FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2006464961> ?p ?o ?g. }

• W2006464961 endingPage "16316" @default.
• W2006464961 startingPage "16311" @default.
• W2006464961 abstract "The inositol 1,4,5-trisphosphate receptor (IP3R) plays an essential role in Ca2+ signaling during lymphocyte activation. Engagement of the T cell or B cell receptor by antigen initiates a signal transduction cascade that leads to tyrosine phosphorylation of IP3R by Src family nonreceptor protein tyrosine kinases, including Fyn. However, the effect of tyrosine phosphorylation on the IP3R and subsequent Ca2+ release is poorly understood. We have identified tyrosine 353 (Tyr353) in the IP3-binding domain of type 1 IP3R (IP3R1) as a phosphorylation site for Fyn both in vitro and in vivo. We have developed a phosphoepitope-specific antibody and shown that IP3R1-Y353 becomes phosphorylated during T cell and B cell activation. Furthermore, tyrosine phosphorylation of IP3R1 increased IP3 binding at low IP3 concentrations (<10 nm). Using wild-type IP3R1 or an IP3R1-Y353F mutant that cannot be tyrosine phosphorylated at Tyr353 or expressed in IP3R-deficient DT40 B cells, we demonstrated that tyrosine phosphorylation of Tyr353 permits prolonged intracellular Ca2+ release during B cell activation. Taken together, these data suggest that one function of tyrosine phosphorylation of IP3R1-Y353 is to enhance Ca2+ signaling in lymphocytes by increasing the sensitivity of IP3R1 to activation by low levels of IP3. The inositol 1,4,5-trisphosphate receptor (IP3R) plays an essential role in Ca2+ signaling during lymphocyte activation. Engagement of the T cell or B cell receptor by antigen initiates a signal transduction cascade that leads to tyrosine phosphorylation of IP3R by Src family nonreceptor protein tyrosine kinases, including Fyn. However, the effect of tyrosine phosphorylation on the IP3R and subsequent Ca2+ release is poorly understood. We have identified tyrosine 353 (Tyr353) in the IP3-binding domain of type 1 IP3R (IP3R1) as a phosphorylation site for Fyn both in vitro and in vivo. We have developed a phosphoepitope-specific antibody and shown that IP3R1-Y353 becomes phosphorylated during T cell and B cell activation. Furthermore, tyrosine phosphorylation of IP3R1 increased IP3 binding at low IP3 concentrations (<10 nm). Using wild-type IP3R1 or an IP3R1-Y353F mutant that cannot be tyrosine phosphorylated at Tyr353 or expressed in IP3R-deficient DT40 B cells, we demonstrated that tyrosine phosphorylation of Tyr353 permits prolonged intracellular Ca2+ release during B cell activation. Taken together, these data suggest that one function of tyrosine phosphorylation of IP3R1-Y353 is to enhance Ca2+ signaling in lymphocytes by increasing the sensitivity of IP3R1 to activation by low levels of IP3. The inositol 1,4,5-trisphosphate receptor (IP3R) 1The abbreviations used are: IP3R, inositol 1,4,5-trisphosphate receptor; PLC, phospholipase C; TCR, T cell receptor; BCR, B cell receptor; GST, glutathione S-transferase; GFP, green fluorescent protein. 1The abbreviations used are: IP3R, inositol 1,4,5-trisphosphate receptor; PLC, phospholipase C; TCR, T cell receptor; BCR, B cell receptor; GST, glutathione S-transferase; GFP, green fluorescent protein. is an intracellular calcium (Ca2+) release channel located on the endoplasmic reticulum of mammalian cells. IP3Rs belong to a family of intracellular Ca2+ release channels that include three major isoforms (IP3R1, IP3R2, and IP3R3) as well as the three forms of the related ryanodine receptors (RyR1, RyR2, and RyR3). Hydrolysis of the minor membrane lipid phosphatidylinositol 4,5-bisphosphate by phospholipase C (PLC) results in the production of diacylglycerol and inositol 1,4,5-trisphosphate (IP3) (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar). IP3 binds to the N-terminal portion of IP3Rs (2Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 328-334Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and causes Ca2+ release from the endoplasmic reticulum via a nonselective cation pore in the C-terminal portion of the channel (3Boehning D. Mak D.-O.D. Foskett J.K. Joseph S.K. J. Biol. Chem. 2001; 276: 13509-13512Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Earlier work from our laboratory has demonstrated that IP3R is required for mobilization of Ca2+ from endoplasmic reticulum stores and Ca2+-dependent antigen-specific T cell proliferation via interleukin-2 production in the Jurkat T cell lymphoma line (4Jayaraman T. Ondriasova E. Ondrias K. Harnick D. Marks A.R. Proc. Natl. Acad. Sci., U. S. A. 1995; 92: 6007-6011Crossref PubMed Scopus (131) Google Scholar).Following the engagement of the T cell receptor (TCR) by antigen-presenting complexes, a cascade of tyrosine kinase activation is initiated. The kinases involved include the Src family nonreceptor tyrosine kinases Lck and Fyn, which phosphorylate the TCR (5Kennedy J.S. Raab M. Rudd C.E. Cell Calcium. 1999; 26: 227-235Crossref PubMed Scopus (20) Google Scholar). Recruitment of other kinases and adaptor proteins by the phosphorylated TCR ζ-chain leads to the phosphorylation and activation of PLC-γ1 to produce IP3 (6Koretzky G.A. Myung P.S. Nat. Rev. Immunol. 2001; 1: 95-107Crossref PubMed Scopus (121) Google Scholar). During T cell activation, Fyn regulates PLC-γ1 activity via these down-stream events and phosphorylates IP3R1 in T lymphocytes (7Jayaraman T. Ondrias K. Ondriasova E. Marks A.R. Science. 1996; 272: 1492-1494Crossref PubMed Scopus (202) Google Scholar). Fyn-mediated tyrosine phosphorylation increases IP3R1 open probability by reducing Ca2+-dependent inhibition of the channel (7Jayaraman T. Ondrias K. Ondriasova E. Marks A.R. Science. 1996; 272: 1492-1494Crossref PubMed Scopus (202) Google Scholar). Consistent with these findings, T cells from Fyn knockout mice show reduced Ca2+ release (8Appleby M.W. Gross J.A. Cooke M.P. Levin S.D. Qian X. Perlmutter R.M. Cell. 1992; 70: 751-763Abstract Full Text PDF PubMed Scopus (441) Google Scholar) and reduced IP3R1 tyrosine phosphorylation in response to TCR ligation (7Jayaraman T. Ondrias K. Ondriasova E. Marks A.R. Science. 1996; 272: 1492-1494Crossref PubMed Scopus (202) Google Scholar). The pleiotropic effects of Fyn on the Ca2+ signaling cascade are complex, and the precise contribution of IP3R1 tyrosine phosphorylation to the Ca2+ signaling events that result in T cell activation and proliferation has not been defined.We have now identified a Fyn-phosphorylated tyrosine residue in IP3R1 (Tyr353) and shown that phosphorylation of Tyr353 increases the binding affinity of IP3 to the IP3R at low levels of IP3 (<10 nm), consistent with the previously reported increases in IP3-activated channel activity in response to Fyn phosphorylation (7Jayaraman T. Ondrias K. Ondriasova E. Marks A.R. Science. 1996; 272: 1492-1494Crossref PubMed Scopus (202) Google Scholar). DT40 IP3R-deficient B cells stably expressing a Tyr353-nonphosphorylatable IP3R1 mutant (IP3R1-Y353F) demonstrated altered Ca2+ release from intracellular stores in response to B cell receptor activation.MATERIALS AND METHODSCell Culture and Transfection—Chicken DT40 B cells were cultured in RPMI 1640 containing 10% fetal bovine serum, 1% chicken serum, 50 μm β-mercaptoethanol, penicillin, and streptomycin at 37 °C in a humidified incubator with 5% CO2. For transfection, 2 × 107 cells in logarithmic phase growth were mixed with 30 μg of linearized plasmid cDNA in 400 μl of cytomix medium (9Cui J. Bian J.S. Kagan A. McDonald T.V. J. Biol. Chem. 2002; 277: 47175-47183Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Electroporation was performed in a 4-mm cuvette using Gene Pulser II apparatus (Bio-Rad) at 350 V, 975 microfarads. For selection of stably transfected clones, 2 mg/ml G418 was added into the culture medium 48 h after transfection, and the cells were incubated for 10–14 days without disturbance. JE6.1 cells were cultured in RPMI 1640 with 10% fetal bovine serum. HEK293 cells were kept in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and transfected with plasmid cDNA as described (10Cui J. Kagan A. Qin D. Mathew J. Melman Y.F. McDonald T.V. J. Biol. Chem. 2001; 276: 17244-17251Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).DNA Constructs—Full-length human T cell type 1 IP3R cDNA (11Harnick D.H. Jayaraman T. Go L. Ma Y. Mulieri P. Marks A.R. J. Biol. Chem. 1995; 270: 2833-2840Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) was cloned into the pcDNA3.1(-) vector using XhoI/KpnI sites. All mutants were generated by PCR using the QuikChange site-directed mutagenesis kit (Stratagene). The human T cell Fyn was cloned by reverse transcriptase-PCR using total RNA from the Jurkat-TAG cell line and the following primers with EcoRI/BamHI sites: 5′-GGGGAATTCAGGCTGTGTGCAATGTAAGG-3′ and 5′-CGCGGATCCTTACAGGTTTTCACCAGGTTGG-3′. The cDNA was cloned into p3×FLAG-CMV-10 vector (Sigma) to provide an N-terminal FLAG epitope. Fusion of enhanced green fluorescent protein (GFP) with the N terminus of IP3R1 was obtained by introducing the XhoI/SacII IP3R1 fragment from pcDNA3.1(-) into the pEGFPC3 vector. A series of GST fusion constructs that include different regions of IP3R1 was created by PCR and cloned in-frame into the pGEX-4T1 vector. Purification of the GST fusion proteins was conducted using methods recommended by the manufacturer (Amersham Biosciences). All constructs were verified by automated fluorescent DNA sequence analysis.Cell Stimulation, Immunoprecipitation and Immunoblotting— 2 × 108 Jurkat cells were stimulated with 3 μg/ml anti-CD3 monoclonal antibody (OKT-3) on ice for 10 min, followed by 15 μg/ml goat antimouse IgG for 3 min at 37 °C. DT40 cells were stimulated with 10 μg/ml anti-chicken IgM monoclonal antibody (M-4) for 2 min at 37 °C. The cells used for immunoprecipitation were rinsed once with phosphate-buffered saline and lysed in lysis buffer: 150 mm NaCl, 25 mm Tris-HCl, pH 7.5, 5 mm EDTA, 1% Nonidet P-40, 0.4% deoxycholic acid, Complete EDTA-free protease inhibitor tablets (Roche Applied Science), 1 mm Na3VO4, 0.2 mm phenylmethylsulfonyl fluoride, 1 mm NaF. Lysates were incubated with antibodies for 2 h, and the immune complex was isolated by the addition of protein A-Sepharose for 1 h at 4 °C. Precipitated proteins were then eluted with Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes for immunoblotting with appropriate antibodies.[3H]IP3 Binding and in Vitro Kinase Assay—Binding studies were performed at 4 °C in a total volume of 100 μl. Immunoprecipitated IP3R1 bound to protein A-Sepharose beads was washed once with binding buffer containing 50 mm Tris, pH 8.3, 1 mm EDTA and incubated with 9 nm [3H]IP3 and varying amounts of unlabeled IP3 for 30 min. Reactions were terminated by centrifugation, washed twice with binding buffer, and suspended in scintillation fluid for determination of bound [3H]IP3. The specific binding was defined as total binding minus nonspecific binding that was measured in the presence of 0.01 and 1 μm unlabeled IP3. Equilibrium competition binding curves were fitted assuming a single class of binding site using nonlinear curve fitting equations (Origin Software). For in vitro Fyn phosphorylation, GST fusion proteins bound to glutathione-Sepharose or immunoprecipitates bound to protein A-Sepharose beads were incubated for 10 min at room temperature in 20 μl of kinase buffer (25 mm HEPES, pH 7.1, 10 mm MgCl2, 5 mm MnCl2, 0.5 mm EGTA, 1 mm Na3VO4, 1 mm dithiothreitol, 100 μm MgATP) in the presence of 5 units of active Fyn (Upstate Biotechnology) with or without 10% [γ-32P]ATP. The reaction products were analyzed by autoradiography or immunoblotting using antibodies against phosphotyrosine.Cytosolic Ca2+Measurement—2 × 106 cells were loaded for 30 min at 37 °C with 2 μm Fura-2-acetylmethoxy ester and 0.05% Pluronic F-127 in 400 μl of growth medium. The cells were washed twice and resuspended in 2 ml of calcium-free Hanks' balanced salt solution containing 1 mm EGTA, 20 mm HEPES, and 0.03% bovine serum albumin. Fluorescence of the stirred cell suspension was measured ratiometrically at 25 °C by emission at 510 nm and excitation at 340 nm and 380 nm using a fluorospectrophotometer (PTI). Data were fitted to pulse equations using Origin software, from which the first derivative was determined.Antibodies—The following peptides were used to generate specific antibodies against the C terminus of IP3R1 and phospho-Tyr353 IP3R1, respectively: (C)-RIGLLGHPPHMNVNPQQPA (12Wojcikiewicz R.J. Luo S.G. J. Biol. Chem. 1998; 273: 5670-5677Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) and (C)-QEKM-VpYSLVS. Polyclonal rabbit antibodies were generated at Zymed Laboratories Inc. using the PolyQuik protocol followed by affinity chromatography against epitope peptides. The antibody against the C terminus of IP3R1 is specific for IP3R1; it does not react with either recombinant IP3R2 or recombinant IP3R3 (data not shown). The following commercial antibodies were also used for immunoblotting and stimulation purposes: mouse monoclonal anti-phosphotyrosine 4G10 (Upstate Biotechnology), anti-GFP (BD Biosciences), mouse anti-chicken IgM M-4 (Southern Biotech), anti-mouse IgG (Sigma), and monoclonal anti-CD3 antibody OKT-3 (Ortho Biotech).RESULTS AND DISCUSSIONFyn Phosphorylates IP3R1 at Tyr353 in Vitro—We have demonstrated previously that IP3R1 is phosphorylated by the tyrosine kinase Fyn during T cell activation (7Jayaraman T. Ondrias K. Ondriasova E. Marks A.R. Science. 1996; 272: 1492-1494Crossref PubMed Scopus (202) Google Scholar). We initially studied two predicted phosphorylation sites for Src family tyrosine kinases, based on a consensus motif for these kinases, (R/K)X2,3(D/E)X2,3Y (13Songyang Z. Carraway K.L. Eck M.J. Harrison S.C. Feldman R.A. Mohammadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantley L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (841) Google Scholar). These sites were Tyr482 (in the IP3-binding domain) and Tyr2617 (in the C-terminal cytosolic domain) (11Harnick D.H. Jayaraman T. Go L. Ma Y. Mulieri P. Marks A.R. J. Biol. Chem. 1995; 270: 2833-2840Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Mutation of either site or both sites to phenylalanine, however, had no significant effect on the tyrosine phosphorylation pattern in response to Fyn of either full-length recombinant IP3R1 containing these mutations or of GST fusion protein fragments of IP3R1 encompassing these residues (data not shown). To find an authentic tyrosine phosphorylation site, fragments of IP3R1 (other than those encompassing transmembrane domain regions) were fused to GST, and the purified fusion proteins were used as substrates for in vitro Fyn phosphorylation (Fig. 1A). Fragment 305–447 showed the strongest phosphorylation signal. According to the consensus sequence given above, this fragment contains a potential site for Src kinase family phosphorylation, Tyr353. Mutagenesis of tyrosine to phenylalanine showed that Tyr353 is the only site in this fragment phosphorylated by Fyn. The Y353F mutant eliminated the phosphorylation signal detected either by autoradiography using radiolabeled [γ-32P]ATP or by the anti-phosphotyrosine antibody 4G10 (Fig. 1B). Tyr353 is located in the IP3-binding domain and is conserved throughout all three types of IP3R (data not shown). Based on the 2.2-Å crystal structure of the IP3-binding core of mouse IP3R1 (14Bosanac I. Alattia J.-R. Mal T.K. Chan J. Talarico S. Tong F.K. Tong K.I. Yoshikawa F. Furuichi T. Iwai M. Michikawa T. Mikoshiba K. Ikura M. Nature. 2002; 420: 696-700Crossref PubMed Scopus (274) Google Scholar), Tyr353 is located at the beginning of the β7 β-strand, immediately adjacent to the SI splice variant region (see Fig. 6). The structure indicates that Tyr353 is highly likely to be present on the exposed surface of the IP3-binding core, suggesting that Tyr353 is capable of being phosphorylated by cytosolic tyrosine kinases.Fig. 6Location of Tyr353 in the IP3-binding core region of IP3R1. The 2.2-Å crystal structure of the IP3-binding core of mouse IP3R1 in complex with IP3 is shown as published previously by Bosanac et al. (Fig. 2A of Ref. 14Bosanac I. Alattia J.-R. Mal T.K. Chan J. Talarico S. Tong F.K. Tong K.I. Yoshikawa F. Furuichi T. Iwai M. Michikawa T. Mikoshiba K. Ikura M. Nature. 2002; 420: 696-700Crossref PubMed Scopus (274) Google Scholar). The IP3 molecule (shown in purple) is bound at the interface between the α-domain (green) and the β-domain (olive), and the locations of residue Tyr353, the SI splice region, the CaI-Ca2+-binding region, and IP3 are marked. The crystal structure is reprinted by permission from Macmillan Publishers Ltd. ((2002) Nature420, 696–700).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next expressed full-length wild-type IP3R1 and IP3R1-Y353F in HEK293 cells, and immunoprecipitated IP3R1 using a specific C-terminal antibody. The Y353F mutant, however, was still tyrosine-phosphorylated by Fyn in vitro, although the signal was reduced (Fig. 1C). Thus, in full-length IP3R1, other tyrosine residues besides Tyr353 can also be phosphorylated by Fyn in vitro. However, Fyn phosphorylation of the GST fusion protein fragment of IP3R1 containing Tyr353 was much stronger than that of any other fragments, suggesting that Tyr353 may be preferentially Fyn phosphorylated in vivo (Fig. 1A).Fyn Phosphorylates IP3R1 at Tyr353 in Vivo—To analyze tyrosine phosphorylation of IP3R1 in vivo, we prepared mutants of T cell Fyn with defective kinase activity (K296M) or with constitutive kinase activity (Y528F) (15Fusaki N. Semba K. Katagiri T. Suzuki G. Matsuda S. Yamamoto T. Int. Immunol. 1994; 6: 1245-1255Crossref PubMed Scopus (34) Google Scholar) and co-expressed these active Fyn (Fyn(+)) or inactive Fyn (Fyn(-)) constructs with IP3R1 in HEK293 cells. Fig. 2A shows that when coexpressed with Fyn(+), the N-terminal (1–611) portion of wild-type IP3R1 is tyrosine phosphorylated, but the phosphorylation of the Y353F N-terminal mutant is undetectable. Fig. 2B shows that both wild-type and Y353F full-length IP3R1 are phosphorylated by Fyn in vivo, consistent with our in vitro data in Fig. 1. To determine whether Tyr353 is phosphorylated in full-length IP3R1, we generated a phosphoepitope-specific antibody (anti-IP3R1-pY353). Anti-IP3R1-pY353 only recognized wild-type full-length IP3R1 when co-expressed with Fyn(+). IP3R1-Y353F co-expressed with Fyn(+), wild-type IP3R1, or IP3R1-Y353F co-expressed with Fyn(-) could not be detected by anti-IP3R1-pY353 antibody (Fig. 2C). These results confirmed that Tyr353 is an authentic site of phosphorylation by Fyn but not the only site in full-length IP3R1 phosphorylated by Fyn.Fig. 2In vivo phosphorylation of Tyr353 of IP3R1 by co-expression with constitutively active Fyn (Fyn(+)) or inactive Fyn (Fyn(-)) in HEK293 cells. Immunoprecipitates (IP) were analyzed by immunoblotting with general anti-P-Tyr antibody 4G10 (A and B) or antibody specific for phosphorylated Tyr353 of IP3R1 (p-Y353) (C). A, tyrosine phosphorylation by Fyn(+) of a protein consisting of GFP fused to the N terminus of an IP3R1 fragment, amino acids 1–611 (GFP-NT). The band migrating near 50 kDa in all three lanes represents the heavy chain of mouse IgG. B, tyrosine phosphorylation by Fyn(+) and Fyn(-) of full-length IP3R1 (without GFP fusion). C, tyrosine phosphorylation at Tyr353 of full-length IP3R1. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The anti-IP3R1-pY353 antibody was next used to determine whether Tyr353 was phosphorylated in antigen-stimulated lymphocytes. The pY353 level in JE6.1 Jurkat T cells and DT40 B cells were detected by activating cells through ligation of the TCR or B cell receptor (BCR), respectively. As shown in Fig. 3, Tyr353 of IP3R1 was phosphorylated in both activated T and B lymphocytes; in contrast, only very weak signals were detected in the nonstimulated control cells. We have observed that IP3R1 immunoprecipitated from activated and nonactivated lymphocytes and subsequently immunoblotted with general antiphosphotyrosine antibody (4G10) shows no significant increase in total phosphotyrosine content (data not shown). This suggests that other tyrosine residues on IP3R1 (Fig. 1A) are not specifically phosphorylated in response to antigen-induced lymphocyte activation. Although these studies do not exclude the possibility that other tyrosine residues on IP3R1 are regulated by signaling mechanisms, our experiments using anti-IP3R1-pY353 antibody show that Tyr353 is phosphorylated during activation via TCR or BCR ligation.Fig. 3Tyr353 is phosphorylated in response to TCR or BCR stimulation. JE6.1 cells or DT40 cells were stimulated by 3 μg/ml OKT-3 or 10 μg/ml M-4 for 2–3 min at 37 °C, respectively. IP3R1 was immunoprecipitated by antibody against the C terminus of IP3R1 and detected by specific antibody against phospho-Tyr353 of IP3R1 (upper panel). The membrane was stripped and reprobed with anti-IP3R1 antibody (lower panel). The graph shows the relative phosphorylation level at Tyr353 in activated versus nonactivated lymphocytes, which was determined by densitometric quantitation of the anti-phospho-Tyr353 immunoblot signal normalized to the anti-IP3R1 signal. The IP3R1-Y353 phosphorylation level of activated JE6.1 cells was taken as 100%. In comparison, nonactivated JE6.1 cells had 31%, activated DT40 cells had 120%, and nonactivated DT40 cells had 14% phosphorylation of IP3R1-Y353.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Phosphorylation of Tyr353 Modulates Affinity of IP3 Binding to the IP3R—Because Tyr353 is located in the IP3-binding domain of IP3R1, we investigated the effects of phosphorylation of Tyr353 on IP3 binding. Equal amounts of immunoprecipitated proteins from transfected HEK293 cells (measured by immunoblotting) were used in an equilibrium [3H]IP3 competition binding assay (Fig. 4A). Fig. 4B compares the [3H]IP3 specifically bound to phosphorylated or nonphosphorylated IP3R1 at an IP3 concentration of 9.1 nm. Phosphorylation of Tyr353 on IP3R1 (via co-transfection of constitutively active Fyn with IP3R1) significantly increased the affinity of IP3 binding. As shown in Fig. 4C, Fyn-phosphorylated IP3R1 exhibited increased [3H]IP3 binding at low unlabeled IP3 concentrations (less than 10 nm), whereas when the unlabeled IP3 concentration in the assay was above 100 nm, no significant differences were observed between phosphorylated wild-type IP3R1, the IP3R1-Y353F mutant, or nonphosphorylated wild-type IP3R1. In the case of phosphorylated wild-type IP3R1, for IP3 binding, the EC50 = 8.45 ± 1.36 nm (n = 7, p < 0.05 compared with nonphosphorylated wild-type IP3R1 or phosphorylated IP3R1-Y353F). The EC50 values of nonphosphorylated wild-type IP3R1 or phosphorylated IP3R1-Y353F were 19.34 ± 1.55 nm (n = 7) and 14.44 ± 1.22 nm (n = 5), respectively. These results are comparable with published Kd values for IP3 binding for native or recombinant IP3R1 (16Riley A.M. Morris S.A. Nerou E.P. Correa V. Potter B.V.L. Taylor C.W. J. Biol. Chem. 2002; 277: 40290-40295Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 17Kaznacheyeva E. Lupu V.D. Bezprozvanny I. J. Gen. Physiol. 1998; 111: 847-856Crossref PubMed Scopus (62) Google Scholar, 18Kaftan E.J. Ehrlich B.E. Watras J. J. Gen. Physiol. 1997; 110: 529-538Crossref PubMed Scopus (134) Google Scholar). These data suggest that the channels are more sensitive to low levels of IP3 when Tyr353 is phosphorylated. According to the known structure of the IP3-binding domain, Tyr353 is located in one of the regions required to form the IP3-binding site, although it is not one of the residues that directly interacts with IP3 (2Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 328-334Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 14Bosanac I. Alattia J.-R. Mal T.K. Chan J. Talarico S. Tong F.K. Tong K.I. Yoshikawa F. Furuichi T. Iwai M. Michikawa T. Mikoshiba K. Ikura M. Nature. 2002; 420: 696-700Crossref PubMed Scopus (274) Google Scholar, 19Yoshikawa F. Morita M. Monkawa T. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1996; 271: 18277-18284Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Phosphorylation at Tyr353 could influence the conformation of the IP3-binding region in an allosteric manner. Another possibility is that phosphorylation of Tyr353 may regulate channel activity by modification of the coupling between IP3 and Ca2+ binding to the channel, as Tyr353 is also situated in close proximity to two known sites of Ca2+ binding to the IP3R (14Bosanac I. Alattia J.-R. Mal T.K. Chan J. Talarico S. Tong F.K. Tong K.I. Yoshikawa F. Furuichi T. Iwai M. Michikawa T. Mikoshiba K. Ikura M. Nature. 2002; 420: 696-700Crossref PubMed Scopus (274) Google Scholar). Further experiments are required to test these hypotheses.Fig. 4Phosphorylation at Tyr353 of IP3R1 increases the affinity of IP3 binding.A, Western blot of the phosphotyrosine level and protein level of IP3R1 used in the binding assay. B, specific [3H]IP3 (9.1 nm) binding to immunoprecipitated wild-type IP3R1 or IP3R1-Y353F. The asterisk denotes p < 0.05 in comparison with the wild type coexpressed with Fyn(+)). C, competition of [3H]IP3 binding to IP3R1 by varying amounts of unlabeled IP3. Values are normalized to 100% of maximum specific binding. Results are the means ± S.E. from 5–7 independent experiments. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of Tyr353 Phosphorylation on BCR-induced Calcium Release from Intracellular Calcium Stores—To address down-stream signaling consequences of Tyr353 phosphorylation, we examined cytosolic Ca2+ transients in DT40 cells in response to BCR activation. We generated DT40 B cell lines that express only wild-type IP3R1 or IP3R1-Y353F using the triple IP3R-deficient DT40 cell line, which lacks all three types of IP3R (20Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (374) Google Scholar). Expression of recombinant IP3R1 at levels comparable with that of IP3R1 in wild-type DT40 cells was shown by immunoblotting (Fig. 5A). Fura-2-loaded cells were stimulated with anti-chicken IgM, M-4, and the increase in intracellular Ca2+ concentration was measured using an extracellular solution of 1 mm EGTA in Hanks' balanced salt solution as a Ca2+-free buffer. As shown in Fig. 5B, the initial rising phase of the Ca2+ transient was similar between wild-type and IP3R1-Y353F cells. However, the decay of the Ca2+ transient in wild-type IP3R1 cells was significantly slower than that in IP3R1-Y353F cells. To compare the Ca2+ release of wild-type IP3R1 cells and IP3R1-Y353F cells, the fluorescent ratio data were fitted to a curve and normalized. The inset of Fig. 5B shows that the increased duration of the Ca2+ transient in wild-type IP3R1 cells is caused by a prolonged decay phase. The first derivative of the Ca2+ transient is shown in Fig. 5C. In external Ca2+ conditions that preclude store-operated Ca2+ entry, the first derivative reflects the kinetics of IP3R1 channel activity and endoplasmic reticulum Ca2+ release. The maximum rate of release is determined primarily by the kinetics of channel activation, whereas the maximum rate of decay reflects the kinetics of channel inactivation. The first derivative reveals that the main difference between wild-type and IP3R1-Y353F mutant channels is enhanced inactivation of the channel in the tyrosine phosphorylation-deficient mutant (time constant = 0.135 ± 0.011 for wild-type IP3R1, 0.266 ± 0.020 for IP3R1-Y353F, p < 0.01, n = 4). This suggests that one role of tyrosine phosphorylation of IP3R1 at Tyr353 is to decrease inactivation of the channel, particularly as IP3 levels are falling following the initial activation of the channel. This finding is consistent with our results showing that tyrosine phosphorylation of IP3R1-Y353 enhances IP3 binding when [IP3] < 10 nm. Indeed, the inset of Fig. 5C shows that the maximum rate of decay of IP3R1-Y353F is significantly faster than that of wild-type IP3R1.Fig. 5Effects of the Y353F mutation of IP3R1 on intracellular Ca2+ release by BCR-induced B cell activation.A, expression level of IP3R1 in wild-type DT40 cells; triple IP3R-deficient DT40 cells and triple IP3R-deficient DT40 cells stably transfected with wild-type IP3R1 or IP3R1-Y353F. B, intracellular Ca2+ response in Fura-2-loaded DT40 cells in Ca2+-free Hanks' balanced salt solution monitored by spectrofluorometry after stimulation with M-4 anti-chicken IgM monoclonal antibody (5 μg/ml). Inset shows the fitting curves for the Ca2+ transient. C, first derivative of Ca2+ transient in A. Inset shows the normalized maximum decay rate. The asterisk denotes p < 0.05 in comparison with the wild-type IP3R1, n = 4.View Large Image Figure ViewerDownload Hi-res image" @default.
• W2006464961 created "2016-06-24" @default.
• W2006464961 creator A5001611184 @default.
• W2006464961 creator A5008339680 @default.
• W2006464961 creator A5025609995 @default.
• W2006464961 creator A5033764248 @default.
• W2006464961 creator A5062239858 @default.
• W2006464961 creator A5072691544 @default.
• W2006464961 date "2004-04-01" @default.
• W2006464961 modified "2023-10-14" @default.
• W2006464961 title "Regulation of the Type 1 Inositol 1,4,5-Trisphosphate Receptor by Phosphorylation at Tyrosine 353" @default.
• W2006464961 cites W1486474082 @default.
• W2006464961 cites W1800193450 @default.
• W2006464961 cites W1974125844 @default.
• W2006464961 cites W1980166477 @default.
• W2006464961 cites W1994580024 @default.
• W2006464961 cites W1996162251 @default.
• W2006464961 cites W1996602476 @default.
• W2006464961 cites W1997901402 @default.
• W2006464961 cites W2000334213 @default.
• W2006464961 cites W2001907971 @default.
• W2006464961 cites W2015084021 @default.
• W2006464961 cites W2020828897 @default.
• W2006464961 cites W2025081899 @default.
• W2006464961 cites W2026907934 @default.
• W2006464961 cites W2035920388 @default.
• W2006464961 cites W2041873532 @default.
• W2006464961 cites W2055307687 @default.
• W2006464961 cites W2061221873 @default.
• W2006464961 cites W2073545060 @default.
• W2006464961 cites W2134613573 @default.
• W2006464961 cites W2143843444 @default.
• W2006464961 cites W2163248783 @default.
• W2006464961 cites W2165742826 @default.
• W2006464961 doi "https://doi.org/10.1074/jbc.m400206200" @default.
• W2006464961 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14761954" @default.
• W2006464961 hasPublicationYear "2004" @default.
• W2006464961 type Work @default.
• W2006464961 sameAs 2006464961 @default.
• W2006464961 citedByCount "66" @default.
• W2006464961 countsByYear W20064649612012 @default.
• W2006464961 countsByYear W20064649612013 @default.
• W2006464961 countsByYear W20064649612014 @default.
• W2006464961 countsByYear W20064649612015 @default.
• W2006464961 countsByYear W20064649612016 @default.
• W2006464961 countsByYear W20064649612017 @default.
• W2006464961 countsByYear W20064649612018 @default.
• W2006464961 countsByYear W20064649612019 @default.
• W2006464961 countsByYear W20064649612020 @default.
• W2006464961 countsByYear W20064649612022 @default.
• W2006464961 countsByYear W20064649612023 @default.
• W2006464961 crossrefType "journal-article" @default.
• W2006464961 hasAuthorship W2006464961A5001611184 @default.
• W2006464961 hasAuthorship W2006464961A5008339680 @default.
• W2006464961 hasAuthorship W2006464961A5025609995 @default.
• W2006464961 hasAuthorship W2006464961A5033764248 @default.
• W2006464961 hasAuthorship W2006464961A5062239858 @default.
• W2006464961 hasAuthorship W2006464961A5072691544 @default.
• W2006464961 hasBestOaLocation W20064649611 @default.
• W2006464961 hasConcept C11960822 @default.
• W2006464961 hasConcept C152624196 @default.
• W2006464961 hasConcept C170493617 @default.
• W2006464961 hasConcept C185592680 @default.
• W2006464961 hasConcept C2776165026 @default.
• W2006464961 hasConcept C2777427919 @default.
• W2006464961 hasConcept C2777553839 @default.
• W2006464961 hasConcept C55493867 @default.
• W2006464961 hasConcept C86803240 @default.
• W2006464961 hasConcept C95444343 @default.
• W2006464961 hasConceptScore W2006464961C11960822 @default.
• W2006464961 hasConceptScore W2006464961C152624196 @default.
• W2006464961 hasConceptScore W2006464961C170493617 @default.
• W2006464961 hasConceptScore W2006464961C185592680 @default.
• W2006464961 hasConceptScore W2006464961C2776165026 @default.
• W2006464961 hasConceptScore W2006464961C2777427919 @default.
• W2006464961 hasConceptScore W2006464961C2777553839 @default.
• W2006464961 hasConceptScore W2006464961C55493867 @default.
• W2006464961 hasConceptScore W2006464961C86803240 @default.
• W2006464961 hasConceptScore W2006464961C95444343 @default.
• W2006464961 hasIssue "16" @default.
• W2006464961 hasLocation W20064649611 @default.
• W2006464961 hasOpenAccess W2006464961 @default.
• W2006464961 hasPrimaryLocation W20064649611 @default.
• W2006464961 hasRelatedWork W1966018982 @default.
• W2006464961 hasRelatedWork W1987307749 @default.
• W2006464961 hasRelatedWork W2016352783 @default.
• W2006464961 hasRelatedWork W2022006194 @default.
• W2006464961 hasRelatedWork W2030033027 @default.
• W2006464961 hasRelatedWork W2033441570 @default.
• W2006464961 hasRelatedWork W2085026676 @default.
• W2006464961 hasRelatedWork W2088802228 @default.
• W2006464961 hasRelatedWork W2102847281 @default.
• W2006464961 hasRelatedWork W4252597221 @default.
• W2006464961 hasVolume "279" @default.
• W2006464961 isParatext "false" @default.
• W2006464961 isRetracted "false" @default.
• W2006464961 magId "2006464961" @default.
• W2006464961 workType "article" @default.