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• W2072194715 abstract "The Drosophila phototransduction cascade terminates in the opening of an ion channel, designated transient receptor potential (TRP). TRP has been shown to become phosphorylated in vitro, suggesting regulation of the ion channel through posttranslational modification. However, except for one phosphorylation site, Ser982, which was analyzed by functional in vivo studies (Popescu, D. C., Ham, A. J., and Shieh, B. H. (2006) J. Neurosci. 26, 8570–8577), nothing is known about the role of TRP phosphorylation in vivo. Here, we report the identification of 21 TRP phosphorylation sites by a mass spectrometry approach. 20 phosphorylation sites are located in the C-terminal portion of the channel, and one site is located near the N terminus. All 21 phosphorylation sites were also identified in the inaCP209 mutant, indicating that phosphorylation of TRP at these sites occurred independently from the eye-enriched protein kinase C. Relative quantification of phosphopeptides revealed that at least seven phosphorylation sites were predominantly phosphorylated in the light, whereas one site, Ser936, was predominantly phosphorylated in the dark. We show that TRP phosphorylated at Ser936 was located in the rhabomere. Light-dependent changes in the phosphorylation state of this site occurred within minutes. The dephosphorylation of TRP at Ser936 required activation of the phototransduction cascade. The Drosophila phototransduction cascade terminates in the opening of an ion channel, designated transient receptor potential (TRP). TRP has been shown to become phosphorylated in vitro, suggesting regulation of the ion channel through posttranslational modification. However, except for one phosphorylation site, Ser982, which was analyzed by functional in vivo studies (Popescu, D. C., Ham, A. J., and Shieh, B. H. (2006) J. Neurosci. 26, 8570–8577), nothing is known about the role of TRP phosphorylation in vivo. Here, we report the identification of 21 TRP phosphorylation sites by a mass spectrometry approach. 20 phosphorylation sites are located in the C-terminal portion of the channel, and one site is located near the N terminus. All 21 phosphorylation sites were also identified in the inaCP209 mutant, indicating that phosphorylation of TRP at these sites occurred independently from the eye-enriched protein kinase C. Relative quantification of phosphopeptides revealed that at least seven phosphorylation sites were predominantly phosphorylated in the light, whereas one site, Ser936, was predominantly phosphorylated in the dark. We show that TRP phosphorylated at Ser936 was located in the rhabomere. Light-dependent changes in the phosphorylation state of this site occurred within minutes. The dephosphorylation of TRP at Ser936 required activation of the phototransduction cascade. Reversible protein phosphorylation is a well established mechanism for regulating the activity of ion channels. Typically, the pattern of phosphorylation of ion channels is complex, involving several phosphorylation sites with consensus sequences for a number of protein kinases, such as protein kinase C (PKC), 3The abbreviations used are: PKCprotein kinase CACNacetonitrileFAformic acidMSmass spectrometryLCliquid chromatographyMS/MStandem mass spectrometryTRPtransient receptor potentialTRPLtransient receptor potential-likeSer(P)936-TRPTRP phosphorylated at serine 936HPLChigh performance liquid chromatographyUPLCultraperformance liquid chromatographyXICextracted ion chromatogrameye-PKCeye-enriched protein kinase CPBSphosphate-buffered saline. protein kinase A, calcium/calmodulin-dependent protein kinase, or casein kinase, which phosphorylate serine and threonine residues, as well as kinases phosphorylating tyrosine residues. For example, in the major delayed rectifier K+ channel Kv2.1, expressed in most central neurons, 16 phosphorylation sites have been identified by mass spectrometry, a subset of which contributes to graded modulation of voltage-dependent gating (2Park K.S. Mohapatra D.P. Misonou H. Trimmer J.S. Science. 2006; 313: 976-979Crossref PubMed Scopus (231) Google Scholar). protein kinase C acetonitrile formic acid mass spectrometry liquid chromatography tandem mass spectrometry transient receptor potential transient receptor potential-like TRP phosphorylated at serine 936 high performance liquid chromatography ultraperformance liquid chromatography extracted ion chromatogram eye-enriched protein kinase C phosphate-buffered saline. Transient receptor potential (TRP) channels constitute a protein family of about 30 unique homologs that are assigned to seven subfamilies on the basis of sequence homology: canonical TRPC, vanilloid TRPV, melastatin TRPM, polycystin TRPP, mucolipin TRPML, and ankyrin transmembrane proteins TRPA and NOMPC-like TRPN (3Montell C. Sci. STKE. 2005; 2005: re3PubMed Google Scholar, 4Clapham D.E. Nature. 2003; 426: 517-524Crossref PubMed Scopus (2161) Google Scholar). The founding member of this protein family is the Drosophila TRP channel, which, together with its homolog TRP-like (TRPL), is located in the rhabdomeral photoreceptor membrane of the fly compound eye and represents the major light-sensitive ion channel in this phospholipase C-mediated visual transduction cascade (5Minke B. Parnas M. Annu. Rev. Physiol. 2006; 68: 649-684Crossref PubMed Scopus (64) Google Scholar). Phosphorylation of several TRP channels has been described. Among the vertebrate TRPC channels, TRPC3 and TRPC6 are inhibited by phosphorylation events mediated by protein kinase C and protein kinase G (6Zhang L. Saffen D. J. Biol. Chem. 2001; 276: 13331-13339Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 7Venkatachalam K. Zheng F. Gill D.L. J. Biol. Chem. 2003; 278: 29031-29040Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 8Kwan H.Y. Huang Y. Yao X. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2625-2630Crossref PubMed Scopus (145) Google Scholar). In contrast, Src kinase activity is required for the activation of TRPC3 by diacylglycerol (9Vazquez G. Wedel B.J. Kawasaki B.T. Bird G.S. Putney Jr., J.W. J. Biol. Chem. 2004; 279: 40521-40528Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and Fyn kinase phosphorylates and thereby increases the activity of TRPC6 (10Hisatsune C. Kuroda Y. Nakamura K. Inoue T. Nakamura T. Michikawa T. Mizutani A. Mikoshiba K. J. Biol. Chem. 2004; 279: 18887-18894Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Abolition of the putative protein kinase C phosphorylation site Thr635 in the S4/S5 linker region of TRPC3 by mutation results in increased channel activity and was found to underlie the phenotype of moonwalker mice, which is caused by loss of Purkinje cells (11Becker E.B. Oliver P.L. Glitsch M.D. Banks G.T. Achilli F. Hardy A. Nolan P.M. Fisher E.M. Davies K.E. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 6706-6711Crossref PubMed Scopus (159) Google Scholar). The regulation of the capsaicin- and heat-sensitive TRPV1 channel through phosphorylation of serine residues by protein kinase C is also well established (12Bhave G. Hu H.J. Glauner K.S. Zhu W. Wang H. Brasier D.J. Oxford G.S. Gereau 4th, R.W. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12480-12485Crossref PubMed Scopus (368) Google Scholar, 13Premkumar L.S. Ahern G.P. Nature. 2000; 408: 985-990Crossref PubMed Scopus (721) Google Scholar, 14Numazaki M. Tominaga T. Toyooka H. Tominaga M. J. Biol. Chem. 2002; 277: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). Phosphorylation of TRPV1 sensitizes this channel to capsaicin, heat, and other agonists. Besides protein kinase C, calcium/calmodulin-dependent kinase and protein kinase A were implicated in phosphorylation of TRPV1 (15Jung J. Shin J.S. Lee S.Y. Hwang S.W. Koo J. Cho H. Oh U. J. Biol. Chem. 2004; 279: 7048-7054Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 16Rathee P.K. Distler C. Obreja O. Neuhuber W. Wang G.K. Wang S.Y. Nau C. Kress M. J. Neurosci. 2002; 22: 4740-4745Crossref PubMed Google Scholar). The first TRP channel shown to become phosphorylated again was the Drosophila TRP channel. This channel is part of a signaling complex assembled by the INAD scaffold protein together with phospholipase C and an eye-enriched protein kinase C (eye-PKC) encoded by the inaC gene. It was shown initially for the larger fly Calliphora vicina and later also for Drosophila that the addition of ATP to the isolated signaling complex resulted in phosphorylation of TRP and INAD, suggesting that these two proteins of the signaling complex are targets of the associated protein kinase C (17Huber A. Sander P. Paulsen R. J. Biol. Chem. 1996; 271: 11710-11717Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 18Huber A. Sander P. Bähner M. Paulsen R. FEBS Lett. 1998; 425: 317-322Crossref PubMed Scopus (76) Google Scholar, 19Liu M. Parker L.L. Wadzinski B.E. Shieh B.H. J. Biol. Chem. 2000; 275: 12194-12199Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). A detailed in vitro analysis of TRP phosphorylation by Popescu and colleagues (1Popescu D.C. Ham A.J. Shieh B.H. J. Neurosci. 2006; 26: 8570-8577Crossref PubMed Scopus (49) Google Scholar) revealed a PKC target site in TRP, Ser982. This site was confirmed as a bona fide phosphorylation site in vivo by mass spectrometry. Mutation of Ser982 to Ala resulted in a defect in deactivation of the photoresponse. However, this deactivation defect was observed only upon very intense illumination with white light, whereas a deactivation defect in the eye-PKC null mutant was observed also at much lower light intensities. This difference in phenotype and the observation of multiple phosphorylation sites for different protein kinases in vertebrate TRP channels suggest that additional phosphorylation sites are involved in the regulation of Drosophila TRP. Assuming that reversible phosphorylation of Drosophila TRP should be regulated by light and darkness, which trigger activation and deactivation of this ion channel, respectively, we sought to identify light-regulated phosphorylation sites using quantitative mass spectrometry. Although seven of the identified phosphorylation sites displayed enhanced phosphorylation in the light, a single phosphorylation site, Ser936, was predominantly phosphorylated in the dark and became dephosphorylated upon illumination. We show that changes in the phosphorylation state of this site were detected within minutes after switching to another light condition. Light-triggered dephosphorylation of Ser936 required activation of the phototransduction cascade, suggesting that an increase in intracellular Ca2+ may regulate the dephosphorylation of this site. The following strains and mutants of Drosophila melanogaster were used: w Oregon R, yw;;trpP343 (20Pak W.L. Breakefield X. Neurogenetics: Genetic Approaches to the Nervous System. Elsevier, Amsterdam1979: 67-99Google Scholar), w;;trpP365 (21Yoon J. Ben-Ami H.C. Hong Y.S. Park S. Strong L.L. Bowman J. Geng C. Baek K. Minke B. Pak W.L. J. Neurosci. 2000; 20: 649-659Crossref PubMed Google Scholar), yw;;ninaE17 (22O'Tousa J.E. Baehr W. Martin R.L. Hirsh J. Pak W.L. Applebury M.L. Cell. 1985; 40: 839-850Abstract Full Text PDF PubMed Scopus (383) Google Scholar), w;Gαq1 (23Scott K. Becker A. Sun Y. Hardy R. Zuker C. Neuron. 1995; 15: 919-927Abstract Full Text PDF PubMed Scopus (163) Google Scholar), w,norpAP24 (24Bloomquist B.T. Shortridge R.D. Schneuwly S. Perdew M. Montell C. Steller H. Rubin G. Pak W.L. Cell. 1988; 54: 723-733Abstract Full Text PDF PubMed Scopus (517) Google Scholar), and w;inaCP209 (25Smith D.P. Ranganathan R. Hardy R.W. Marx J. Tsuchida T. Zuker C.S. Science. 1991; 254: 1478-1484Crossref PubMed Scopus (216) Google Scholar). Flies were reared at 25 °C. For all experiments, 1–5-day-old flies were used. Flies were illuminated with an 18-watt fluorescent lamp, 2400 lux, unless noted otherwise. For mass spectrometry experiments, flies were illuminated with white light or were kept in the dark overnight before they were subjected to immunoprecipitation. For analyses of the TRP phosphorylation state at Ser936 in different mutants, flies were kept in the dark for 12–18 h and were then illuminated with white light for 1 h and vice versa before they were subjected to Western blot analyses. To analyze the action spectrum of TRP dephosphorylation at Ser936, flies were kept in the dark for 12–18 h and were then illuminated with white light (2400 lux; see above), blue light (acrylic glass wide band filter transmitting light between 310 and 490 nm, 30 lux), green light (acrylic glass wide band filter transmitting light between 460 and 610 nm, 140 lux), orange light (acrylic glass cut-off filter transmitting light >560 nm, 1300 Lux), or red light (acrylic glass cut-off filter transmitting light >630 nm, 270 lux) or were kept in the dark for 1 h before they were subjected to Western blot analyses. For investigation of the time course of TRP phosphorylation and dephosphorylation at Ser936, flies were kept in the dark or were illuminated with white light 12–18 h before the experiment and were then subjected to the opposite light condition for different periods of time. For immunolocalization of TRP or Ser(P)936-TRP, flies were illuminated with white light, kept in the dark for 12–18 h, or illuminated with white light plus UV (150-watt xenon high pressure lamp, 20,000 lux) for 1 h before they were subjected to immunocytochemistry. Dark-kept flies were dissected under dim red light (Schott RG 630, cold light source KL1500), whereas flies kept in white or colored light were dissected under these light conditions. Fly heads from wild type or inaCP209 flies were separated from bodies by freezing in liquid nitrogen and vigorous vortexing. Heads were passed through a 45-mesh sieve holding back the bodies and were collected on a 25-mesh sieve (Neolab). Approximately 300 wild-type or inaCP209 heads were homogenized in 0.5 ml of extraction buffer supplemented with protease and phosphatase inhibitors (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Triton X-100, 50 μm (4-amidinophenyl)-methanesulfonyl fluoride hydrochloride monohydrate, 10 μg/ml aprotinin, leupeptin, and pepstatin A, 10 mm benzamide, 10 mm sodium fluoride, 1 mm orthovanadate, 10 mm β-glycerophosphate, 500 nm cantharidine, 10 mm sodium pyrophosphate) using a micropestle (Roth). Head homogenates were extracted on ice for 30 min. The mixture was centrifuged for 10 min at 12,000 × g, and the supernatant was used for immunoprecipitation. Immunoprecipitation of the INAD signaling complex from Drosophila head extracts was performed with a monoclonal α-TRP antibody (MAb83F6, Developmental Studies Hybridoma Bank, University of Iowa). Head extracts were incubated with 50 μl of protein G-agarose beads (Pierce) and 6 μg of α-TRP antibody for 1 h at 4 °C. The beads were collected by centrifugation at 10,000 × g for 1 min at 4 °C and washed four times, with 0.5 ml of ice-cold extraction buffer. Precipitated proteins were eluted from protein G-agarose beads with 25 μl of 2× SDS sample buffer (150 mm Tris, pH 6.8, 1.2% (w/v) SDS, 30% (w/v) glycerol, 15% (v/v) 2-mercaptoethanol, 0.02% (w/v) bromphenol blue) at 80 °C for 10 min. The eluate was subjected to SDS-PAGE, and protein bands were visualized by colloidal Coomassie Blue staining (Roth). Proteins were in-gel digested using either trypsin or chymotrypsin (Roche Applied Science) according to Shevchenko et al. (26Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7830) Google Scholar). For chymotryptic digests, a modified incubation buffer was used (100 mm Tris-HCl, pH 7.6, 10 mm CaCl2). After digestion, the gel pieces were extracted with 50% acetonitrile (ACN), 50% 0.1% formic acid (FA) (v/v) for 15 min. The supernatant was collected, and the gel pieces were covered with 5% FA for 15 min before the same volume of ACN was added. After incubation for 10 min, the supernatant was collected. The pooled supernatants were then dried in a vacuum centrifuge and stored at −20 °C. Dried samples were dissolved in 0.1% FA. Nano-LC-ESI-MS/MS experiments were performed on an ACQUITY nano-UPLC system (Waters) coupled to an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific). Tryptic and chymotryptic digests of TRP were concentrated and desalted on a precolumn (2 cm × 180 μm, Symmetry C18, 5-μm particle size, Waters) and separated on a 20 cm × 75 μm BEH 130 C18 reversed phase column (1.7 μm particle size, Waters). Gradient elution was performed from 1% ACN to 50% ACN in 0.1% FA within 60 min. The LTQ-Orbitrap was operated under the control of XCalibur 2.0.7 software. Survey spectra (m/z = 300–1800) were detected in the Orbitrap at a resolution of 60,000 at m/z = 400. Data-dependent tandem mass spectra were generated for the five most abundant peptide precursors in the linear ion trap. For all measurements using the Orbitrap detector, internal calibration was performed using lock-mass ions from ambient air as described in Ref. 27Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2832) Google Scholar. MascotTM 2.2 (Matrix Science) and Sequest (Thermo Fisher Scientific) search engines were used for protein identification. Spectra were searched against the Drosophila subset of the NCBI protein sequence data base downloaded as FASTA-formatted sequences from the NCBI FTP site. Search parameters specified trypsin or chymotrypsin as the cleaving enzyme, allowing four missed cleavages (including cleavage before Pro), a 3 ppm mass tolerance for peptide precursors, and 0.5 Da tolerance for fragment ions. Carbamidomethylation of cysteine residues was set as a fixed modification, and Ser, Thr, and Tyr phosphorylation, methionine oxidation, and N-terminal acetylation of proteins were allowed as variable modifications. Phosphopeptide MS/MS spectra sequence assignments and phosphorylated residues were verified manually. Quantification of light-dependent TRP phosphorylation sites was accomplished by a label-free nano-HPLC-MS method. For each light condition (light or dark), five (tryptic digests) or three (chymotryptic digests) independent experiments and a technical replicate for each experiment were performed. The commercially available SIEVE 1.2 software (Thermo Fisher Scientific) was used for alignment of chromatographic peaks as well as detection, deisotoping, and peak area determination of MS1 features (peptides) over multiple LC-MS/MS runs. The SIEVE software uses the ChromAlign algorithm (28Sadygov R.G. Maroto F.M. Hühmer A.F. Anal. Chem. 2006; 78: 8207-8217Crossref PubMed Scopus (88) Google Scholar) to perform chromatographic alignment between LC-MS/MS runs (e.g. see supplemental Fig. S1). After completion of the alignment step, peptide peak areas were calculated by SIEVE based on extracted ion chromatograms (XICs) of each MS feature (peptide). Peak areas of TRP phosphopeptides were extracted from the SIEVE result table for normalization. Phosphopeptide identifications were based on (a) accurate mass and retention time and (b) assignment of phosphorylated residues in the corresponding MS/MS spectra of the Mascot and Sequest search results. Only phosphopeptides that were present in all biological and technical replicates of at least one light condition (light or dark) were considered for quantification. Normalization of peak areas in all tryptic or chymotryptic LC-MS/MS runs was achieved by using unphosphorylated TRP peptides as omnipresent internal standards as described (29Ruse C.I. Willard B. Jin J.P. Haas T. Kinter M. Bond M. Anal. Chem. 2002; 74: 1658-1664Crossref PubMed Scopus (80) Google Scholar, 30Steen H. Jebanathirajah J.A. Springer M. Kirschner M.W. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 3948-3953Crossref PubMed Scopus (182) Google Scholar). Normalization peptides (reference peptides) were chosen by the following criteria: (a) peptides were present in every LC-MS/MS run, (b) they were not prone to miscleavages and oxidation, and (c) they showed peak area variations among different LC-MS/MS runs that reflected the general variations of the majority of the peptides (30Steen H. Jebanathirajah J.A. Springer M. Kirschner M.W. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 3948-3953Crossref PubMed Scopus (182) Google Scholar). Four reference peptides (NNYEILK, YILAPDSEGAK, GIDPYFPR, and DIFSSLAK) were used for normalization of phosphopeptides derived from tryptic digests, and two reference peptides (QIISERADTEW and ILEEYQGTDKF) were used for the chymotryptic digests. To obtain normalization factors for each experiment, the peak area of a reference peptide was divided by the mean of the peak areas of this reference peptide in the sample set. The peak area of the phosphopeptides of each experiment was then divided by each of the four (tryptic digests) or two (chymotryptic digests) normalization factors, and the resulting values were averaged to obtain the normalized peak areas of the phosphopeptides. The mean of normalized peak areas for every phosphopeptide over all LC-MS/MS runs of one light condition (light or dark) was calculated, and the higher value was set to 100%. An unpaired t test was performed for statistical evaluation of the quantification results. A synthetic phosphopeptide, NH2-CADEVpSLADD-CONH2, including Ser(P)936, was synthesized and injected into rabbits in order to produce antisera (Pineda Antibody Service, Germany). For affinity purification, the phosphopeptide was conjugated to epoxy-activated Sepharose 6B (GE Healthcare) (10 mg of phosphopeptide per 0.5 g of Sepharose) overnight in 2 ml of coupling buffer (0.2 m NaHCO3/Na2CO3, pH 10). After conjugation, the resin was washed once with 2.5 ml of coupling buffer, and residual active groups were blocked with 3 ml of 1 m ethanolamine, pH 8.0, overnight. The resin was washed five times with 2.5 ml of 1× PBS (137 m NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 2 mm KH2PO4), pH 7.0, and packed into a C10/10 column with adaptor AC 10 (GE Healthcare). Unspecifically bound protein was washed off with 15 ml of 1× PBS, pH 7.0, 10 ml of 1× PBS, pH 3.0, and 15 ml of 1× PBS, pH 7.0. 15 ml of antiserum diluted in 30 ml of 1× PBS, pH 7.0, were passed over the column for 2 h, the column was washed with 10 ml of 1× PBS, pH 7.0, and bound antibody was eluted with 1× PBS, pH 3.0. Eluted fractions were adjusted to pH 7.0. The resulting antibody recognized TRP phosphorylated at Ser936 (Ser(P)936-TRP) and hereafter is referred to as α-Ser(P)936. For Western blot analyses, fly heads were homogenized in 1× SDS-PAGE extraction buffer (4% SDS, 1 mm EDTA, 75 mm Tris/HCl, pH 6.8), and extraction was carried out for 10 min at room temperature. The extracts were centrifuged at 16,000 × g at 22 °C for 10 min. Supernatants were subjected to SDS-PAGE according to Laemmli (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207472) Google Scholar), using 8% polyacrylamide gels. Proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad). The membrane was then blocked for 1 h with 5% skim milk in TBS-T (50 mm Tris/HCl, pH 7.3, 150 mm NaCl, 0.1% Tween 20). The ECL Plus Western blotting analysis system (GE Healthcare) was used for signal detection with x-ray film. The primary antibodies used for Western blot analysis were α-Ser(P)936 and α-TRP (MAb83F6, Developmental Studies Hybridoma Bank). When needed, the α-Ser(P)936 and α-TRP signals were quantified with ImageJ 1.41o (National Institutes of Health) by determining the integrated density (i.e. the sum of the values of the pixels in a selection) of each band. α-Ser(P)936 integrated density values were divided by α-TRP values for normalization. Immunocytochemistry was carried out as described elsewhere (32Meyer N.E. Oberegelsbacher C. Dürr T.D. Schäfer A. Huber A. Fly. 2008; 2: 384-394Crossref Scopus (10) Google Scholar). Primary antibodies used for immunocytochemistry were α-TRP (MAb83F6, Developmental Studies Hybridoma Bank) and α-Ser(P)936. Secondary antibodies were α-mouse Cy5 (Dianova), α-rabbit Cy5 (Dianova), α-mouse AlexaFluor 488 (MoBiTec), and AlexaFluor 546-coupled phalloidin (Invitrogen). The sections were examined with the AxioImager.Z1m microscope (objective, EC Plan-Neofluar ×40/1.3 Oil, Zeiss) with the ApoTome module (Zeiss) and documented with the AxioCam MRm (Zeiss). To identify in vivo TRP phosphorylation sites, an MS-based analysis was carried out. Drosophila head extracts were prepared from either dark- or light-adapted flies. The TRP ion channel was purified by immunoprecipitation with a monoclonal α-TRP antibody and size fractionation by SDS-PAGE. The gel was stained with colloidal Coomassie Blue, and the TRP band at 150 kDa was excised and subjected to in-gel trypsin or chymotrypsin digestion. A representative gel is shown in Fig. 1A. TRP peptides from tryptic or chymotryptic digests were analyzed by LC-MS/MS and identified by data base searches using the Mascot and Sequest search engines, allowing protein modifications, such as oxidation of methionines, acetylation, and specifically phosphorylation. In total, a sequence coverage of 89% of the TRP protein was obtained. This includes 66% sequence coverage from tryptic peptides and 74% sequence coverage from chymotryptic peptides (Fig. 1B). Nano-LC-MS/MS analysis revealed 20 phosphorylated serine and threonine residues that were located in the C terminus of the ion channel, including the previously identified phosphorylation site Ser982 (1Popescu D.C. Ham A.J. Shieh B.H. J. Neurosci. 2006; 26: 8570-8577Crossref PubMed Scopus (49) Google Scholar). A single phosphorylated serine residue (Ser15) was identified in the N terminus of TRP (Fig. 1, B and D). The phosphorylation sites were unambiguously identified on the basis of fragmentation spectra (LC-MS/MS analysis). A representative fragmentation spectrum (MS/MS spectrum) for the identification of phosphorylated Ser936 is shown in Fig. 1C. Fragmentation spectra for the other phosphorylation sites are included in the supplement (supplemental Fig. S2). To investigate a possible physiological function of the identified TRP phosphorylation sites, we analyzed their light dependence. In order to obtain conclusive data on the light dependence of TRP phosphorylation sites, we quantified the amount of phosphopeptides present in samples isolated from flies kept in the dark or under white light illumination using a label-free mass spectrometry approach. Only phosphopeptides that were detected in all nano-LC-MS/MS runs of at least one light condition with a signal intensity of at least 10,000 counts were subjected to quantification. 17 of the 21 identified phosphorylation sites were located on phosphopeptides that met these criteria and were quantified. At least three independent nano-LC-MS/MS runs and three corresponding technical replicates were used for quantification. Alignment of nano-LC-MS/MS runs, construction of XICs, and calculation of peak areas were accomplished by SIEVE 1.2 software (Thermo Fisher Scientific), as described under “Experimental Procedures.” A representative XIC of a peptide comprising the Ser936 phosphorylation site is shown in Fig. 2A. Results of the relative quantification of tryptic and chymotryptic phosphopeptides are shown in Fig. 2, B and C, respectively. When possible, the most abundant peptide variant, phosphorylated at a single, unambiguously assigned site was chosen to determine the light dependence of the respective site. However, for some phosphorylation sites, the peptide variants that could be quantified reliably comprised more than one possible phosphorylation site. In most cases, these regioisomers could not be separated by nano-UPLC and were therefore quantified together. In these cases, the number of phosphorylations present on the respective peptides is given as nxP in Fig. 2, B and C, as well as in supplemental Fig. S3, A and B. Quantification revealed that there are significant light-dependent changes in the phosphorylation state of at least eight sites. Five phosphorylation sites, Thr849, Thr864, Ser872, Ser964, and Ser982, were present as unique sites on phosphopeptides that showed significant up-regulation in illuminated flies (Fig. 2, B and C). One phosphorylation site, Ser717, could only be quantified as a doubly phosphorylated chymotryptic peptide (Fig. 2C). Because the other phosphorylation site on the corresponding tryptic peptide, Ser721, showed no significant light-dependent changes (Fig. 2B), we infer that Ser717 is predominantly phosphorylated in the light. A peptide containing either phosphorylated Ser961 or Thr963 was found at a significantly higher level in the light (Fig. 2C). Although these two sites were unambiguously identified by their fragmentation spectra, they could only be quantified together, and it is not possible to tell whether the phosphorylation of Ser961 or Thr963 or of both is up-regulated in the light. Finally, a single site, Ser936, was predominantly detected in its phosphorylated state in dark-adapted flies (Fig. 2, A and B). In order to determine which phosphorylation sites are substrates of eye-PKC, we isolated TRP from the eye-PKC null mutant inaCP209 and analyzed its phosphorylation state by LC-MS/MS. Surprisingly, all phosphopeptides that were observed in TRP isolated from wild type flies were also identified in the inaC mutant, including the peptide comprising Ser982. Moreover, the light dependence of the phosphorylation of these sites observed in wild type flies was similar in the inaC mutant, as revealed by quantitative MS analysis (supplemental Fig. S2 and Fig. 2). This suggests that the phosphorylation sites identified here are not substrates of eye-PKC, or they are phosphorylated by at least one other kinase. In the following experiments, we foc" @default.
• W2072194715 created "2016-06-24" @default.
• W2072194715 creator A5005557027 @default.
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• W2072194715 date "2010-05-01" @default.
• W2072194715 modified "2023-10-17" @default.
• W2072194715 title "Light-dependent Phosphorylation of the Drosophila Transient Receptor Potential Ion Channel" @default.
• W2072194715 cites W1492591187 @default.
• W2072194715 cites W1506809239 @default.
• W2072194715 cites W1883732752 @default.
• W2072194715 cites W1968152658 @default.
• W2072194715 cites W1968532742 @default.
• W2072194715 cites W1970042814 @default.
• W2072194715 cites W1972815989 @default.
• W2072194715 cites W1974003226 @default.
• W2072194715 cites W1975297292 @default.
• W2072194715 cites W1981055094 @default.
• W2072194715 cites W1984033377 @default.
• W2072194715 cites W1984764741 @default.
• W2072194715 cites W1986235007 @default.
• W2072194715 cites W1989868645 @default.
• W2072194715 cites W1993489871 @default.
• W2072194715 cites W1995692886 @default.
• W2072194715 cites W2000152564 @default.
• W2072194715 cites W2006842412 @default.
• W2072194715 cites W2022208918 @default.
• W2072194715 cites W2022864334 @default.
• W2072194715 cites W2040664792 @default.
• W2072194715 cites W2040878362 @default.
• W2072194715 cites W2043217287 @default.
• W2072194715 cites W2045626430 @default.
• W2072194715 cites W2049225095 @default.
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• W2072194715 cites W2070391349 @default.
• W2072194715 cites W2078594804 @default.
• W2072194715 cites W2079036036 @default.
• W2072194715 cites W2087329385 @default.
• W2072194715 cites W2089650056 @default.
• W2072194715 cites W2091515478 @default.
• W2072194715 cites W2091618466 @default.
• W2072194715 cites W2092882644 @default.
• W2072194715 cites W2097663185 @default.
• W2072194715 cites W2100837269 @default.
• W2072194715 cites W2104726963 @default.
• W2072194715 cites W2110211303 @default.
• W2072194715 cites W2117327695 @default.
• W2072194715 cites W2117528871 @default.
• W2072194715 cites W2124923401 @default.
• W2072194715 cites W2131998466 @default.
• W2072194715 cites W2139451963 @default.
• W2072194715 cites W2140946052 @default.
• W2072194715 cites W2147599690 @default.
• W2072194715 cites W2150691484 @default.
• W2072194715 cites W2159900956 @default.
• W2072194715 cites W2166037338 @default.
• W2072194715 cites W2170991485 @default.
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