Scholar, 34Kawasaki B.T. Liao Y. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A... 2006; 103: 335-340Google Scholar, 35Odell A.F. Scott J.L. Van Helden D.F. J. Biol. Chem... 2005; 280: 37974-37987Google Scholar, 36Vazquez G. Wedel B.J. Kawasaki B.T. Bird G.S. Putney Jr., J.W. J. Biol. Chem... 2004; 279: 40521-40528Google Scholar, 37Zhang X. Huang J. McNaughton P.A. EMBO J.. 2005; 24: 4211-4223Google Scholar). However, both the involvement of SFKs and the role of tyrosine 253 in the activation of TRPV4 were subsequently contradicted by others (23Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 396-401Google Scholar). 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 Tyr110 residue in the stimulus-specific modulation of TRPV4 channel function, and contribute to the understanding of the polymodal nature of TRPV4 activation. Plasmids—TRPV4 with a C-terminal FLAG tag was generated from a pcDNA3-based plasmid containing the mouse Trpv4 cDNA (6Wissenbach U. Bodding M. Freichel M. Flockerzi V. FEBS Lett... 2000; 485: 127-134Google Scholar). 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 Ca2+ imaging with HeLa cells, TRPV4 was expressed from the pCAGGS/IRES-GFP vector (6Wissenbach U. Bodding M. Freichel M. Flockerzi V. FEBS Lett... 2000; 485: 127-134Google Scholar). 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 Tyr110 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-transfected with appropriate pLXSN-based plasmid and helper plasmids. Transduction of MDCK cells was carried out in the presence of 8 μg/ml Polybrene (Sigma). TRPV4-expressing cells were selected with 0.25 mg/ml geneticin (Invitrogen), and analyzed by Western blotting and immunofluorescence. Reagents—PMA, 4αPDD, bisindolylmaleimide I hydrochloride (BIM I) (Sigma), and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (Calbiochem) were dissolved in dimethyl sulfoxide at 2–5 mm. Ruthenium red (Latoxan) was dissolved in water at 10 mm. Immunoprecipitations and Western Blotting—Cells were washed with ice-cold phosphate-buffered saline and lysed in the immunoprecipitation buffer (20 mm Tris, pH 7.5, 1% Triton X-100, 50 mm NaCl, 50 mm sodium fluoride, 15 mm Na4P2O7, 0.1 mm EDTA) supplemented with 1 mm sodium orthovanadate and protease inhibitor mixture (Roche). The lysates were cleared by centrifugation at 125,000 × g for 30 min at 4 °C, and incubated with the anti-FLAG M2 resin (Sigma). The bound proteins were washed five times with the immunoprecipitation buffer and analyzed by Western blotting with the following antibodies: mouse anti-FLAG, mouse anti-actin (clone AC-15), rabbit anti-phosphotyrosine (all Sigma), mouse anti-v-src (clone 327) (Calbiochem), mouse anti-V5 (Serotec), mouse anti-phosphotyrosine (clone 4G10) (Upstate), and rabbit anti-phospho-Src (Tyr416) (Cell Signaling). Enzyme-linked Immunosorbent Assay—The assay was performed as previously described (38Wegierski T. Hill K. Schaefer M. Walz G. EMBO J.. 2006; 25: 5659-5669Google Scholar). 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 α-V5 antibody (1.33 μg/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 α-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 × 6-cm precast 4–12% BisTris gel (Invitrogen) under MOPS buffer conditions. After colloidal Coomassie G-250 staining (39Neuhoff V. Stamm R. Eibl H. Electrophoresis.. 1985; 6: 427-448Google Scholar) bands corresponding to TRPV4 were excised and processed for mass spectrometric detection as previously described (40Schindler J. Lewandrowski U. Sickmann A. Friauf E. J. Proteome Res... 2008; 7: 432-442Google Scholar). An in-gel proteolytic digest was performed with 12.5 ng/μl trypsin (Promega) overnight at 37 °C. Peptides were extracted from the gel slices with 15 μl 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-μm inner diameter × 2-cm length precolumn (Ace C18, 5 μm 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-μm inner diameter × 150-mm length (Ace C18, 3 μm 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™ scores >39 (significance threshold p < 0.05) were considered for further manual evaluation regarding the range of ion series with a focus on the correct annotation of phosphorylation sites. Ca2+ Imaging—HeLa, HEK 293T (transfected with FuGENE 6), or MDCK cells were grown on 30-mm round coverslips. The cells were incubated with 2 μm 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 ×40/1.3 oil immersion objective) and perfused with isotonic solution (150 mm NaCl, 6 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 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 Ca2+-free solutions, Ca2+ 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, τ=6×η×Q/(b×h2)(Eq. 1) where τ is shear stress (dyne/cm2); η is fluid viscosity (0.69 cP); Q, flow rate (ml/s); b, chamber width (0.2 cm); h, chamber height (0.02 cm).FIGURE 6Murine TRPV4 stably expressed in MDCK cells mediates responses to PMA in PKC- and SFK-dependent manner. A, Fura-2 calcium imaging of MDCK cells stably expressing mouse TRPV4-FLAG WT, Y110F, or empty pLXSN vector. Cells were stimulated with 1 μm PMA and subsequently with HTS at 35 °C, as indicated with bars above the graphs. Shown are traces of representative measurements (the black line denotes mean 340/380 ratio for all cells recorded in the measurement, the gray area indicates 1 S.D.), and mean amplitudes of Ca2+ transients with S.E. of n independent measurements (n indicated in brackets above the error bars) in response to PMA treatment. B, MDCK cells expressing TRPV4-FLAG WT were treated with 1 μm PMA, HTS, and 50 μm ATP in Ca2+-free solution (Ca2+ replaced by 5 mm EGTA). Shown is a representative trace (repeated 4 times). C–E, representative traces and statistical analysis of mean amplitudes (depicted as above) of Ca2+ responses to 1 μm PMA in MDCK cells expressing TRPV4-FLAG WT with or without the following chemical inhibitors in the bath solution: C,1 μm ruthenium red (RR); D, 100 nm BIM I; and E,1 μm PP2. The inhibitors were also present during the incubation of cells with Fura-2 AM (RR was used at 100 nm during incubation).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 [Ca2+]i in HEK 293T cells was performed as described in Ref. 41Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem... 1985; 260: 3440-3450Google Scholar, using the following equation, [Ca2+]i=β×Kd×[(R−Rmin)/(Rmax−R)](Eq. 2) where β is the ratio of fluorescence emission intensity at 380 nm excitation in Ca2+-depleting and Ca2+-saturating conditions, R is 340/380 ratio at any time, Rmin is the minimum ratio in Ca2+-depleting conditions (5 mm EGTA, 2 μm ionomycin), Rmax is the maximum ratio in Ca2+-saturating conditions (5 mm Ca2+, 2 μm ionomycin), and Kd is the Ca2+ dissociation constant of fura-2 (220 nm). Immunofluorescence—Cells grown on glass coverslips were fixed in 3.7% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked in 2% horse serum, and incubated either with rabbit anti-TRPV4 antibody (38Wegierski T. Hill K. Schaefer M. Walz G. EMBO J.. 2006; 25: 5659-5669Google Scholar) followed by anti-rabbit Cy3-coupled antibody (Jackson ImmunoResearch) or with anti-FLAG antibody followed by anti-mouse Cy3-coupled antibody. Nuclei were stained with 1 μg/ml Hoechst 33342 (Molecular Probes). Images were taken with Zeiss LSM 510 confocal microscope using C-Apochromat ×63/1.2 W (for Fig. 3B) or Plan-Neofluar ×100/1.3 oil (for Fig. 5B) objectives.FIGURE 5Steady state levels and localization of TRPV4-FLAG WT and Y110F proteins stably expressed in MDCK cells. A, the levels of TRPV4-FLAG proteins in lysates from MDCK cells were analyzed by Western blotting (WB) and compared with actin levels. The lysates from MDCK cells expressing pLXSN empty vector were analyzed as control. B, confocal fluorescence microscopy of immunostained MDCK cells stably expressing TRPV4-FLAG WT (left) and Y110F (right) proteins. TRPV4 was detected with anti-FLAG antibodies. Scale bars denote 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Chemical Cross-linking—Cross-linking of TRPV4 with bis(sulfosuccinimidyl)suberate (BS3) (Pierce) was performed as described previously (42Wang Y. Fu X. Gaiser S. Kottgen M. Kramer-Zucker A. Walz G. Wegierski T. J. Biol. Chem... 2007; 282: 36561-36570Google Scholar). Statistical Analysis—Statistical significance was calculated with one-sample t test (for Fig. 2) or unpaired t test (for Figs. 4, 6, supplemental S2, and S4). Unless otherwise stated, n refers to one independent measurement. Significance in figures was depicted as follows: N.S., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. 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 FLAG-tagged 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 Tyr110 and Tyr805 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 Tyr253, a previously reported target site of SKFs during hypotonic cell swelling (30Xu H. Zhao H. Tian W. Yoshida K. Roullet J.B. Cohen D.M. J. Biol. Chem... 2003; 278: 11520-11527Google Scholar). We next extended our analysis o" @default.
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• W2016087379 date "2009-01-01" @default.
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• W2016087379 title "Tyrosine Phosphorylation Modulates the Activity of TRPV4 in Response to Defined Stimuli" @default.
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• W2016087379 doi "https://doi.org/10.1074/jbc.m805357200" @default.
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