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Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 44, Issue of October 29, pp. 46242–46252, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor* Received for publication, May 26, 2004, and in revised form, August 5, 2004 Published, JBC Papers in Press, August 11, 2004, DOI 10.1074/jbc.M405849200 Larry E. Wagner II‡, Wen-Hong Li§ , Suresh K. Joseph , and David I. Yule‡** From the ‡Department of Pharmacology and Physiology, University of Rochester, Rochester, New York 14642, the §Departments of Cell Biology and Biochemistry, University of Texas, Southwestern Medical Center, Dallas, Texas 75390-9039, and the Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Inositol 1,4,5-trisphosphate receptors are intracellular ion Regulation of Ca release through inositol 1,4,5- trisphosphate receptors (InsP R) has important conse- channels that function to couple the activation of cell surface quences for defining the particular spatio-temporal receptors for neurotransmitters, hormones, and growth fac- properties of intracellular Ca signals. In this study, tors to the initiation of intracellular Ca release (1). Three regulation of Ca release by phosphorylation of type 1 genes have been cloned that encode distinct proteins of a InsP R (InsP R-1) was investigated by constructing 3 3 molecular mass of 300 kDa, named the type 1 (InsP R-1), “phosphomimetic” charge mutations in the functionally type 2 (InsP R-2), and type 3 (InsP R-3) InsP Rs (2–4). In 3 3 3 important phosphorylation sites of both the S2 and addition, multiple receptor proteins with distinct tissue dis- S2 InsP R-1 splice variants. Ca release was investi- tributions are produced by alternate splicing of the type 1 gated following expression in Dt-40 3ko cells devoid of receptor gene (5, 6). Most cells express multiple isoforms of endogenous InsP R. In cells expressing either the InsP R (7). Furthermore, the expression level and comple- S1755E S2 or S1589E/S1755E S2 InsP R-1, InsP -in- 3 3 ment of receptors differ in individual tissues, and this to- duced Ca release was markedly enhanced compared gether with regulation of the activity of the channel is with nonphosphorylatable S2 S1755A and S2 S1589A/ 2 thought to be a major determinant of the rich diversity of S1755A mutants. Ca release through the S2 S1589E/ Ca signaling events observed in cells (7, 8). S1755E InsP R-1 was enhanced 8-fold over wild type The functional channel is formed co-translationally by the and 50-fold when compared with the nonphosphorylat- tetrameric association of four individual receptor subunits (9, able S2 S1589A/S1755A mutant. In cells expressing S2 10). Each subunit has a binding site for InsP toward the N InsP R-1 with single mutations in either S1589E or 3 terminus formed by a cluster of positively charged amino acids S1755E, the sensitivity of Ca release was enhanced thought to coordinate the negatively charged phosphate groups 3-fold; sensitivity was midway between the wild type of InsP (11, 12). The C terminus of each subunit is postulated and the double glutamate mutation. Paradoxically, for- skolin treatment of cells expressing either single Ser/ to span intracellular membranes six times and forms a single Glu mutation failed to further enhance Ca release. cation-selective pore (13, 14). In addition, this region signals The sensitivity of Ca release in cells expressing S2 retention of the protein to the endoplasmic reticulum (15, 16). S1755E InsP R-1 was comparable with the sensitivity of Although the InsP -binding pocket and channel pore are highly S2 S1589E/S1755E InsP R-1. In contrast, mutation of 3 conserved between InsP R family members, the intervening S2 S1589E InsP R-1 resulted in a receptor with compa- 3 sequence between the binding region and pore is more diver- rable sensitivity to wild type cells. Expression of S2 gent and consists of the so-called “regulatory and coupling” or S1589E/S1755E InsP R-1 resulted in robust Ca oscilla- “modulatory” domain. This region, consisting of 1600 amino tions when cells were stimulated with concentrations of 2 acids, is thought to be important in modulating the Ca re- -IgM antibody that were threshold for stimulation in lease properties of the InsP R. Indeed, Ca release through S2 wild type InsP R-1-expressing cells. However, at the InsP R is markedly influenced by many factors, most im- higher concentrations of -IgM antibody, Ca oscilla- portantly by Ca itself (17). InsP R activity is also influenced tions of a similar period and magnitude were initiated in through interaction with numerous factors such as proteins, cells expressing either wild type or S2 phosphomimetic adenine nucleotides, and phosphorylation and in particular by mutations. Thus, regulation by phosphorylation of the cyclic nucleotide-dependent kinases (8). functional sensitivity of InsP R-1 appears to define the Two protein kinase A (PKA) consensus sites (RRXS) at Ser- threshold at which oscillations are initiated but not the 1589 and Ser-1755 are present in the InsP R-1 (5, 18), and the frequency or amplitude of the signal when established. 3 most recent studies suggest that phosphorylation of these sites results in a marked enhancement of Ca release (19–22). * This work was supported in part by National Institutes of Health Most interestingly, these sites are conserved through evolution Grants RO1-DK54568, R01-DE14756, PO1 DE13539 (to D. I. Y.), and RO1-DK34804 (to S. K. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 The abbreviations used are: InsP R-1, inositol 1,4,5-trisphosphate U.S.C. Section 1734 solely to indicate this fact. receptor type 1; [Ca ] , intracellular calcium concentration; InsP , i 3 Supported by Research Grant I-1510 from the Robert A. Welch inositol 1,4,5-trisphosphate; CCh, carbamylcholine (carbachol); PKA, Foundation and a Career Development award from the American Dia- cAMP-dependent protein kinase; PKG, cGMP dependent protein ki- betes Association. nase; 8-Br cGMP, 8-bromoguanosine-3,5- cyclic monophosphate; ** To whom correspondence should be addressed. Tel.: 585-273-2154; ci-IP /PM, caged isopropylidene inositol trisphosphate; WT, wild type; Fax: 585-670-0394; E-mail: [email protected]. cm-IP , caged methoxymethylene inositol trisphosphate. 46242 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. / Phosphoregulation of S2 InsP R-1 46243 used for the mutagenesis reaction flanked the restriction sites RsrII from Drosophila to humans in InsP R-1, but corresponding and KasI. Following mutation, the resulting fragments were cut with sites are not present in either the InsP R-2 or InsP R-3. It 3 3 RsrII and KasI and inserted into the InsP R-1 backbone at the corre- should be noted, however, that other regions, which are pres- sponding sites. The mutations were confirmed by Big Dye fluorescent ently not defined, appear to function as PKA phosphorylation sequencing. Mutated receptor DNAs were excised from MXT-1 by using sites in InsP R-2 and InsP R-3. Several reports have demon- 3 3 EcoRI and ligated into the mammalian expression vector pGW (pro- strated biochemically that both Ser-1589 and Ser-1755 can be vided by Dr. David Yue, The Johns Hopkins University). Orientation was confirmed by using restriction enzyme digestion. Mutants were phosphorylated in InsP R-1 (18, 23, 24); however, these studies named based on the splice variant, either S2 or S2 followed by the have not provided a consensus as to which sites are important amino acid present at position 1589 and 1755. Thus, a mutation in S2 physiologically in tissue that expresses either of the two major InsP R-1 S1755E is designated,“S2 SE,” and in InsP R-1 S2 S1755A 3 3 splice variants of InsP R-1. is designated “S2 SA”. Similarly, an S2 InsP R-1 with mutations in A recent study (19) from our laboratory has elucidated the both S1589E and S1755E is designated “S2 EE”. The numbering of particular sites that are functionally important for the phos- residues is based on the full-length rat InsP R-1. Transfection of Dt-40 Cells—Dt-40 cells lacking all three InsP re- phoregulation of the two major splice variants of the InsP R-1. 3 ceptor subtypes were transfected by using electroporation at 350 V and These studies were performed by using mutational analysis by 950 microfarads (4-mm gap cuvette). 2  10 cells were co-transfected substituting alanine for the serine residues present at individ- with 25 g of the InsP R-1 cDNA, 25 g of the muscarinic type 3 (m3R) ual putative phosphorylation sites. Although phosphorylation receptor DNA, and 4 g of the red fluorescent protein plasmid pH- of both neuronal (S2) and peripheral (S2) forms of InsP R-1 3 cRed1-N1 (Clontech). Cells were incubated with DNA in 500 lof by PKA resulted in enhanced Ca release, mutational analy- Opti-MEM media (Invitrogen) on ice for 10 min. The cell/DNA mixture was electroporated, incubated on ice for 30 min, increased to 5 ml with sis indicated that only phosphorylation of Ser-1755 was func- Opti-MEM, and placed in a 5% CO incubator at 39 °C for 5 h. The cells tionally important in the neuronal S2 InsP R-1. In contrast, were then centrifuged and resuspended in 12 ml of complete RPMI both Ser-1589 and Ser-1755 appeared to be phosphorylated and media (Invitrogen). Transfection efficiency was typically 20%. Exper- significant in the peripheral S2 form of InsP R-1. In addition, iments were performed within 32 h of transfection. although the S2 form of the receptor was subject to direct Transfection of HEK-293 Cells and Assessment of Phosphorylation of phosphoregulation by cGMP-dependent protein kinase (PKG), S2 InsP R-1—HEK-293 cells were plated onto 25-cm culture flasks and allowed to grow to near-confluency. Cells were transfected with 5 the S2 form was not influenced by activation of PKG. These g of each S2 InsP R-1 DNA construct by using the LipofectAMINE data represent one of the few major differences reported for the reagent (Invitrogen) as per the manufacturer’s instructions. The follow- regulation of the two major splice variants of the InsP R-1. ing day, batches of cells were treated in the presence or absence of 20 M In the present study, we have constructed charge mutations, forskolin for 10 min, aspirated from flasks, lysed, and immunoprecipi- substituting glutamate residues for the serine residues in the tated with a polyclonal -InsP R-1 antibody that recognizes amino functionally important phosphorylation sites in both S2 and acids 2731–2749 of InsP R-1. Immunoprecipitates were separated on 5% SDS gels transferred to nitrocellulose and then probed with either S2 variants of the InsP R-1. These mutations are predicted to the -InsP R-1 antibody or a polyclonal antibody that recognizes the mimic phosphorylation and to allow the assessment of the phosphorylated state of Ser-1755 (28) (-phospho-Ser-1755), kindly pro- functional effects of phosphorylation of InsP R-1. Most impor- vided by Dr. S. Snyder. Blots that were probed with -phospho-Ser- tantly, the Ca release properties of phosphomimetic muta- 1755 were stripped and reprobed with the -InsP R-1 antisera to con- tions are predicted to be essentially independent of cell type- firm the presence and relative quantity of the InsP R-1. specific factors, including the expression of accessory proteins Digital Imaging of [Ca ] —Transfected Dt-40 3ko cells were washed once in a HEPES-buffered physiological saline solution (HEPES-PSS) such as protein A-kinase anchoring proteins. These effects containing (in mM) 5.5 glucose, 137 NaCl, 0.56 MgCl , 4.7 KCl, 1 should also be unambiguously specific to InsP R and thus Na HPO , 10 HEPES (pH 7.4), 1.2 CaCl , and 1% w/v bovine serum 2 2 4 2 independent of confounding PKA effects on other Ca -han- albumin. Cells were then resuspended in bovine serum albumin dling machinery. This latter consideration has historically HEPES-PSS with 1 M Fura-2 (AM), placed on a 15-mm glass coverslip plagued the functional assessment of PKA phosphorylation of in a low volume perfusion chamber, and allowed to adhere for 30 min at InsP R. These mutations have allowed us to define the relative room temperature. Cells were perfused continuously for 10 min with HEPES-PSS before experimentation to allow complete Fura-2 de-ester- sensitivity of Ca release of the phosphomimetic mutations ification. A field of cells for each experiment was chosen that contained and to confirm which sites are important in each splice variant. a wide range of transfection efficiency based upon the intensity of red The present study has also addressed whether phosphorylation fluorescence emitted when excited at 560 nm. Individual cells that had of individual sites is permissive or additive in each splice var- emission gray levels between 1500 and 2500 were subsequently chosen iant, and we have investigated the consequences of phospho- to standardize expression levels. [Ca ] imaging was performed essen- rylating InsP R-1 on Ca oscillations, the physiological pat- tially as described previously by using an inverted epifluorescence Nikon microscope with a 40 oil immersion objective lens (numerical tern of Ca signaling in nonelectrically excitable cells. aperture, 1.3) (19). Cells were excited alternately with light at 340 and MATERIALS AND METHODS 380  10 nm bandpass filters (Chroma, Rockingham, VT) using a The acetoxymethyl esters of Fura-2 and Fluo-4 were purchased from monochrometer (TILL Photonics, Pleasanton, CA). Fluorescence im- Molecular Probes (Eugene, OR). Cell-permeable cyclic nucleotides and ages were captured and digitized with a digital camera driven by TILL forskolin were purchased from Biomol (Plymouth Meeting, PA). All Photonics software. Images were captured every 2 s with an exposure of other chemicals were purchased from Sigma. The Dt-40 cells lacking 2 ms and 4 by 4 binning. 340/380 ratio images were calculated online InsP R (Dt-40 3ko) were kindly provided by Dr. Kurosaki (Kansai and stored immediately to a hard disk. Medical University, Japan) and were maintained as described Flash Photolysis—Transfected cells were simultaneously loaded with previously (25–27). the visible wavelength indicator Fluo-4 and a cell-permeable form of Production of Mutations—The rat S2 InsP R-1 in the expression caged inositol trisphosphate (ci-IP /PM) for 30 min. ci-IP /PM is a 3 3 plasmid pIRES-GFP was digested with the restriction endonuclease homologue of cm-IP /PM. The 2- and 3-hydroxyls of the inositol ring are SalI. The overhang created by digestion was blunted by using T4 protected by an isopropylidene group in ci-IP /PM and are protected by polymerase. An EcoRI linker was then ligated onto the blunted ends of a methoxymethylene group in cm-IP /PM (29). Like cm-IP /PM, ci- 3 3 the construct. The entire receptor DNA was excised from the plasmid IP /PM diffuses across cell membranes, and the PM group is hydrolyzed using EcoRI and ligated into the plasmid MXT-1. The region containing by cellular esterases, and Ca release can be induced upon photo- the S2 splice variant and potential PKA phosphorylation sites was uncaging as i-IP is liberated from the cage and acts in a similar fashion excised from its backbone in pCDNA 3.1 by RsrII and KasI and ligated to InsP at InsP R. A further period of 30 min was allowed for 3 3 into the InsP R construct in MXT-1. The potential PKA phosphoryla- de-esterification of both dye and cage. Cells were illuminated at 488 tion sites Ser-1589 and Ser-1755 were mutated, individually in both splice variants and together in the S2 splice variant, to alanines or glutamates by using sequential PCR mutagenesis. The outside primers W.-H. Li, unpublished data. / 46244 Phosphoregulation of S2 InsP R-1 10 nm and fluorescence collected through a 525  25-nm bandpass filter charge (0.5–5 ms). No increase in [Ca ] was observed follow- and captured using the Till Photonics imaging suite. These traces are ing the longest UV discharge in cells either not loaded with displayed as % F/F , where F is the recorded fluorescence, and F is the o o ci-IP /PM or not expressing HcRed. Fig. 1, B and C, shows mean of the initial 10 sequential frames. Photolytic release was per- traces from typical experiments in individual Dt-40 3ko cells formed as described previously by using a pulsed xenon arc lamp (Till expressing S2 EE or S2 AA (Fig. 1, B and C, respectively, Photonics). A high intensity (0.5–5 ms duration; 80 J) discharge of UV light (360  7.5 nm) was reflected onto the plane of focus by using a and pooled data in Fig. 1F). In cells expressing S2 EE, Ca DM400 dichroic mirror and Nikon 40 oil immersion objective, 1.3 NA. release as defined by a 0.05% F/F increase in initial fluo-4 Frequency Distribution of Ca Oscillations—Cells transfected with fluorescence was observed in 60% of cells when exposed to UV WT and mutant S2 isoforms were stimulated with varying concentra- discharge for 0.5 ms. Subsequent exposure to UV light for 1.25 tions of an -IgM antibody (Southern Biotechnology Associates, Inc., ms elicited a more robust increase in [Ca ] in all cells. Fi- Birmingham, AL). Infrequent Ca oscillations were produced presum- i ably through B cell receptor cross-linking, activation of phospholipase nally, photo-release following a 5-ms flash, in general evoked a C-, and subsequent production of InsP . The frequency of Ca oscil- 3 further increase in the magnitude of Ca release. In contrast, lations was determined by selecting individual peaks that displayed an an elevation of intracellular Ca was never observed under increase in ratio units greater than 0.05 and are listed as frequency in identical conditions following a 0.5-ms UV flash in cells ex- milliHertz (number of oscillations in 1000 s). pressing S2 AA. A significant Ca release was only observed Concentration-Response Relationships—Normalized F concentra- in 50% of cells following a 1.25-ms flash, and the majority of tion-response relationships were fit with the following logistic Equation 1, cells only responded to the longest uncaging duration, albeit with a smaller magnitude than in S2 EE-expressing cells Slope F  1/1  C/EC (Eq. 1) exposed to the same stimulus. A similar pattern of sensitivity was observed in Dt-40 3ko where F is the change in fluorescence normalized to the maximal response; C is agent concentration; EC is the concentration where the cells expressing S2 S1755E (S2 SE) and S2 S1755A (S2 response is half of maximum, and Slope is a slope factor related to the SA) as shown in Fig. 1, D and E, respectively, and pooled data Hill coefficient. in Fig. 1G. Cells expressing S2 SE responded more robustly to Statistical Analysis—The effects of treatment were determined by photolysis of ci-IP than cells expressing S2 SA exposed to an normalizing the peak change in fluorescence ratio by stimulation fol- identical stimulus. These data indicate that serine to gluta- lowing forskolin or 8-Br-cGMP exposure to that of stimulation in control mate mutations at the functionally important phosphorylation HEPES-PSS. Thus, pooled data represent a normalized fold increase over control for the treated trial. In all cases where statistical signifi- sites in both splice variants of InsP R-1 are “phosphomimetic,” cance is indicated, two-tailed heteroscedastic t tests were performed. p i.e. charge mutations mimic phosphorylation in that these con- values 0.05 were considered to indicate statistical significance and structs display an apparent increased functional sensitivity are denoted by an asterisk in the figures. to InsP . Sensitivity of S2 InsP R-1 and Phosphorylation Site Mu- RESULTS AND DISCUSSION 3 tants—Experiments were next performed to determine the rel- Phosphomimetic Mutations in Functionally Important Phos- ative sensitivity of Ca release through the phosphomimetic phorylation Sites in Both S2 and S2 InsP R-1 Result in mutations of the S2 InsP R-1 with respect to the wild type 2 3 Enhanced Ca Release—Phosphorylation of proteins results and phosphorylation-deficient mutants. Dt-40 3ko cells were in the addition of net negative charge to the phosphoacceptor transfected with cDNAs encoding m3R, HcRed together with residue. In the case of PKA phosphoregulation, the functional the InsP R-1 construct of interest. Stimulation with the mus- effects of phosphorylation are thought to occur as the negative carinic agonist CCh results in robust increases in [Ca ] in charge added to the serine or (less frequently) threonine resi- transfected cells through the G -coupled stimulation of q/11 dues neutralizes the positive charge of basic arginine or lysine phospholipase C- and subsequent formation of InsP . The residues present upstream in the classical RRX(S/T) consensus magnitude of the initial peak provides a good estimation of the motif (30). This charge neutralization, in turn, is thought to extent of Ca release as this parameter in Dt-40 cells, like result in a conformational change in the protein. A common many cells, is essentially independent of Ca influx. Further- approach employed to investigate the functional effects of phos- more, in Dt-40 cells the [Ca ] response to stimulation with phorylation is to construct phosphomimetic mutations whereby CCh does not appreciably desensitize, and thus the effects of glutamic or aspartic acid residues are substituted at the phos- multiple concentrations of agonist can be assessed in a single phoacceptor site (31, 32). The rationale for this strategy is that cell. Individual HcRed-expressing cells were stimulated with the negatively charged side chain of the substituted acidic increasing concentrations of CCh (1 nM to1 M) for 60 s followed amino acid will mimic, to an extent, the addition of a phosphate by a 5-min wash between applications of agonist. In each case, moiety to the protein. these experiments were performed on multiple cells, express- To investigate the consequences of InsP R-1 phosphoryla- ing a narrow range of HcRed fluorescence and from multiple tion, we analyzed in Dt-40 3ko cells the Ca release properties batches of transfected cells to minimize variation because of of phosphomimetic mutations in the functionally important expression level of m3R and InsP R-1. Concentration-response sites in both S2 and S2 InsP R-1. This cell line provides the 3 relationships were generated by normalizing each initial peak only known InsP R null background (26, 27). In initial exper- to the maximum response in the individual cell and subse- iments, a comparison was made between the sensitivity of Ca quently averaging the pool of cells expressing a particular release by InsP R-1 phosphomimetic mutations versus non- construct. Fig. 2A shows a typical example of this experimental phosphorylatable alanine mutations at the sites we reported procedure performed on Dt-40 3ko cells expressing S2 EE. A previously (19) to be relevant. Dt-40 3ko cells were transfected significant increase in Ca was detected in the majority of with DNA encoding HcRed to facilitate identification of trans- S2 EE-expressing cells following stimulation with 1 nM CCh, fected cells and either S2 S1589E/S1755E InsP R-1 (S2 EE) and the peak response occurred following stimulation between or S2 S1589A/S1755A (S2 AA). Ca release was monitored following flash photolysis of ci-IP , a cell-permeable form of 10 and 50 nM CCh. In contrast, as shown in Fig. 2, B and C, Ca release following CCh stimulation in the wild type S2 caged InsP . This experimental paradigm provides a relatively direct assessment of the effects of InsP R-1 phosphorylation on InsP R-1 or S2 AA was considerably less sensitive to CCh 3 3 stimulation. Analysis of the pooled data indicated that the the Ca release process. The amount of ci-IP photo-released was controlled by varying the duration of the UV flash dis- magnitude of the peak response to any of these constructs was / Phosphoregulation of S2 InsP R-1 46245 FIG.1. Flash photolysis of caged InsP reveals that phosphomimetic InsP R-1 exhibits enhanced sensitivity to InsP . A represents 3 3 3 a schematic of amino acids 1586–1755 of the S2 InsP R-1 showing the S2 splice site and potential PKA/PKG phosphorylation sites. Dt-40 3ko cells were transfected with either phosphomimetic mutations of InsP R-1 (B and D) or nonphosphorylatable mutations (C and E). Experiments in B and C are representative traces from cells transfected with the S2 InsP R-1 and in D and E with S2 InsP R-1. The cells were loaded with 3 3 the visible wavelength Ca indicator fluo-4 and the cell-permeable caged InsP R analogue ci-IP -PM as described under “Experimental 3 3 Procedures.” Increasing iIP concentrations were generated by varying the duration (energy magnitude) of UV flash exposure from 0.5 to 5 mss. B, cells transfected with S2 EE constructs respond at a lower threshold of UV light exposure and with more robust elevations in [Ca ] than cells transfected with S2 AA mutations that are nonphosphorylatable (C). Similarly, as shown in D,S2 SE mutations are more sensitive than S2 SA mutations. The pooled data, expressed as fold increase in the F/F fluorescence, are shown in F (S2 constructs) and G (S2 constructs). The magnitude of responses to 1.25- and 2.5-ms exposure to UV light was significantly greater in phosphomimetic mutations in each splice variant, p 0.005. The values in parentheses indicate the number of cells responding above a 0.05% F/F threshold in relation to the number of cells tested. not significantly different (maximum peak  response: S2 mutant (EC 4.3  1.2 nM CCh) was 7.5-fold more sensitive WT 0.9  0.1 ratio units; S2 AA 0.67 0.1 ratio units, when compared with WT S2 InsP R-1 (EC 32.6  7.5 nM) 3 50 and S2 EE 1.13  0.1 ratio units), suggesting that the and some 50-fold more sensitive than the nonphosphorylatable efficacy of Ca release was essentially unaltered. However, S2 AA mutant (EC 229.5  14.6 nM). These data provide when an estimate of the relative sensitivity was made by fitting strong evidence that the S2 EE construct is more sensitive to the normalized concentration-response relationships for each stimulation by InsP and present evidence that phosphoryla- construct (Fig. 2D), CCh-induced Ca release in the S2 EE tion of the InsP R-1 results in marked regulation of channel 3 / 46246 Phosphoregulation of S2 InsP R-1 intermediate sensitivity of the wild type S2 InsP R-1 relative to the phosphomimetic S2 InsP R-1 receptor. Although we have demonstrated that each site can be phos- phorylated, it is not definitively known whether the functional effects of phosphorylating individual sites are independent and additive or alternatively if the full effect is seen following phosphorylation of an individual site. Thus, experiments were next performed to assess the sensitivity of single phosphomi- metic mutations within each phosphorylation site. Concentra- tion-response relationships for CCh-induced Ca release were constructed for Dt-40 3ko cells expressing either S2 S1755E InsP R-1 (S2 SE) or S2 S1589E InsP R-1 (S2 ES). The 3 3 data were analyzed as described previously for Fig. 2D. Fig. 3A shows the fit for the normalized concentration-response rela- tionship for S2 SE and S2 ES and for comparison also shows the fit for S2 EE and S2 WT (Fig. 3A, dotted lines; data from Fig. 2D). Once again the maximal initial peak responses in either mutant were not significantly altered from wild type (S2 ES 0.54  0.1  ratio units; S2 SE 0.78  0.1 ratio units); however, the sensitivity of each of these mutants was significantly shifted, such that the EC for CCh-induced Ca release was enhanced 3-fold over the response in wild type for either mutant (EC for CCh-induced release: S2 ES 12.4  0.5 nM;S2 SE 13.5  1nM). These data are summarized in Table I. The enhanced apparent sensitivity of individual phosphomi- metic mutants was essentially equal and intermediate between the sensitivity of the S2 WT and S2 EE mutations. These data are consistent with phosphorylation of individual sites being functionally additive. To address this possibility directly, experiments were performed evaluating the effects of activat- ing endogenous PKA in cells expressing S2 SE or S2 ES to mimic prior phosphorylation of an individual site. As shown in Fig. 3B, and reported previously, activation of PKA by incuba- tion with forskolin results in a dramatic potentiation of Ca release in Dt-40 3ko cells expressing S2 WT (19). Most sur- prisingly, although the sensitivity to CCh was enhanced, as evidenced by the low concentration of CCh necessary to evoke 2 2 threshold Ca release, no potentiation of Ca release follow- ing forskolin incubation was observed in Dt-40 3ko cells ex- pressing either S2 ES (Fig. 3C; pooled data in Fig. 3F), S2 SE (Fig. 3D; pooled data in Fig. 3F), or S2 EE (Fig. 3E; pooled data in Fig. 3F). Thus, although each phosphorylation site in S2 InsP R-1 can be phosphorylated and mimicking phospho- rylation of both sites leads to a receptor with enhanced sensi- tivity relative to phosphorylation of an individual site, phos- phorylation of both sites in situ does not appear to occur and FIG.2. InsP sensitivity of S2 InsP R-1 and phosphomimetic 3 3 therefore is not functionally additive. Although these data may mutations. Concentration-response relationships for CCh-induced seem paradoxical, a possible explanation, consistent with all Ca signals were examined in Dt-40 3ko cells expressing m3 receptors the observations is that the initial phosphorylation of either and InsP R-1 constructs. A, cells transfected with S2 EE were stim- ulated with increasing concentrations of CCh as indicated (n 9 cells). Ser-1589 or Ser-1755 leads to a conformational change in the A similar paradigm was performed for cells expressing S2 WT (n 10) receptor that now precludes the phosphorylation of the addi- (B) and for cells expressing S2 AA constructs (n 9) (C). The mag- 2 tional site. To test this hypothesis, experiments were per- nitude of the initial peak (as an indicator of Ca release) for each formed to determine the phosphorylation state of the various response was normalized to the maximum response in each cell. The pooled data and the fit that describes each concentration-response mutants after raising cAMP levels. These experiments were relationship for each construct is shown in D and illustrates that the performed in HEK-293 cells because of the high transfection sensitivity of Ca release was greatest in S2 EE followed by S2 WT efficiency and low endogenous levels of InsP R-1 (18, 33). HEK- and S2 AA. 293 cells were transfected with the constructs as indicated in Fig. 4, incubated in the presence or absence of forskolin for 10 min, then pelletted, and lysed. Following incubation of the function. This profound regulation could be expected to have lysates with -InsP R-1 antibody, the immune complexes were major consequences for calcium signaling events in peripheral tissue such as liver, testis, and smooth muscle which express captured and separated on SDS gels, and the phosphorylation status of Ser-1755 was determined by Western blotting with an the S2 InsP R-1 (5, 6). The observation that S2 AA is relatively less sensitive than S2 WT is consistent with the antibody that specifically recognizes this phosphorylated resi- due in InsP R-1 (28). As shown in Fig. 4A, wild type S2 possibility that a proportion of the wild type receptor is consti- tutively phosphorylated in Dt-40 cells, thus contributing to the InsP R-1 was robustly phosphorylated after forskolin incuba- 3 / Phosphoregulation of S2 InsP R-1 46247 FIG.3. Functional sensitivity of single phosphomimetic mutations in S2 InsP R-1. A, concentration-response relationships were generated exactly as shown in Fig. 2D and as described under “Experimental Procedures.” Cells expressing either S2 SE (n 6) or S2 ES (n 5) displayed intermediate sensitivity between S2 WT and S2 EE constructs. Dotted lines indicate S2 WT and S2 EE for comparison (data from Fig. 2D). B, threshold CCh-stimulated Ca release is markedly potentiated by activation of PKA following forskolin treatment in cells expressing S2 WT. In contrast, no potentiation of threshold CCh-stimulated Ca release is observed in cells expressing S2 SE in D,S2ES in C,orS2 EE in E. Pooled data are shown in F; potentiation by forskolin is only seen in S2 WT. Numbers in parentheses indicate the number of analyzed cells. tion, whereas no phosphorylation was detected in wild type cells transfected with S2 EE, S2 SE or, most importantly, S2 AA or untransfected HEK-293 cells. Similarly, in Fig. 4B, the S2 ES construct. These data are strongly supportive of a marked increase in phosphorylation could be detected in S2 the contention that the initial phosphorylation of one site pre- InsP R-1, whereas no phosphorylation could be detected in cludes further phosphorylation at the additional residue as 3 / 46248 Phosphoregulation of S2 InsP R-1 TABLE I Summary of the effects of phosphorylation by PKA or PKG of the S2/S2 splice variants of InsP R-1 and phosphoregulatory mutations at Ser-1589 and Ser-1755 expressed in Dt40 3 knock out cells The data are derived from this paper and Ref. 19. NT, not tested. S2 InsP R-1 S2 InsP R-1 3 3 Amino acid at 1589/1755 CCh EC (M)/(fold 2 CCh EC (M)/(fold 2 50 50 Ca release after PKA/PKG activation Ca release after PKA/PKG activation change from WT) change from WT) Wild-type SS 32.1  4.2/(1) Enhanced/enhanced 32.6  7.5/(1) Enhanced/no change AS NT Enhanced/enhanced NT Enhanced/no change SA 116.3  3.0/(0.3) No change/no change NT Enhanced/no change AA NT NT/NT 229.5  14.6/(0.14) No change/no change SE 3.6  0.2/(8.9) No change/no change 13.5  1.1/(2.4) No change/NT ES 23.7  1.2/(1.3) Enhanced/enhanced 12.4  0.5/(2.6) No change/NT EE NT NT/NT 4.3  1.2/(7.6) No change/NT the InsP R-1 (34–36) and is therefore supportive of our conten- tion that the initial CCh-stimulated [Ca ] peak is a good indicator of InsP R function. In a similar fashion to the S2 InsP R-1, mutation of Ser-1755 to either alanine or glutamic acid did not significantly alter the maximal initial peak upon CCh stimulation (maximum peak  response: S2 WT 0.61  0.1  ratio units; S2 SE 0.59  0.1  ratio units; S2 SA 0.84  0.2  ratio units) but did, however, significantly affect the apparent sensitivity of Ca release as shown in Fig. 5A. The S2 SE mutant exhibited a similar EC for CCh- induced Ca release as the S2 EE mutation (EC :S2 SE 3.6  0.2 nM CCh; S2 EE 4.3 nM), being 9-fold more sensitive than S2 WT and 32-fold more sensitive than the nonphosphorylatable S2 SA mutation (EC S2 SA 116.3  3nM CCh). These data are summarized in Table I. In contrast to the S2 InsP R-1 S1755E construct, mutation of Ser-1589 to glutamate in the S2 form of InsP R-1 did not significantly affect the apparent sensitivity of Ca release as shown in Fig. 5B. Both the maximum peak response to CCh and the sensitivity of the receptor were very similar to that of S2 WT InsP R-1 (maximum peak  response 0.59  0.2 ratio units; EC 23.7  1.2 nM CCh). However, it is formally FIG.4. Phosphorylation of S2 InsP R-1 mutants. The phospho- possible that phosphorylation of Ser-1589 is only functionally rylation status of S2 InsP R-1 constructs was assessed as described important following phosphorylation of Ser-1755 in S2 under “Materials and Methods.” A, incubation with 20 M forskolin InsP R-1. To test this idea, experiments were performed acti- results in phosphorylation of Ser-1755 in S2 InsP R-1. No phospho- rylation is seen in S2 AA-transfected cells or untransfected HEK-293 vating PKA with forskolin in Dt-40 3ko cells expressing S2 cells (UN). B, forskolin treatment results in phosphorylation of Ser- ES or SE mutants. Treatment with forskolin resulted in a 1755 in S2 InsP R-1 but not S2 EE, S2ES, or S2 SE. The lack of marked potentiation of CCh-induced Ca release in cells ex- phosphorylation of Ser-1755 in the S2 ES mutant is interpreted as pressing either S2 WT or S2 ES (Fig. 6, A and B, respec- indicating that mimicking phosphorylation of Ser-1589 by phosphomi- metic substitution precludes the phosphorylation of Ser-1755 consistent tively, and pooled data in D) presumably as Ser-1755 was with the functional data shown in Fig. 3. IP, immunoprecipitation; WB, phosphorylated. Although cells expressing S2 SE were more Western blot. sensitive to CCh, no further potentiation was observed follow- ing forskolin incubation (Fig. 6C and pooled data in Fig. 6D). suggested by the functional data and indicate that physiologi- These data indicate that it is unlikely that phosphorylation of cally only phosphorylation of a single residue is functionally Ser-1755 is permissive for any functional effect of phosphoryl- relevant in the S2 splice variant of InsP R-1. ating Ser-1589 in S2 InsP R-1. 3 3 Sensitivity of S2 InsP R-1 and Phosphorylation Site Mu- Effect of PKG Activation on S2 InsP R-1 and Phosphomi- 3 3 tants—The sensitivity of Ca release via the neuronal S2 metic Mutants—We have reported previously that PKG activa- InsP R-1 was next assessed by using a similar experimental tion results in direct phosphoregulation of only the neuronal paradigm to that used for the S2 form of the receptor. Al- S2 form of InsP R-1 (19). In addition, PKG regulation of Ca though biochemically both Ser-1589 and Ser-1755 are equally release only occurs by phosphorylation of Ser-1755. In contrast susceptible to phosphorylation by PKA in S2 InsP R-1 (18), to these data, a recent report (18) has shown that Ser-1589 and only Ser-1755 appears to be functionally relevant in terms of not Ser-1755 is phosphorylated upon activation of PKG. It modulating Ca release (19). Fig. 5A shows the fits for the should be noted that these experiments were performed by normalized concentration-response relationships for CCh-in- using the mouse S1/S2 splice variant of InsP R-1, whereas duced Ca release in cells expressing S2 WT, phosphomi- our functional experiments utilized rat S1/S2 InsP R-1. The metic S2 SE, and S2 SA. The apparent sensitivity of CCh- species and splice variant differences aside, the reported dif- induced Ca release in cells expressing S2 WT was ferences are difficult to reconcile with data demonstrating that essentially identical to S2 WT InsP R-1 (EC for CCh-in- mimicking phosphorylation of Ser-1589 in S2 InsP R-1 does 3 50 3 2 2 duced Ca release; S2 InsP R-1 32.1  4.2 nM versus S2 not influence Ca release (Figs. 5 and 6). Despite these differ- 32.6  7.5 nM). These data are in agreement with the published ences, we performed experiments to define further the phos- literature that indicates that InsP binding and InsP -induced phorylation sites important for regulation of Ca release by 3 3 calcium release is identical in the two major splice variants of PKG. Fig. 7A shows a representative trace from cells express- / Phosphoregulation of S2 InsP R-1 46249 FIG.5. Functional sensitivity of S2 InsP R-1. Concentration-response relationships were generated exactly as described previously. A, the normalized relationships for S2 WT (n 11), S2 SE (n 6), and S2 SA (n 5) are shown. The fits illustrate the increased sensitivity of the S2 SE relative to S2 WT and S2 SA constructs. B, the nor- malized concentration-response relation- ship for S2 ES is shown with S2 SE and S2 WT (dotted lines for compari- son). These data indicate that the sensi- tivity of S2 ES is similar to S2 WT. ing S2 WT InsP R-1 thus illustrating the marked potentia- phosphomimetic constructs used in this study reveal the intrin- tion of Ca release upon specific activation of PKG by 8-Br- sic, functionally important sites in a manner independent of cGMP. A similar striking potentiation of Ca release was also the particular cellular context because any targeting step is observed in cells expressing S2 ES (Fig. 7B and pooled data circumvented. These sites and the functional consequences of Fig. 7D), again presumably as Ser-1755 is phosphorylated fol- phosphoregulation are thus likely a general property of the lowing incubation with 8-Br-cGMP. In contrast, in cells ex- InsP R-1. In addition, the particular sites in S2 InsP R-1 are 3 3 pressing S2 SE no effect on CCh-induced Ca release follow- entirely consistent with our earlier study (19) of the sites func- ing activation of PKG was observed (Fig. 7C and pooled data in tionally important in enhanced Ca release following PKA or Fig. 7D). These data provide evidence that it is unlikely that PKG phosphoregulation, and this reinforces the view that phosphorylation of Ser-1589 by PKG either in isolation or fol- phosphorylation of S2 InsP R-1 Ser-1589 has no significant lowing phosphorylation of Ser-1755 plays a role in modulation role, at least in terms of Ca release. 2 2 Ca release through S2 InsP R-1. However, we cannot rule Effect of InsP R Phosphorylation on Ca Signaling 3 3 out that phosphorylation of this residue impacts other pro- Events—In many nonelectrically excitable cell types, the phys- cesses, such as protein interactions or clustering important for iological mode of Ca signaling is through the generation of InsP R-1 function. Ca oscillations (1, 38). Moreover, it is a generally held view The specific pattern of phosphorylation occurring upon stim- that the spatial and temporal properties of Ca oscillations ulation of PKA or PKG could be cell type-specific as, for exam- make an important contribution to defining the fidelity and ple, in the case of PKA as a result of the targeting through specificity of Ca signaling. In many current models address- protein A-kinase anchoring proteins, as has been demonstrated ing the mechanism underlying Ca oscillations, a key feature recently (37) for InsP R. Alternatively, cell-specific effects is the regulation of Ca release through InsP R. We therefore 3 3 could conceivably occur through restricted access of the kinase next designed experiments to assess the consequences of to its substrate. Notwithstanding the general importance of InsP R-1 phosphorylation (expressed in isolation) on the initi- kinase targeting for efficient, localized phosphorylation, the ation and generation of Ca oscillations. In preliminary ex- / 46250 Phosphoregulation of S2 InsP R-1 FIG.7. Effect of PKG activation in single phosphomimetic con- structs. Threshold CCh-stimulated Ca release is markedly potenti- FIG.6. Effects of PKA phosphorylation on S2 InsP R-1 single ated by activation of PKG following 8-Br-cGMP treatment in S2 WT glutamate substitution constructs. Threshold CCh-stimulated Ca (A)orS2 ES-expressing cells (B). C, in contrast. no potentiation of release is markedly potentiated by activation of PKA following forskolin 2 threshold CCh-stimulated Ca release is observed in S2 SE-express- treatment in S2 WT-expressing cells (A)orS2 ES (B). C, in contrast, ing cells. D, pooled data illustrate that S2 ES has properties identical no potentiation of threshold CCh-stimulated Ca release is observed in to S2 WT, and thus phosphorylation of this residue following activa- cells expressing S2 SE. D, pooled data illustrate that S2 ES has 2 tion of PKG is unlikely to impact Ca release. Numbers in parentheses properties identical to S2 WT, and thus phosphorylation of this resi- indicate the number of analyzed cells. due is unlikely to impact Ca release. Numbers in parentheses indicate the number of analyzed cells. in Fig. 8. Stimulation with 250 ng/ml -IgM proved to be a periments we failed to initiate Ca oscillations with reproduc- threshold concentration in S2 WT-expressing cells. This de- ible characteristics with CCh in m3R-transfected cells (data not gree of stimulation generally resulted in a single small increase shown). Therefore, we chose to stimulate cells with an -IgM in [Ca ] after a long latency (Fig. 8A, left panel, and pooled antibody and to initiate Ca oscillations through activation of data in Fig. 8, C–E). An identical stimulus in cells expressing the endogenous B cell receptor, phospholipase C- activation, S2 EE, in contrast, resulted in repetitive Ca transients and the formation of InsP . Dt-40 3ko cells expressing either following a much shorter latency, consistent with the increased S2 WT or S2 EE to mimic PKA phosphorylation of the apparent sensitivity of the S2 EE constructs (Fig. 8A, right InsP R-1 were stimulated with various concentrations of -IgM panel, and pooled data Fig. 8, C–E). Most interestingly, stim- antibody as illustrated by the selection of representative traces ulation of S2 WT-expressing cells with 500 ng/ml -IgM an- / Phosphoregulation of S2 InsP R-1 46251 FIG.8. Comparison of Ca oscillations in Dt-40 3ko cells expressing S2 WT or phosphomimetic S2 constructs. Dt-40 3ko cells were transfected with either S2 WT (left panel)orS2 EE construct (middle panel). A, cells were stimulated with a threshold concentration (250 ng/ml) of -IgM antibody to stimulate the B cell receptor. In S2 WT-expressing cells, this resulted in a single Ca transient after a long latency (A, left panel). In contrast, multiple Ca transients were elicited in S2 EE-expressing cells following a shorter latency. B, cells were stimulated with 500 ng/ml -IgM antibody. Cells transfected with either S2 WT or S2 EE exhibited multiple Ca transients of similar frequency, although the initial peak was generally larger and latency shorter in S2 EE-expressing cells. C, the pooled data from cells stimulated with various concentrations of -IgM for frequency of oscillations is shown; D, is shown for latency; E, is shown for the magnitude of the initial peak. Numbers in parentheses indicate the number of analyzed cells. tibody resulted in Ca oscillations with similar frequency to exhibiting a lower threshold for activation by InsP infusion cells expressing S2 EE (Fig. 8B and pooled data C–E). How- (40) or agonist activation (41). ever, the latency before the initiation of an increase in Ca These data using phosphomimetic mutations of InsP R-1 was significantly shorter in S2 EE-expressing cells. In addi- splice variants are in broad agreement with a number of stud- tion, the magnitude of the initial transient was also signifi- ies that have reported increased sensitivity of InsP R-1 activity cantly larger in S2 EE-expressing cells when compared with following phosphoregulation by PKA (19–22, 41, 42). The most WT. Stimulation with 1 g/ml -IgM antibody resulted in tran- important consequence of this increased sensitivity appears to sients that were indistinguishable in terms of latency, fre- be in defining the threshold where a cell will respond to a quency, or initial peak magnitude in S2 EE- or S2 WT- stimulus. Given the almost ubiquitous expression of various expressing cells (pooled data Fig. 8, C–E). Thus, PKA-mediated forms of InsP R-1, this is likely a generally important phenom- phosphorylation, by increasing the sensitivity of the InsP R-1 enon. A number of mechanisms are plausible to explain the to InsP , may define the threshold at which cells begin to increased sensitivity of InsP -induced Ca release. These in- 3 3 oscillate but does not markedly influence the temporal proper- clude modulation of InsP binding, an idea supported by meas- ties of Ca oscillations when initiated. This latter observation urements of InsP R binding in hepatocytes (42) (presumably presumably reflects the fact that the frequency of oscillations is S2 InsP R-1 and InsP R-2). In these studies the apparent 3 3 primarily defined by mechanisms such as Ca feedback (39) affinity of InsP binding was enhanced 2-fold at resting 2 2 rather than the absolute sensitivity of the InsP R-1 to InsP [Ca ], and the [Ca ] necessary for half-maximal stimulation 3 3 within a defined range. These data are largely consistent with of InsP binding was reduced. In contrast, a study of recombi- data from hepatocytes where PKA activation resulted in cells nant S1 S2 InsP R-1 expressed in SF9 cells and reconsti- 3 / 46252 Phosphoregulation of S2 InsP R-1 14. Boehning, D., Mak, D. O., Foskett, J. K., and Joseph, S. K. (2001) J. Biol. tuted into lipid bilayers has reported a similar increase in Chem. 276, 13509–13512 InsP R sensitivity to InsP but that the bell-shaped Ca sen- 3 3 15. Galvan, D. L., and Mignery, G. A. (2002) J. Biol. Chem. 277, 48248–48260 16. Parker, A. K., Gergely, F. V., and Taylor, C. W. (2004) J. Biol. Chem. 279, sitivity of channel opening is not altered following phosphoryl- 23797–23805 ation of the InsP R-1 (20). These findings suggest that modu- 17. Taylor, C. W., and Laude, A. J. (2002) Cell Calcium 32, 321–334 lation of the Ca sensitivity of channel activity is unlikely to 18. Soulsby, M. D., Alzayady, K., Xu, Q., and Wojcikiewicz, R. J. (2004) FEBS Lett. 557, 181–184 account for the increased apparent sensitivity of the receptor, 19. Wagner, L. E., II, Li, W. H., and Yule, D. I. (2003) J. Biol. Chem. 278, at least in this form of the InsP R-1. A number of alternative 45811–45817 mechanisms for altering InsP R-1 sensitivity are conceivable. 20. Tang, T. S., Tu, H., Wang, Z., and Bezprozvanny, I. (2003) J. Neurosci. 23, 403–415 For example, phosphorylation of the receptor could alter the 21. Nakade, S., Rhee, S. K., Hamanaka, H., and Mikoshiba, K. (1994) J. Biol. gating of the channel directly. In addition, phosphorylation Chem. 269, 6735–6742 22. Wojcikiewicz, R. J., and Luo, S. G. (1998) J. Biol. Chem. 273, 5670–5677 might secondarily modulate the receptor by regulation of the 23. Ferris, C. D., Cameron, A. M., Bredt, D. S., Huganir, R. L., and Snyder, S. H. association of regulatory factors such as proteins or adenine (1991) Biochem. Biophys. Res. Commun. 175, 192–198 nucleotides (43–45). Indeed, a precedent for this type of regu- 24. Haug, L. S., Jensen, V., Hvalby, O., Walaas, S. I., and Ostvold, A. C. (1999) J. Biol. Chem. 274, 7467–7473 lation exists because PKA phosphorylation has been shown to 25. Kubista, H., Hawkins, T., and Moss, S. E. (1998) Biochim. Biophys. Acta 1448, alter the association of calmodulin with the S2 form of 299–310 26. Sugawara, H., Kurosaki, M., Takata, M., and Kurosaki, T. (1997) EMBO J. 16, InsP R-1 (36). 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Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

Wagner, Larry E.; Li, Wen-Hong; Joseph, Suresh K.; Yule, David I.
Journal of Biological Chemistry – Oct 1, 2004
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 44, Issue of October 29, pp. 46242–46252, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor* Received for publication, May 26, 2004, and in revised form, August 5, 2004 Published, JBC Papers in Press, August 11, 2004, DOI 10.1074/jbc.M405849200 Larry E. Wagner II‡, Wen-Hong Li§ , Suresh K. Joseph , and David I. Yule‡** From the ‡Department of Pharmacology and Physiology, University of Rochester, Rochester, New York 14642, the §Departments of Cell Biology and Biochemistry, University of Texas, Southwestern Medical Center, Dallas, Texas 75390-9039, and the Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Inositol 1,4,5-trisphosphate receptors are intracellular ion Regulation of Ca release through inositol 1,4,5- trisphosphate receptors (InsP R) has important conse- channels that function to couple the activation of cell surface quences for defining the particular spatio-temporal receptors for neurotransmitters, hormones, and growth fac- properties of intracellular Ca signals. In this study, tors to the initiation of intracellular Ca release (1). Three regulation of Ca release by phosphorylation of type 1 genes have been cloned that encode distinct proteins of a InsP R (InsP R-1) was investigated by constructing 3 3 molecular mass of 300 kDa, named the type 1 (InsP R-1), “phosphomimetic” charge mutations in the functionally type 2 (InsP R-2), and type 3 (InsP R-3) InsP Rs (2–4). In 3 3 3 important phosphorylation sites of both the S2 and addition, multiple receptor proteins with distinct tissue dis- S2 InsP R-1 splice variants. Ca release was investi- tributions are produced by alternate splicing of the type 1 gated following expression in Dt-40 3ko cells devoid of receptor gene (5, 6). Most cells express multiple isoforms of endogenous InsP R. In cells expressing either the InsP R (7). Furthermore, the expression level and comple- S1755E S2 or S1589E/S1755E S2 InsP R-1, InsP -in- 3 3 ment of receptors differ in individual tissues, and this to- duced Ca release was markedly enhanced compared gether with regulation of the activity of the channel is with nonphosphorylatable S2 S1755A and S2 S1589A/ 2 thought to be a major determinant of the rich diversity of S1755A mutants. Ca release through the S2 S1589E/ Ca signaling events observed in cells (7, 8). S1755E InsP R-1 was enhanced 8-fold over wild type The functional channel is formed co-translationally by the and 50-fold when compared with the nonphosphorylat- tetrameric association of four individual receptor subunits (9, able S2 S1589A/S1755A mutant. In cells expressing S2 10). Each subunit has a binding site for InsP toward the N InsP R-1 with single mutations in either S1589E or 3 terminus formed by a cluster of positively charged amino acids S1755E, the sensitivity of Ca release was enhanced thought to coordinate the negatively charged phosphate groups 3-fold; sensitivity was midway between the wild type of InsP (11, 12). The C terminus of each subunit is postulated and the double glutamate mutation. Paradoxically, for- skolin treatment of cells expressing either single Ser/ to span intracellular membranes six times and forms a single Glu mutation failed to further enhance Ca release. cation-selective pore (13, 14). In addition, this region signals The sensitivity of Ca release in cells expressing S2 retention of the protein to the endoplasmic reticulum (15, 16). S1755E InsP R-1 was comparable with the sensitivity of Although the InsP -binding pocket and channel pore are highly S2 S1589E/S1755E InsP R-1. In contrast, mutation of 3 conserved between InsP R family members, the intervening S2 S1589E InsP R-1 resulted in a receptor with compa- 3 sequence between the binding region and pore is more diver- rable sensitivity to wild type cells. Expression of S2 gent and consists of the so-called “regulatory and coupling” or S1589E/S1755E InsP R-1 resulted in robust Ca oscilla- “modulatory” domain. This region, consisting of 1600 amino tions when cells were stimulated with concentrations of 2 acids, is thought to be important in modulating the Ca re- -IgM antibody that were threshold for stimulation in lease properties of the InsP R. Indeed, Ca release through S2 wild type InsP R-1-expressing cells. However, at the InsP R is markedly influenced by many factors, most im- higher concentrations of -IgM antibody, Ca oscilla- portantly by Ca itself (17). InsP R activity is also influenced tions of a similar period and magnitude were initiated in through interaction with numerous factors such as proteins, cells expressing either wild type or S2 phosphomimetic adenine nucleotides, and phosphorylation and in particular by mutations. Thus, regulation by phosphorylation of the cyclic nucleotide-dependent kinases (8). functional sensitivity of InsP R-1 appears to define the Two protein kinase A (PKA) consensus sites (RRXS) at Ser- threshold at which oscillations are initiated but not the 1589 and Ser-1755 are present in the InsP R-1 (5, 18), and the frequency or amplitude of the signal when established. 3 most recent studies suggest that phosphorylation of these sites results in a marked enhancement of Ca release (19–22). * This work was supported in part by National Institutes of Health Most interestingly, these sites are conserved through evolution Grants RO1-DK54568, R01-DE14756, PO1 DE13539 (to D. I. Y.), and RO1-DK34804 (to S. K. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 The abbreviations used are: InsP R-1, inositol 1,4,5-trisphosphate U.S.C. Section 1734 solely to indicate this fact. receptor type 1; [Ca ] , intracellular calcium concentration; InsP , i 3 Supported by Research Grant I-1510 from the Robert A. Welch inositol 1,4,5-trisphosphate; CCh, carbamylcholine (carbachol); PKA, Foundation and a Career Development award from the American Dia- cAMP-dependent protein kinase; PKG, cGMP dependent protein ki- betes Association. nase; 8-Br cGMP, 8-bromoguanosine-3,5- cyclic monophosphate; ** To whom correspondence should be addressed. Tel.: 585-273-2154; ci-IP /PM, caged isopropylidene inositol trisphosphate; WT, wild type; Fax: 585-670-0394; E-mail: [email protected]. cm-IP , caged methoxymethylene inositol trisphosphate. 46242 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. / Phosphoregulation of S2 InsP R-1 46243 used for the mutagenesis reaction flanked the restriction sites RsrII from Drosophila to humans in InsP R-1, but corresponding and KasI. Following mutation, the resulting fragments were cut with sites are not present in either the InsP R-2 or InsP R-3. It 3 3 RsrII and KasI and inserted into the InsP R-1 backbone at the corre- should be noted, however, that other regions, which are pres- sponding sites. The mutations were confirmed by Big Dye fluorescent ently not defined, appear to function as PKA phosphorylation sequencing. Mutated receptor DNAs were excised from MXT-1 by using sites in InsP R-2 and InsP R-3. Several reports have demon- 3 3 EcoRI and ligated into the mammalian expression vector pGW (pro- strated biochemically that both Ser-1589 and Ser-1755 can be vided by Dr. David Yue, The Johns Hopkins University). Orientation was confirmed by using restriction enzyme digestion. Mutants were phosphorylated in InsP R-1 (18, 23, 24); however, these studies named based on the splice variant, either S2 or S2 followed by the have not provided a consensus as to which sites are important amino acid present at position 1589 and 1755. Thus, a mutation in S2 physiologically in tissue that expresses either of the two major InsP R-1 S1755E is designated,“S2 SE,” and in InsP R-1 S2 S1755A 3 3 splice variants of InsP R-1. is designated “S2 SA”. Similarly, an S2 InsP R-1 with mutations in A recent study (19) from our laboratory has elucidated the both S1589E and S1755E is designated “S2 EE”. The numbering of particular sites that are functionally important for the phos- residues is based on the full-length rat InsP R-1. Transfection of Dt-40 Cells—Dt-40 cells lacking all three InsP re- phoregulation of the two major splice variants of the InsP R-1. 3 ceptor subtypes were transfected by using electroporation at 350 V and These studies were performed by using mutational analysis by 950 microfarads (4-mm gap cuvette). 2  10 cells were co-transfected substituting alanine for the serine residues present at individ- with 25 g of the InsP R-1 cDNA, 25 g of the muscarinic type 3 (m3R) ual putative phosphorylation sites. Although phosphorylation receptor DNA, and 4 g of the red fluorescent protein plasmid pH- of both neuronal (S2) and peripheral (S2) forms of InsP R-1 3 cRed1-N1 (Clontech). Cells were incubated with DNA in 500 lof by PKA resulted in enhanced Ca release, mutational analy- Opti-MEM media (Invitrogen) on ice for 10 min. The cell/DNA mixture was electroporated, incubated on ice for 30 min, increased to 5 ml with sis indicated that only phosphorylation of Ser-1755 was func- Opti-MEM, and placed in a 5% CO incubator at 39 °C for 5 h. The cells tionally important in the neuronal S2 InsP R-1. In contrast, were then centrifuged and resuspended in 12 ml of complete RPMI both Ser-1589 and Ser-1755 appeared to be phosphorylated and media (Invitrogen). Transfection efficiency was typically 20%. Exper- significant in the peripheral S2 form of InsP R-1. In addition, iments were performed within 32 h of transfection. although the S2 form of the receptor was subject to direct Transfection of HEK-293 Cells and Assessment of Phosphorylation of phosphoregulation by cGMP-dependent protein kinase (PKG), S2 InsP R-1—HEK-293 cells were plated onto 25-cm culture flasks and allowed to grow to near-confluency. Cells were transfected with 5 the S2 form was not influenced by activation of PKG. These g of each S2 InsP R-1 DNA construct by using the LipofectAMINE data represent one of the few major differences reported for the reagent (Invitrogen) as per the manufacturer’s instructions. The follow- regulation of the two major splice variants of the InsP R-1. ing day, batches of cells were treated in the presence or absence of 20 M In the present study, we have constructed charge mutations, forskolin for 10 min, aspirated from flasks, lysed, and immunoprecipi- substituting glutamate residues for the serine residues in the tated with a polyclonal -InsP R-1 antibody that recognizes amino functionally important phosphorylation sites in both S2 and acids 2731–2749 of InsP R-1. Immunoprecipitates were separated on 5% SDS gels transferred to nitrocellulose and then probed with either S2 variants of the InsP R-1. These mutations are predicted to the -InsP R-1 antibody or a polyclonal antibody that recognizes the mimic phosphorylation and to allow the assessment of the phosphorylated state of Ser-1755 (28) (-phospho-Ser-1755), kindly pro- functional effects of phosphorylation of InsP R-1. Most impor- vided by Dr. S. Snyder. Blots that were probed with -phospho-Ser- tantly, the Ca release properties of phosphomimetic muta- 1755 were stripped and reprobed with the -InsP R-1 antisera to con- tions are predicted to be essentially independent of cell type- firm the presence and relative quantity of the InsP R-1. specific factors, including the expression of accessory proteins Digital Imaging of [Ca ] —Transfected Dt-40 3ko cells were washed once in a HEPES-buffered physiological saline solution (HEPES-PSS) such as protein A-kinase anchoring proteins. These effects containing (in mM) 5.5 glucose, 137 NaCl, 0.56 MgCl , 4.7 KCl, 1 should also be unambiguously specific to InsP R and thus Na HPO , 10 HEPES (pH 7.4), 1.2 CaCl , and 1% w/v bovine serum 2 2 4 2 independent of confounding PKA effects on other Ca -han- albumin. Cells were then resuspended in bovine serum albumin dling machinery. This latter consideration has historically HEPES-PSS with 1 M Fura-2 (AM), placed on a 15-mm glass coverslip plagued the functional assessment of PKA phosphorylation of in a low volume perfusion chamber, and allowed to adhere for 30 min at InsP R. These mutations have allowed us to define the relative room temperature. Cells were perfused continuously for 10 min with HEPES-PSS before experimentation to allow complete Fura-2 de-ester- sensitivity of Ca release of the phosphomimetic mutations ification. A field of cells for each experiment was chosen that contained and to confirm which sites are important in each splice variant. a wide range of transfection efficiency based upon the intensity of red The present study has also addressed whether phosphorylation fluorescence emitted when excited at 560 nm. Individual cells that had of individual sites is permissive or additive in each splice var- emission gray levels between 1500 and 2500 were subsequently chosen iant, and we have investigated the consequences of phospho- to standardize expression levels. [Ca ] imaging was performed essen- rylating InsP R-1 on Ca oscillations, the physiological pat- tially as described previously by using an inverted epifluorescence Nikon microscope with a 40 oil immersion objective lens (numerical tern of Ca signaling in nonelectrically excitable cells. aperture, 1.3) (19). Cells were excited alternately with light at 340 and MATERIALS AND METHODS 380  10 nm bandpass filters (Chroma, Rockingham, VT) using a The acetoxymethyl esters of Fura-2 and Fluo-4 were purchased from monochrometer (TILL Photonics, Pleasanton, CA). Fluorescence im- Molecular Probes (Eugene, OR). Cell-permeable cyclic nucleotides and ages were captured and digitized with a digital camera driven by TILL forskolin were purchased from Biomol (Plymouth Meeting, PA). All Photonics software. Images were captured every 2 s with an exposure of other chemicals were purchased from Sigma. The Dt-40 cells lacking 2 ms and 4 by 4 binning. 340/380 ratio images were calculated online InsP R (Dt-40 3ko) were kindly provided by Dr. Kurosaki (Kansai and stored immediately to a hard disk. Medical University, Japan) and were maintained as described Flash Photolysis—Transfected cells were simultaneously loaded with previously (25–27). the visible wavelength indicator Fluo-4 and a cell-permeable form of Production of Mutations—The rat S2 InsP R-1 in the expression caged inositol trisphosphate (ci-IP /PM) for 30 min. ci-IP /PM is a 3 3 plasmid pIRES-GFP was digested with the restriction endonuclease homologue of cm-IP /PM. The 2- and 3-hydroxyls of the inositol ring are SalI. The overhang created by digestion was blunted by using T4 protected by an isopropylidene group in ci-IP /PM and are protected by polymerase. An EcoRI linker was then ligated onto the blunted ends of a methoxymethylene group in cm-IP /PM (29). Like cm-IP /PM, ci- 3 3 the construct. The entire receptor DNA was excised from the plasmid IP /PM diffuses across cell membranes, and the PM group is hydrolyzed using EcoRI and ligated into the plasmid MXT-1. The region containing by cellular esterases, and Ca release can be induced upon photo- the S2 splice variant and potential PKA phosphorylation sites was uncaging as i-IP is liberated from the cage and acts in a similar fashion excised from its backbone in pCDNA 3.1 by RsrII and KasI and ligated to InsP at InsP R. A further period of 30 min was allowed for 3 3 into the InsP R construct in MXT-1. The potential PKA phosphoryla- de-esterification of both dye and cage. Cells were illuminated at 488 tion sites Ser-1589 and Ser-1755 were mutated, individually in both splice variants and together in the S2 splice variant, to alanines or glutamates by using sequential PCR mutagenesis. The outside primers W.-H. Li, unpublished data. / 46244 Phosphoregulation of S2 InsP R-1 10 nm and fluorescence collected through a 525  25-nm bandpass filter charge (0.5–5 ms). No increase in [Ca ] was observed follow- and captured using the Till Photonics imaging suite. These traces are ing the longest UV discharge in cells either not loaded with displayed as % F/F , where F is the recorded fluorescence, and F is the o o ci-IP /PM or not expressing HcRed. Fig. 1, B and C, shows mean of the initial 10 sequential frames. Photolytic release was per- traces from typical experiments in individual Dt-40 3ko cells formed as described previously by using a pulsed xenon arc lamp (Till expressing S2 EE or S2 AA (Fig. 1, B and C, respectively, Photonics). A high intensity (0.5–5 ms duration; 80 J) discharge of UV light (360  7.5 nm) was reflected onto the plane of focus by using a and pooled data in Fig. 1F). In cells expressing S2 EE, Ca DM400 dichroic mirror and Nikon 40 oil immersion objective, 1.3 NA. release as defined by a 0.05% F/F increase in initial fluo-4 Frequency Distribution of Ca Oscillations—Cells transfected with fluorescence was observed in 60% of cells when exposed to UV WT and mutant S2 isoforms were stimulated with varying concentra- discharge for 0.5 ms. Subsequent exposure to UV light for 1.25 tions of an -IgM antibody (Southern Biotechnology Associates, Inc., ms elicited a more robust increase in [Ca ] in all cells. Fi- Birmingham, AL). Infrequent Ca oscillations were produced presum- i ably through B cell receptor cross-linking, activation of phospholipase nally, photo-release following a 5-ms flash, in general evoked a C-, and subsequent production of InsP . The frequency of Ca oscil- 3 further increase in the magnitude of Ca release. In contrast, lations was determined by selecting individual peaks that displayed an an elevation of intracellular Ca was never observed under increase in ratio units greater than 0.05 and are listed as frequency in identical conditions following a 0.5-ms UV flash in cells ex- milliHertz (number of oscillations in 1000 s). pressing S2 AA. A significant Ca release was only observed Concentration-Response Relationships—Normalized F concentra- in 50% of cells following a 1.25-ms flash, and the majority of tion-response relationships were fit with the following logistic Equation 1, cells only responded to the longest uncaging duration, albeit with a smaller magnitude than in S2 EE-expressing cells Slope F  1/1  C/EC (Eq. 1) exposed to the same stimulus. A similar pattern of sensitivity was observed in Dt-40 3ko where F is the change in fluorescence normalized to the maximal response; C is agent concentration; EC is the concentration where the cells expressing S2 S1755E (S2 SE) and S2 S1755A (S2 response is half of maximum, and Slope is a slope factor related to the SA) as shown in Fig. 1, D and E, respectively, and pooled data Hill coefficient. in Fig. 1G. Cells expressing S2 SE responded more robustly to Statistical Analysis—The effects of treatment were determined by photolysis of ci-IP than cells expressing S2 SA exposed to an normalizing the peak change in fluorescence ratio by stimulation fol- identical stimulus. These data indicate that serine to gluta- lowing forskolin or 8-Br-cGMP exposure to that of stimulation in control mate mutations at the functionally important phosphorylation HEPES-PSS. Thus, pooled data represent a normalized fold increase over control for the treated trial. In all cases where statistical signifi- sites in both splice variants of InsP R-1 are “phosphomimetic,” cance is indicated, two-tailed heteroscedastic t tests were performed. p i.e. charge mutations mimic phosphorylation in that these con- values 0.05 were considered to indicate statistical significance and structs display an apparent increased functional sensitivity are denoted by an asterisk in the figures. to InsP . Sensitivity of S2 InsP R-1 and Phosphorylation Site Mu- RESULTS AND DISCUSSION 3 tants—Experiments were next performed to determine the rel- Phosphomimetic Mutations in Functionally Important Phos- ative sensitivity of Ca release through the phosphomimetic phorylation Sites in Both S2 and S2 InsP R-1 Result in mutations of the S2 InsP R-1 with respect to the wild type 2 3 Enhanced Ca Release—Phosphorylation of proteins results and phosphorylation-deficient mutants. Dt-40 3ko cells were in the addition of net negative charge to the phosphoacceptor transfected with cDNAs encoding m3R, HcRed together with residue. In the case of PKA phosphoregulation, the functional the InsP R-1 construct of interest. Stimulation with the mus- effects of phosphorylation are thought to occur as the negative carinic agonist CCh results in robust increases in [Ca ] in charge added to the serine or (less frequently) threonine resi- transfected cells through the G -coupled stimulation of q/11 dues neutralizes the positive charge of basic arginine or lysine phospholipase C- and subsequent formation of InsP . The residues present upstream in the classical RRX(S/T) consensus magnitude of the initial peak provides a good estimation of the motif (30). This charge neutralization, in turn, is thought to extent of Ca release as this parameter in Dt-40 cells, like result in a conformational change in the protein. A common many cells, is essentially independent of Ca influx. Further- approach employed to investigate the functional effects of phos- more, in Dt-40 cells the [Ca ] response to stimulation with phorylation is to construct phosphomimetic mutations whereby CCh does not appreciably desensitize, and thus the effects of glutamic or aspartic acid residues are substituted at the phos- multiple concentrations of agonist can be assessed in a single phoacceptor site (31, 32). The rationale for this strategy is that cell. Individual HcRed-expressing cells were stimulated with the negatively charged side chain of the substituted acidic increasing concentrations of CCh (1 nM to1 M) for 60 s followed amino acid will mimic, to an extent, the addition of a phosphate by a 5-min wash between applications of agonist. In each case, moiety to the protein. these experiments were performed on multiple cells, express- To investigate the consequences of InsP R-1 phosphoryla- ing a narrow range of HcRed fluorescence and from multiple tion, we analyzed in Dt-40 3ko cells the Ca release properties batches of transfected cells to minimize variation because of of phosphomimetic mutations in the functionally important expression level of m3R and InsP R-1. Concentration-response sites in both S2 and S2 InsP R-1. This cell line provides the 3 relationships were generated by normalizing each initial peak only known InsP R null background (26, 27). In initial exper- to the maximum response in the individual cell and subse- iments, a comparison was made between the sensitivity of Ca quently averaging the pool of cells expressing a particular release by InsP R-1 phosphomimetic mutations versus non- construct. Fig. 2A shows a typical example of this experimental phosphorylatable alanine mutations at the sites we reported procedure performed on Dt-40 3ko cells expressing S2 EE. A previously (19) to be relevant. Dt-40 3ko cells were transfected significant increase in Ca was detected in the majority of with DNA encoding HcRed to facilitate identification of trans- S2 EE-expressing cells following stimulation with 1 nM CCh, fected cells and either S2 S1589E/S1755E InsP R-1 (S2 EE) and the peak response occurred following stimulation between or S2 S1589A/S1755A (S2 AA). Ca release was monitored following flash photolysis of ci-IP , a cell-permeable form of 10 and 50 nM CCh. In contrast, as shown in Fig. 2, B and C, Ca release following CCh stimulation in the wild type S2 caged InsP . This experimental paradigm provides a relatively direct assessment of the effects of InsP R-1 phosphorylation on InsP R-1 or S2 AA was considerably less sensitive to CCh 3 3 stimulation. Analysis of the pooled data indicated that the the Ca release process. The amount of ci-IP photo-released was controlled by varying the duration of the UV flash dis- magnitude of the peak response to any of these constructs was / Phosphoregulation of S2 InsP R-1 46245 FIG.1. Flash photolysis of caged InsP reveals that phosphomimetic InsP R-1 exhibits enhanced sensitivity to InsP . A represents 3 3 3 a schematic of amino acids 1586–1755 of the S2 InsP R-1 showing the S2 splice site and potential PKA/PKG phosphorylation sites. Dt-40 3ko cells were transfected with either phosphomimetic mutations of InsP R-1 (B and D) or nonphosphorylatable mutations (C and E). Experiments in B and C are representative traces from cells transfected with the S2 InsP R-1 and in D and E with S2 InsP R-1. The cells were loaded with 3 3 the visible wavelength Ca indicator fluo-4 and the cell-permeable caged InsP R analogue ci-IP -PM as described under “Experimental 3 3 Procedures.” Increasing iIP concentrations were generated by varying the duration (energy magnitude) of UV flash exposure from 0.5 to 5 mss. B, cells transfected with S2 EE constructs respond at a lower threshold of UV light exposure and with more robust elevations in [Ca ] than cells transfected with S2 AA mutations that are nonphosphorylatable (C). Similarly, as shown in D,S2 SE mutations are more sensitive than S2 SA mutations. The pooled data, expressed as fold increase in the F/F fluorescence, are shown in F (S2 constructs) and G (S2 constructs). The magnitude of responses to 1.25- and 2.5-ms exposure to UV light was significantly greater in phosphomimetic mutations in each splice variant, p 0.005. The values in parentheses indicate the number of cells responding above a 0.05% F/F threshold in relation to the number of cells tested. not significantly different (maximum peak  response: S2 mutant (EC 4.3  1.2 nM CCh) was 7.5-fold more sensitive WT 0.9  0.1 ratio units; S2 AA 0.67 0.1 ratio units, when compared with WT S2 InsP R-1 (EC 32.6  7.5 nM) 3 50 and S2 EE 1.13  0.1 ratio units), suggesting that the and some 50-fold more sensitive than the nonphosphorylatable efficacy of Ca release was essentially unaltered. However, S2 AA mutant (EC 229.5  14.6 nM). These data provide when an estimate of the relative sensitivity was made by fitting strong evidence that the S2 EE construct is more sensitive to the normalized concentration-response relationships for each stimulation by InsP and present evidence that phosphoryla- construct (Fig. 2D), CCh-induced Ca release in the S2 EE tion of the InsP R-1 results in marked regulation of channel 3 / 46246 Phosphoregulation of S2 InsP R-1 intermediate sensitivity of the wild type S2 InsP R-1 relative to the phosphomimetic S2 InsP R-1 receptor. Although we have demonstrated that each site can be phos- phorylated, it is not definitively known whether the functional effects of phosphorylating individual sites are independent and additive or alternatively if the full effect is seen following phosphorylation of an individual site. Thus, experiments were next performed to assess the sensitivity of single phosphomi- metic mutations within each phosphorylation site. Concentra- tion-response relationships for CCh-induced Ca release were constructed for Dt-40 3ko cells expressing either S2 S1755E InsP R-1 (S2 SE) or S2 S1589E InsP R-1 (S2 ES). The 3 3 data were analyzed as described previously for Fig. 2D. Fig. 3A shows the fit for the normalized concentration-response rela- tionship for S2 SE and S2 ES and for comparison also shows the fit for S2 EE and S2 WT (Fig. 3A, dotted lines; data from Fig. 2D). Once again the maximal initial peak responses in either mutant were not significantly altered from wild type (S2 ES 0.54  0.1  ratio units; S2 SE 0.78  0.1 ratio units); however, the sensitivity of each of these mutants was significantly shifted, such that the EC for CCh-induced Ca release was enhanced 3-fold over the response in wild type for either mutant (EC for CCh-induced release: S2 ES 12.4  0.5 nM;S2 SE 13.5  1nM). These data are summarized in Table I. The enhanced apparent sensitivity of individual phosphomi- metic mutants was essentially equal and intermediate between the sensitivity of the S2 WT and S2 EE mutations. These data are consistent with phosphorylation of individual sites being functionally additive. To address this possibility directly, experiments were performed evaluating the effects of activat- ing endogenous PKA in cells expressing S2 SE or S2 ES to mimic prior phosphorylation of an individual site. As shown in Fig. 3B, and reported previously, activation of PKA by incuba- tion with forskolin results in a dramatic potentiation of Ca release in Dt-40 3ko cells expressing S2 WT (19). Most sur- prisingly, although the sensitivity to CCh was enhanced, as evidenced by the low concentration of CCh necessary to evoke 2 2 threshold Ca release, no potentiation of Ca release follow- ing forskolin incubation was observed in Dt-40 3ko cells ex- pressing either S2 ES (Fig. 3C; pooled data in Fig. 3F), S2 SE (Fig. 3D; pooled data in Fig. 3F), or S2 EE (Fig. 3E; pooled data in Fig. 3F). Thus, although each phosphorylation site in S2 InsP R-1 can be phosphorylated and mimicking phospho- rylation of both sites leads to a receptor with enhanced sensi- tivity relative to phosphorylation of an individual site, phos- phorylation of both sites in situ does not appear to occur and FIG.2. InsP sensitivity of S2 InsP R-1 and phosphomimetic 3 3 therefore is not functionally additive. Although these data may mutations. Concentration-response relationships for CCh-induced seem paradoxical, a possible explanation, consistent with all Ca signals were examined in Dt-40 3ko cells expressing m3 receptors the observations is that the initial phosphorylation of either and InsP R-1 constructs. A, cells transfected with S2 EE were stim- ulated with increasing concentrations of CCh as indicated (n 9 cells). Ser-1589 or Ser-1755 leads to a conformational change in the A similar paradigm was performed for cells expressing S2 WT (n 10) receptor that now precludes the phosphorylation of the addi- (B) and for cells expressing S2 AA constructs (n 9) (C). The mag- 2 tional site. To test this hypothesis, experiments were per- nitude of the initial peak (as an indicator of Ca release) for each formed to determine the phosphorylation state of the various response was normalized to the maximum response in each cell. The pooled data and the fit that describes each concentration-response mutants after raising cAMP levels. These experiments were relationship for each construct is shown in D and illustrates that the performed in HEK-293 cells because of the high transfection sensitivity of Ca release was greatest in S2 EE followed by S2 WT efficiency and low endogenous levels of InsP R-1 (18, 33). HEK- and S2 AA. 293 cells were transfected with the constructs as indicated in Fig. 4, incubated in the presence or absence of forskolin for 10 min, then pelletted, and lysed. Following incubation of the function. This profound regulation could be expected to have lysates with -InsP R-1 antibody, the immune complexes were major consequences for calcium signaling events in peripheral tissue such as liver, testis, and smooth muscle which express captured and separated on SDS gels, and the phosphorylation status of Ser-1755 was determined by Western blotting with an the S2 InsP R-1 (5, 6). The observation that S2 AA is relatively less sensitive than S2 WT is consistent with the antibody that specifically recognizes this phosphorylated resi- due in InsP R-1 (28). As shown in Fig. 4A, wild type S2 possibility that a proportion of the wild type receptor is consti- tutively phosphorylated in Dt-40 cells, thus contributing to the InsP R-1 was robustly phosphorylated after forskolin incuba- 3 / Phosphoregulation of S2 InsP R-1 46247 FIG.3. Functional sensitivity of single phosphomimetic mutations in S2 InsP R-1. A, concentration-response relationships were generated exactly as shown in Fig. 2D and as described under “Experimental Procedures.” Cells expressing either S2 SE (n 6) or S2 ES (n 5) displayed intermediate sensitivity between S2 WT and S2 EE constructs. Dotted lines indicate S2 WT and S2 EE for comparison (data from Fig. 2D). B, threshold CCh-stimulated Ca release is markedly potentiated by activation of PKA following forskolin treatment in cells expressing S2 WT. In contrast, no potentiation of threshold CCh-stimulated Ca release is observed in cells expressing S2 SE in D,S2ES in C,orS2 EE in E. Pooled data are shown in F; potentiation by forskolin is only seen in S2 WT. Numbers in parentheses indicate the number of analyzed cells. tion, whereas no phosphorylation was detected in wild type cells transfected with S2 EE, S2 SE or, most importantly, S2 AA or untransfected HEK-293 cells. Similarly, in Fig. 4B, the S2 ES construct. These data are strongly supportive of a marked increase in phosphorylation could be detected in S2 the contention that the initial phosphorylation of one site pre- InsP R-1, whereas no phosphorylation could be detected in cludes further phosphorylation at the additional residue as 3 / 46248 Phosphoregulation of S2 InsP R-1 TABLE I Summary of the effects of phosphorylation by PKA or PKG of the S2/S2 splice variants of InsP R-1 and phosphoregulatory mutations at Ser-1589 and Ser-1755 expressed in Dt40 3 knock out cells The data are derived from this paper and Ref. 19. NT, not tested. S2 InsP R-1 S2 InsP R-1 3 3 Amino acid at 1589/1755 CCh EC (M)/(fold 2 CCh EC (M)/(fold 2 50 50 Ca release after PKA/PKG activation Ca release after PKA/PKG activation change from WT) change from WT) Wild-type SS 32.1  4.2/(1) Enhanced/enhanced 32.6  7.5/(1) Enhanced/no change AS NT Enhanced/enhanced NT Enhanced/no change SA 116.3  3.0/(0.3) No change/no change NT Enhanced/no change AA NT NT/NT 229.5  14.6/(0.14) No change/no change SE 3.6  0.2/(8.9) No change/no change 13.5  1.1/(2.4) No change/NT ES 23.7  1.2/(1.3) Enhanced/enhanced 12.4  0.5/(2.6) No change/NT EE NT NT/NT 4.3  1.2/(7.6) No change/NT the InsP R-1 (34–36) and is therefore supportive of our conten- tion that the initial CCh-stimulated [Ca ] peak is a good indicator of InsP R function. In a similar fashion to the S2 InsP R-1, mutation of Ser-1755 to either alanine or glutamic acid did not significantly alter the maximal initial peak upon CCh stimulation (maximum peak  response: S2 WT 0.61  0.1  ratio units; S2 SE 0.59  0.1  ratio units; S2 SA 0.84  0.2  ratio units) but did, however, significantly affect the apparent sensitivity of Ca release as shown in Fig. 5A. The S2 SE mutant exhibited a similar EC for CCh- induced Ca release as the S2 EE mutation (EC :S2 SE 3.6  0.2 nM CCh; S2 EE 4.3 nM), being 9-fold more sensitive than S2 WT and 32-fold more sensitive than the nonphosphorylatable S2 SA mutation (EC S2 SA 116.3  3nM CCh). These data are summarized in Table I. In contrast to the S2 InsP R-1 S1755E construct, mutation of Ser-1589 to glutamate in the S2 form of InsP R-1 did not significantly affect the apparent sensitivity of Ca release as shown in Fig. 5B. Both the maximum peak response to CCh and the sensitivity of the receptor were very similar to that of S2 WT InsP R-1 (maximum peak  response 0.59  0.2 ratio units; EC 23.7  1.2 nM CCh). However, it is formally FIG.4. Phosphorylation of S2 InsP R-1 mutants. The phospho- possible that phosphorylation of Ser-1589 is only functionally rylation status of S2 InsP R-1 constructs was assessed as described important following phosphorylation of Ser-1755 in S2 under “Materials and Methods.” A, incubation with 20 M forskolin InsP R-1. To test this idea, experiments were performed acti- results in phosphorylation of Ser-1755 in S2 InsP R-1. No phospho- rylation is seen in S2 AA-transfected cells or untransfected HEK-293 vating PKA with forskolin in Dt-40 3ko cells expressing S2 cells (UN). B, forskolin treatment results in phosphorylation of Ser- ES or SE mutants. Treatment with forskolin resulted in a 1755 in S2 InsP R-1 but not S2 EE, S2ES, or S2 SE. The lack of marked potentiation of CCh-induced Ca release in cells ex- phosphorylation of Ser-1755 in the S2 ES mutant is interpreted as pressing either S2 WT or S2 ES (Fig. 6, A and B, respec- indicating that mimicking phosphorylation of Ser-1589 by phosphomi- metic substitution precludes the phosphorylation of Ser-1755 consistent tively, and pooled data in D) presumably as Ser-1755 was with the functional data shown in Fig. 3. IP, immunoprecipitation; WB, phosphorylated. Although cells expressing S2 SE were more Western blot. sensitive to CCh, no further potentiation was observed follow- ing forskolin incubation (Fig. 6C and pooled data in Fig. 6D). suggested by the functional data and indicate that physiologi- These data indicate that it is unlikely that phosphorylation of cally only phosphorylation of a single residue is functionally Ser-1755 is permissive for any functional effect of phosphoryl- relevant in the S2 splice variant of InsP R-1. ating Ser-1589 in S2 InsP R-1. 3 3 Sensitivity of S2 InsP R-1 and Phosphorylation Site Mu- Effect of PKG Activation on S2 InsP R-1 and Phosphomi- 3 3 tants—The sensitivity of Ca release via the neuronal S2 metic Mutants—We have reported previously that PKG activa- InsP R-1 was next assessed by using a similar experimental tion results in direct phosphoregulation of only the neuronal paradigm to that used for the S2 form of the receptor. Al- S2 form of InsP R-1 (19). In addition, PKG regulation of Ca though biochemically both Ser-1589 and Ser-1755 are equally release only occurs by phosphorylation of Ser-1755. In contrast susceptible to phosphorylation by PKA in S2 InsP R-1 (18), to these data, a recent report (18) has shown that Ser-1589 and only Ser-1755 appears to be functionally relevant in terms of not Ser-1755 is phosphorylated upon activation of PKG. It modulating Ca release (19). Fig. 5A shows the fits for the should be noted that these experiments were performed by normalized concentration-response relationships for CCh-in- using the mouse S1/S2 splice variant of InsP R-1, whereas duced Ca release in cells expressing S2 WT, phosphomi- our functional experiments utilized rat S1/S2 InsP R-1. The metic S2 SE, and S2 SA. The apparent sensitivity of CCh- species and splice variant differences aside, the reported dif- induced Ca release in cells expressing S2 WT was ferences are difficult to reconcile with data demonstrating that essentially identical to S2 WT InsP R-1 (EC for CCh-in- mimicking phosphorylation of Ser-1589 in S2 InsP R-1 does 3 50 3 2 2 duced Ca release; S2 InsP R-1 32.1  4.2 nM versus S2 not influence Ca release (Figs. 5 and 6). Despite these differ- 32.6  7.5 nM). These data are in agreement with the published ences, we performed experiments to define further the phos- literature that indicates that InsP binding and InsP -induced phorylation sites important for regulation of Ca release by 3 3 calcium release is identical in the two major splice variants of PKG. Fig. 7A shows a representative trace from cells express- / Phosphoregulation of S2 InsP R-1 46249 FIG.5. Functional sensitivity of S2 InsP R-1. Concentration-response relationships were generated exactly as described previously. A, the normalized relationships for S2 WT (n 11), S2 SE (n 6), and S2 SA (n 5) are shown. The fits illustrate the increased sensitivity of the S2 SE relative to S2 WT and S2 SA constructs. B, the nor- malized concentration-response relation- ship for S2 ES is shown with S2 SE and S2 WT (dotted lines for compari- son). These data indicate that the sensi- tivity of S2 ES is similar to S2 WT. ing S2 WT InsP R-1 thus illustrating the marked potentia- phosphomimetic constructs used in this study reveal the intrin- tion of Ca release upon specific activation of PKG by 8-Br- sic, functionally important sites in a manner independent of cGMP. A similar striking potentiation of Ca release was also the particular cellular context because any targeting step is observed in cells expressing S2 ES (Fig. 7B and pooled data circumvented. These sites and the functional consequences of Fig. 7D), again presumably as Ser-1755 is phosphorylated fol- phosphoregulation are thus likely a general property of the lowing incubation with 8-Br-cGMP. In contrast, in cells ex- InsP R-1. In addition, the particular sites in S2 InsP R-1 are 3 3 pressing S2 SE no effect on CCh-induced Ca release follow- entirely consistent with our earlier study (19) of the sites func- ing activation of PKG was observed (Fig. 7C and pooled data in tionally important in enhanced Ca release following PKA or Fig. 7D). These data provide evidence that it is unlikely that PKG phosphoregulation, and this reinforces the view that phosphorylation of Ser-1589 by PKG either in isolation or fol- phosphorylation of S2 InsP R-1 Ser-1589 has no significant lowing phosphorylation of Ser-1755 plays a role in modulation role, at least in terms of Ca release. 2 2 Ca release through S2 InsP R-1. However, we cannot rule Effect of InsP R Phosphorylation on Ca Signaling 3 3 out that phosphorylation of this residue impacts other pro- Events—In many nonelectrically excitable cell types, the phys- cesses, such as protein interactions or clustering important for iological mode of Ca signaling is through the generation of InsP R-1 function. Ca oscillations (1, 38). Moreover, it is a generally held view The specific pattern of phosphorylation occurring upon stim- that the spatial and temporal properties of Ca oscillations ulation of PKA or PKG could be cell type-specific as, for exam- make an important contribution to defining the fidelity and ple, in the case of PKA as a result of the targeting through specificity of Ca signaling. In many current models address- protein A-kinase anchoring proteins, as has been demonstrated ing the mechanism underlying Ca oscillations, a key feature recently (37) for InsP R. Alternatively, cell-specific effects is the regulation of Ca release through InsP R. We therefore 3 3 could conceivably occur through restricted access of the kinase next designed experiments to assess the consequences of to its substrate. Notwithstanding the general importance of InsP R-1 phosphorylation (expressed in isolation) on the initi- kinase targeting for efficient, localized phosphorylation, the ation and generation of Ca oscillations. In preliminary ex- / 46250 Phosphoregulation of S2 InsP R-1 FIG.7. Effect of PKG activation in single phosphomimetic con- structs. Threshold CCh-stimulated Ca release is markedly potenti- FIG.6. Effects of PKA phosphorylation on S2 InsP R-1 single ated by activation of PKG following 8-Br-cGMP treatment in S2 WT glutamate substitution constructs. Threshold CCh-stimulated Ca (A)orS2 ES-expressing cells (B). C, in contrast. no potentiation of release is markedly potentiated by activation of PKA following forskolin 2 threshold CCh-stimulated Ca release is observed in S2 SE-express- treatment in S2 WT-expressing cells (A)orS2 ES (B). C, in contrast, ing cells. D, pooled data illustrate that S2 ES has properties identical no potentiation of threshold CCh-stimulated Ca release is observed in to S2 WT, and thus phosphorylation of this residue following activa- cells expressing S2 SE. D, pooled data illustrate that S2 ES has 2 tion of PKG is unlikely to impact Ca release. Numbers in parentheses properties identical to S2 WT, and thus phosphorylation of this resi- indicate the number of analyzed cells. due is unlikely to impact Ca release. Numbers in parentheses indicate the number of analyzed cells. in Fig. 8. Stimulation with 250 ng/ml -IgM proved to be a periments we failed to initiate Ca oscillations with reproduc- threshold concentration in S2 WT-expressing cells. This de- ible characteristics with CCh in m3R-transfected cells (data not gree of stimulation generally resulted in a single small increase shown). Therefore, we chose to stimulate cells with an -IgM in [Ca ] after a long latency (Fig. 8A, left panel, and pooled antibody and to initiate Ca oscillations through activation of data in Fig. 8, C–E). An identical stimulus in cells expressing the endogenous B cell receptor, phospholipase C- activation, S2 EE, in contrast, resulted in repetitive Ca transients and the formation of InsP . Dt-40 3ko cells expressing either following a much shorter latency, consistent with the increased S2 WT or S2 EE to mimic PKA phosphorylation of the apparent sensitivity of the S2 EE constructs (Fig. 8A, right InsP R-1 were stimulated with various concentrations of -IgM panel, and pooled data Fig. 8, C–E). Most interestingly, stim- antibody as illustrated by the selection of representative traces ulation of S2 WT-expressing cells with 500 ng/ml -IgM an- / Phosphoregulation of S2 InsP R-1 46251 FIG.8. Comparison of Ca oscillations in Dt-40 3ko cells expressing S2 WT or phosphomimetic S2 constructs. Dt-40 3ko cells were transfected with either S2 WT (left panel)orS2 EE construct (middle panel). A, cells were stimulated with a threshold concentration (250 ng/ml) of -IgM antibody to stimulate the B cell receptor. In S2 WT-expressing cells, this resulted in a single Ca transient after a long latency (A, left panel). In contrast, multiple Ca transients were elicited in S2 EE-expressing cells following a shorter latency. B, cells were stimulated with 500 ng/ml -IgM antibody. Cells transfected with either S2 WT or S2 EE exhibited multiple Ca transients of similar frequency, although the initial peak was generally larger and latency shorter in S2 EE-expressing cells. C, the pooled data from cells stimulated with various concentrations of -IgM for frequency of oscillations is shown; D, is shown for latency; E, is shown for the magnitude of the initial peak. Numbers in parentheses indicate the number of analyzed cells. tibody resulted in Ca oscillations with similar frequency to exhibiting a lower threshold for activation by InsP infusion cells expressing S2 EE (Fig. 8B and pooled data C–E). How- (40) or agonist activation (41). ever, the latency before the initiation of an increase in Ca These data using phosphomimetic mutations of InsP R-1 was significantly shorter in S2 EE-expressing cells. In addi- splice variants are in broad agreement with a number of stud- tion, the magnitude of the initial transient was also signifi- ies that have reported increased sensitivity of InsP R-1 activity cantly larger in S2 EE-expressing cells when compared with following phosphoregulation by PKA (19–22, 41, 42). The most WT. Stimulation with 1 g/ml -IgM antibody resulted in tran- important consequence of this increased sensitivity appears to sients that were indistinguishable in terms of latency, fre- be in defining the threshold where a cell will respond to a quency, or initial peak magnitude in S2 EE- or S2 WT- stimulus. Given the almost ubiquitous expression of various expressing cells (pooled data Fig. 8, C–E). Thus, PKA-mediated forms of InsP R-1, this is likely a generally important phenom- phosphorylation, by increasing the sensitivity of the InsP R-1 enon. A number of mechanisms are plausible to explain the to InsP , may define the threshold at which cells begin to increased sensitivity of InsP -induced Ca release. These in- 3 3 oscillate but does not markedly influence the temporal proper- clude modulation of InsP binding, an idea supported by meas- ties of Ca oscillations when initiated. This latter observation urements of InsP R binding in hepatocytes (42) (presumably presumably reflects the fact that the frequency of oscillations is S2 InsP R-1 and InsP R-2). In these studies the apparent 3 3 primarily defined by mechanisms such as Ca feedback (39) affinity of InsP binding was enhanced 2-fold at resting 2 2 rather than the absolute sensitivity of the InsP R-1 to InsP [Ca ], and the [Ca ] necessary for half-maximal stimulation 3 3 within a defined range. These data are largely consistent with of InsP binding was reduced. In contrast, a study of recombi- data from hepatocytes where PKA activation resulted in cells nant S1 S2 InsP R-1 expressed in SF9 cells and reconsti- 3 / 46252 Phosphoregulation of S2 InsP R-1 14. Boehning, D., Mak, D. O., Foskett, J. K., and Joseph, S. K. (2001) J. Biol. tuted into lipid bilayers has reported a similar increase in Chem. 276, 13509–13512 InsP R sensitivity to InsP but that the bell-shaped Ca sen- 3 3 15. Galvan, D. L., and Mignery, G. A. (2002) J. Biol. Chem. 277, 48248–48260 16. Parker, A. K., Gergely, F. V., and Taylor, C. W. (2004) J. Biol. Chem. 279, sitivity of channel opening is not altered following phosphoryl- 23797–23805 ation of the InsP R-1 (20). These findings suggest that modu- 17. Taylor, C. W., and Laude, A. J. (2002) Cell Calcium 32, 321–334 lation of the Ca sensitivity of channel activity is unlikely to 18. Soulsby, M. D., Alzayady, K., Xu, Q., and Wojcikiewicz, R. J. (2004) FEBS Lett. 557, 181–184 account for the increased apparent sensitivity of the receptor, 19. Wagner, L. E., II, Li, W. H., and Yule, D. I. (2003) J. Biol. Chem. 278, at least in this form of the InsP R-1. A number of alternative 45811–45817 mechanisms for altering InsP R-1 sensitivity are conceivable. 20. Tang, T. S., Tu, H., Wang, Z., and Bezprozvanny, I. (2003) J. Neurosci. 23, 403–415 For example, phosphorylation of the receptor could alter the 21. Nakade, S., Rhee, S. K., Hamanaka, H., and Mikoshiba, K. 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