Calcium mobilization via type III inositol 1,4,5-trisphosphate receptors is not altered by PKA-mediated phosphorylation of serines 916, 934 and 1832

Abstract

Several studies have shown that PKA-mediated phosphorylation of IP₃R1 at serines S¹⁵⁸⁸ and S¹⁷⁵⁵ enhances the receptor's ability to mobilize Ca²⁺. In contrast, much less is known about whether Ca²⁺ mobilization via IP₃R2 and IP₃R3 is regulated by PKA. We report here that IP₃R2 is only very weakly phosphorylated in response to PKA activation and is probably not a physiological substrate for this kinase. IP₃R3, however, is known to be phosphorylated by PKA at three sites (S⁹¹⁶, S⁹³⁴ and S¹⁸³²) and, thus, we examined how phosphorylation of these sites affects Ca²⁺ mobilization in DT40-3KO cells stably expressing either exogenous wild-type or mutant IP₃R3s; an antibody raised against phospho-serine 934 of IP₃R3 was used to demonstrate that the exogenous IP₃R3s are strongly phosphorylated in response to PKA activation. Surprisingly, our data show that IP₃R3-mediated Ca²⁺ mobilization is unaffected by phosphorylation of S⁹¹⁶, S⁹³⁴ and S¹⁸³². In contrast, phosphorylation of exogenous IP₃R1 (monitored with an antibody against phospho-serine 1755) enhances Ca²⁺ mobilization, indicating that DT40-3KO cells have the capacity to respond to phosphorylation of IP₃Rs. Overall, these data suggest that modification of Ca²⁺ flux may not be the primary effect of IP₃R3 phosphorylation by PKA.

1. Introduction

Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are a family of proteins which form Ca²⁺ channels in endoplasmic reticulum (ER) membranes and govern Ca²⁺ release from this organelle by opening in response to IP₃ and Ca²⁺ binding [1-3]. In mammals, type I, II and III IP₃ receptors (IP₃R1, IP₃R2, and IP₃R3) are highly homologous and have the same overall structure; with an N-terminal “ligand-binding domain”, a C-terminal “channel domain”, and an intervening “coupling domain” containing putative binding sites for several regulatory factors [1-3]. Despite these similarities, however, expression of IP₃R types varies between tissues [4, 5] and subtle differences in their functional properties, such as their regulation by IP₃, Ca²⁺ and ATP, are apparent [3, 6-8]. Functional differences between the three receptor types may also arise from their differential phosphorylation by cAMP-dependent protein kinase (PKA) [9-11] but, with the exception of IP₃R1, there is no clear consensus regarding the functional consequences of these phosphorylation events.

PKA phosphorylates IP₃R1 at two sites (S¹⁵⁸⁸ and S¹⁷⁵⁵) [12-14] and studies on IP₃R1 expressed in IP₃R-null DT40-3KO cells (hereafter referred to as 3KO cells) indicate that this phosphorylation enhances IP₃R1-mediated Ca²⁺ release [15, 16], a finding consistent with biophysical studies on purified IP₃R1 reconstituted into lipid bilayers [17]. In contrast, PKA-mediated phosphorylation of IP₃R2 has not been examined in detail, and while IP₃R3 is known to be phosphorylated at three sites (S⁹¹⁶ , and S⁹³⁴ and S¹⁸³²) [18], the effects of phosphorylation at these sites has not been examined. Attempts to ascribe functional effects to PKA-mediated IP₃R2 and IP₃R3 phosphorylation, however, have been made by activating PKA in cells that express relatively high levels of each receptor type. Enhancement of Ca²⁺ signaling in parotid acinar cells, for example, has been attributed to IP₃R2 phosphorylation [19]. In contrast, inhibition of Ca²⁺ signaling has been attributed to phosphorylation of IP₃R3 in pancreatic acinar cells [20, 21]. While these findings suggest that IP₃R2 and IP₃R3 might be differentially affected by PKA, the presence of multiple IP₃R types and other PKA-regulated proteins in these systems makes it impossible to unequivocally attribute effects on Ca²⁺ signaling to the phosphorylation of one particular IP₃R type.

It should be possible to overcome these difficulties using 3KO cells [22], as exogenous wild-type and mutant IP₃Rs can be expressed and analyzed without interference from endogenous receptors [15, 16]. Thus, our initial goal was to define the effects of PKA activation on IP₃R2 and IP₃R3-mediated Ca²⁺ mobilization in 3KO cells. However, as our preliminary studies showed that IP₃R2 is phosphorylated only very weakly by PKA, we focused on IP₃R3. We report here that, surprisingly, phosphorylation of S⁹¹⁶, S⁹³⁴ and S¹⁸³² has no effect on IP₃R3-medicated Ca²⁺ mobilizatior in transfected 3KO cells. In contrast, phosphorylation of IP₃R1 enhances Ca²⁺ signaling, demonstrating that the 3KO cell-based system has the capacity to respond to IP₃R phosphorylation.

2. Materials and methods

2.1 Materials

CT1, CT2 and CT3, affinity purified rabbit antisera specific to the C-termini of IP₃R1, IP₃R2 and IP₃R3, respectively, have been described previously [4]. Anti-IgM (clone M-4), a mouse monoclonal antibody directed against chicken IgM, was from Southern Biotechnology Associates. Trypsin was from Sigma (bovine pancreas; #T4665). TL3, a mouse monoclonal antibody directed against amino acids 22–230 of IP₃R3, was from BD Biosciences. DT40 and 3KO cells [22] were kindly provided by Dr. D. Yule (University of Rochester, NY, U.S.A) with the permission of Dr. T. Kurosaki (Kansai Medical University, Japan).

2.2 IP₃R cDNAs

Mouse SI+/SII+ IP₃R1 (a kind gift from Dr, K. Mikoshiba, The University of Tokyo, Japan), rat IP₃R2 (a kind gift from Dr. G. Mignery, Loyola University, Chicago, IL, U.S.A.) and rat IP₃R3 (a kind gift from Dr. G. Bell, University of Chicago, Chicago, IL, U.S.A.) cDNAs have been described elsewhere [14, 23-25]. To facilitate transfection of 3KO cells, the coding region of wild-type rat IP₃R3 was excised from its original vector backbone [24] with NotI and was ligated into the multiple cloning region of correspondingly cut pcDNA3 (Invitrogen). The mutation of PKA phosphorylation sites was then performed as described [18].

2.3 Generation of phospho-IP₃R antibodies

Anti-pS934 and anti-pS1755 were raised in rabbits as described [4] against the synthetic peptides C-V-L-R-K-Q-phosphoS-V-F and C-S-G-R-R-E-phosphoS-L-T-S, which correspond to amino acids 929-936 of rat IP₃R3 [24] and 1750-1758 of mouse IP₃R1 [25], respectively, with an additional cysteine at the N-termini to facilitate coupling reactions. Crude antisera were affinity-purified on peptide-coupled SulfoLink columns (Pierce). Briefly, columns were incubated with antisera overnight at 4 °C and washed with 1 M guanidine HCl, followed by 50 mM Tris, pH 7.4. Specifically bound antibodies were eluted from the columns with 4.5 M MgCl₂ in 50 mM Tris-HCl, pH 7.4, containing 0.1% bovine serum albumin. The eluate was then dialyzed against 155 mM NaCl in 50 mM Tris-HCl, pH 7.4, for 2 hr at room temperature and then overnight at 4 °C against the same buffer supplemented with 10% glycerol and 0.005% NaN₃.

2.4 Cell culture

DT40 and 3KO cell lines were cultured at 40°C, under 5% CO₂ in L-glutamine-containing RPMI 1640 (Mediatech Inc) supplemented with 1% chicken serum, 10% fetal bovine serum and antibiotics (100 units penicillin and 100 μg streptomycin/ml). Human embryonic kidney (HEK) 293 and African green monkey COS kidney cells were cultured as described [4]. The concentration of DMSO, used as a vehicle for forskolin and 3-isobutyl-1-methylxanthine (IBMX), was less than 1% in all experiments.

2.5 Transfection of IP₃R cDNAs

Stable transfection of 3KO cells was achieved by electroporation at 550 V and 25 μF (4-mm gap cuvette) using a BioRad GenePulser XCell. Briefly, 10 million cells were incubated for 10 min on ice in 800 μl of phosphate buffered saline (PBS) containing 25 μg of IP₃R cDNA linearized in the non-coding region. The mixture was then electroporated, incubated for 10 min on ice, and incubated overnight in 25 ml culture medium. Finally, the cells were centrifuged, re-suspended in 80 ml culture medium supplemented with 2 mg/ml G418, seeded into 96-well plates, and grown for 7-10 days prior to selection of G418-resistant clones for expansion. Transient transfection of HEK and COS cells was performed as described [14].

2.6 IP₃R phosphorylation in intact cells

Phosphorylation of IP₃Rs was analyzed using [³²P]P_i-labeled HEK cells, as described [14], or through immunoblotting of electrophoresed cell lysates or immunoprecipitated IP₃Rs (the ∼200-300 kDa regions of gels are shown). Cell lysates were prepared via solubilization with ice-cold, phosphatase-inhibitor-supplemented lysis buffer [14] and were either probed directly, or phosphorylated IP₃Rs were immunoprecipitated by overnight incubation at 4°C with Protein A–Sepharose CL-4B beads and anti-pS1755 or anti-pS934. Immune complexes were then electrophoresed and probed with IP₃R1-specific CT1 or IP₃R3-specific TL3. To ensure that the conditions for measurement of IP₃R phosphorylation were the same as those used in Ca²⁺ mobilization experiments (see section 2.8), DT40 or transfected 3KO cells were incubated with 3 μM Fura-2AM, washed, and resuspended in Krebs-HEPES buffer (KHB) [26] prior to stimulation.

2.7 Immunofluorescence microscopy

COS cells were seeded on poly-l-lysine-coated glass coverslips (500,000 cells/ 9.6 cm²), transfected with 1 μg IP₃R3^SSS cDNA, stimulated, fixed with 1% paraformaldehyde in PBS for 1 hr at room temperature, washed with PBS, incubated at −20°C for 10 min with methanol, rinsed for 10 sec with ice-cold acetone, and re-hydrated in PBS for 30 min at room temperature. Fixed cells were then incubated for 45 min in blocking buffer (10% goat serum, 0.1% BSA in PBS) before overnight incubation at 4°C in blocking buffer supplemented with primary antibody (TL3 or anti-pS934). Following three washes in PBS, the cells were incubated for 45 min at room temperature in blocking buffer supplemented with secondary antibody (FITC- or TRITC-conjugated goat anti-rabbit IgG, respectively). Finally, the fixed cells were washed three times, stained with 100 ng/ml 4'-6-diamidino-2-phenylindole (DAPI) for 5 min, washed three more times, and coverslips were mounted using Vectashield reagent (Vector Labs Inc). Images were acquired using a Zeiss Axioplan 2 microscope equipped with a 63x oil immersion objective (Plan Apochromat from Zeiss).

2.8 Ca²⁺ mobilization

Five million cells were harvested by centrifugation (2000 x g for 10 min), re-suspended in 1 ml culture medium, and incubated with 3 μM Fura-2AM for 20 min at 37°C. Following three washes with KHB, the cells were incubated for 10 min at 37°C in 1 ml KHB to allow for de-esterfication of Fura-2AM, were washed once more, resuspended in 2 ml KHB, and excited at 340 and 380 nm and emission intensity at 509 nm was recorded with an LS-50B luminescence spectrometer (Perkin-Elmer). Cytoplasmic Ca²⁺ concentration ([Ca²⁺]i) was calculated as described [27] utilizing 0.1% Triton X-100 and 7 mM EGTA to obtain R_max and R_min, respectively. All combined data are presented as mean ± S.E.M from ≥ 3 independent experiments except as noted. Values of “[Ca²⁺]i increase” were calculated by subtracting basal [Ca²⁺]i values from [Ca²⁺]i values recorded ∼60 s after stimulation with anti-IgM or ∼15 s after trypsin, and are expressed as a percentage of maximum; the average response (n ≥ 3) to a maximal dose of stimulus in the absence of forskolin plus IBMX. Statistical significance was then assessed using the unpaired Student's t test; asterisks indicate when p < 0.05.

3 Results

3.1 PKA-mediated phosphorylation of IP₃Rs in intact cells

As our overall goal was to define the effects of PKA-mediated phosphorylation of IP₃Rs on Ca²⁺ signaling in intact cells, we first sought to define which of the receptors are phosphorylated. We, and others, have already shown that IP₃R1 and IP₃R3 are phosphorylated by PKA in intact cells; at Ser¹⁵⁸⁸ and Ser¹⁷⁵⁵ in IP₃R1 [12-14], and Ser⁹¹⁶, Ser⁹³⁴ and Ser¹⁸³² in IP₃R3 [18]. The extent to which IP₃R2 is phosphorylated, however, is not clear. To determine this, we examined exogenous IP₃R2 expressed in [³²P]P_i-labeled HEK cells stimulated with 10 μM forskolin, a condition that results in maximal and equivalent phosphorylation of exogenous IP₃R1 and IP₃R3 [14, 18]. Fig 1A demonstrates that IP₃R2 (lanes 1-2) was only very weakly phosphorylated under conditions that allowed for substantial phosphorylation of IP₃R3 (lanes 3-4) and IP₃R1 [14, 18]. Fig 1B shows that there was ∼90% less radioactivity associated with exogenous IP₃R2 than IP₃R3. Thus, IP₃R2 is a very poor substrate for PKA and we did not examine it further. Rather, we focused on elucidating the functional effects of PKA-dependent IP₃R3 phosphorylation.

Figure 1. Phosphorylation of IP₃Rs in intact cells.

(A) [³²P]P_i-labeled HEK cells transfected with cDNAs encoding wild-type IP₃R2 (lanes 1-2), or IP₃R3 (lanes 3-4) were exposed to 10 μM forskolin for 5 min as indicated, and IP₃Rs were immunoprecipitated with CT2 or CT3, electrophoresed and assessed for Coomassie blue staining capacity (upper panel) and associated radioactivity (lower panel). Negligible staining and radioactivity was obtained from control (vector-transfected) cells (not shown) [14, 18]. (B) Histogram of the data presented in A. Exogenous IP₃R phosphorylation was calculated by subtracting the negligible radioactivity obtained from control cells from that in lanes 1-4 and is expressed as a percentage of the radioactivity associated with IP₃R3 in the presence of forskolin (lane 4). Data shown are representative of three independent experiments.

3.2 Validation of anti-pS934

To facilitate the study of IP₃R3 phosphorylation, we developed an antibody against phospho-S⁹³⁴ of IP₃R3, the predominant site of PKA-mediated phosphorylation [18]. The specificity of this antibody, termed anti-pS934, was demonstrated in immunoblots of cell lysates prepared from HEK cells expressing exogenous wild-type and mutant IP₃R3s (Fig 2A), a system in which we have previously characterized IP₃R3 phosphorylation using [³²P]P_i [18]. Anti-pS934 exhibited minor immunoreactivity towards wild-type IP₃R3 (IP₃R3^SSS; lane 3) and IP₃R3 mutated at S⁹¹⁶ and S¹⁸³² (IP₃R3^ASA; lane 9) in the absence of stimulus, and this was increased substantially under conditions of maximal IP₃R phosphorylation (lanes 4 and 10). Anti-pS934 did not, however, exhibit any immunoreactivity towards IP₃R3 mutated at S⁹³⁴ (IP₃R3^SAS; lanes 7-8), IP₃R3 mutated at S⁹¹⁶, S⁹³⁴, and S¹⁸³² (IP₃R3^AAA; lanes 5-6), or wild-type IP₃R1 (IP₃R1^SS; lanes 11-12) in the absence or presence of forskolin, demonstrating that it is entirely specific towards IP₃R3 phosphorylated at S⁹³⁴. This was confirmed by immunofluorescence microscopy of transfected COS cells. Exogenous IP₃R3^SSS was recognized by TL3 in ∼50% of the cells, as indicated by punctate staining throughout the cytoplasm (panels v and vi), and anti-pS934 produced a similar signal in IP₃R3^SSS-expressing cells after the addition of forskolin (panel viii). Thus, anti-pS934 is specific and is effective in both immunoblots and immunofluorescence (Fig 2), and subsequent studies (Fig 3) show it also to be effective in immunoprecipitations.

Figure 2. Validation of anti-pS934.

(A) HEK cells transfected with empty vector (lanes 1-2), or cDNA encoding wild-type IP₃R3 (IP₃R3^SSS; lanes 3-4), IP₃R3 mutated at S⁹¹⁶, S⁹³⁴, S⁹¹⁶, and S¹⁸³² (IP₃R3^AAA; lanes 5-6), mutated at S⁹³⁴ (IP₃R3^SAS; lanes 7-8), mutated at S⁹¹⁶ and S¹⁸³² (IP₃R3^ASA; lanes 9-10) or wild-type IP₃R1 (IP₃R1^SS; lanes 11-12) were incubated with or without 10 μM forskolin for 5 min as indicated, and lysates were prepared and probed with anti-pS934 (upper panel), TL3 (lower panel; lanes 1-10), or CT1 (lower panel; lanes 11-12). (B) COS cells transfected with cDNA encoding IP₃R3^SSS were incubated without (upper panels) or with (lower panels) 10 μM forskolin for 10 min, fixed, and stained with DAPI to visualize nuclei (panels iii and iv), TL3 to visualize IP₃R3 (panels v and vi), and anti-pS934 to visualize IP₃R3 phosphorylated at S⁹³⁴ (panels vii and viii).

Figure 3. Phosphorylation of IP³R3s expressed in 3KO cells.

(A) DT40 cells (lanes 1-2), 3KO cells (lanes 3-4), or IP₃R3-expressing 3KO cells (lanes 5-12) were exposed to 1 μM forskolin plus 200 μM IBMX for 10 min as indicated, lysates were prepared, and IP₃R3s phosphorylated at S⁹³⁴ were immunoprecipitated with anti-pS934. Total IP₃R3 in lysates prior to immunoprecipitation (upper panel) or phosphorylated IP₃R3 in immunoprecipitates (lower panel) were assessed in immunoblots with TL3. (B) 3KO-SSS cells were exposed to 0-100 μM forskolin in the absence (upper panel) or presence (lower panel) of 200 μM IBMX for 10 min, and IP₃R3 phosphorylation was assessed as in A. (C) IP₃R3^SSS-expressing HEK cells were exposed to 0.01-3.0 μM forskolin for 10 min and IP₃R3 phosphorylation was assessed using anti-pS934 in immunoblots of cell lysates. (D) Traces of typical Ca²⁺ responses to forskolin (added at t = 0.5 min) in 3KO cells (dashed line) and 3KO-SSS cells (solid lines). (E) 3KO-SSS cells were incubated with or without 1μM forskolin plus 200 μM IBMX for 10 min as indicated and levels of IP₃R3 in cell lysates prior to (pre) and following (post) immunoprecipitation of phosphorylated IP₃R3 with anti-pS934 were assessed in immunoblots with TL3. The lack of difference in signal intensity in control lysates pre and post immunoprecipitation (lanes 1 and 2) shows that IP₃R3^SSS is not significantly phosphorylated, whereas the difference in signal intensity in stimulated cells (lanes 3 and 4; a reduction of ∼50%) shows that at least 50% of IP₃R3^SSS is phosphorylated.

3.3 Phosphorylation of IP₃R3s expressed in 3KO cells

To study the effects of phosphorylation on IP₃R3-mediated Ca²⁺ signaling, we chose to use transfected 3KO cells, since they provide a system to examine Ca²⁺ signaling mediated exclusively via exogenous IP₃Rs, free from the interference of endogenous IP₃Rs [15, 16]. As expected, endogenous IP₃R3 was present in DT40 cells (Fig 3A, upper panel, lanes 1-2), but was absent from 3KO cells (lanes 3-4). Stable expression of exogenous IP₃R3^SSS (3KO-SSS, lanes 5-6), IP₃R3^SAS (3KO-SAS, lanes 7-8), IP₃R3^ASA (3KO-ASA, lanes 9-10), and IP₃R3^AAA(3KO-AAA, lanes 11-12) was achieved at levels comparable to, or above, endogenous IP₃R3. To examine whether these exogenous receptors could be phosphorylated, cell lysates were incubated with anti-pS934 and immunoprecipitated IP₃R3s were visualized with TL3 (Fig 3A, lower panel). This showed that IP₃R3^SSS (lane 6) and IP₃R3^ASA (lane 10) were phosphorylated at S⁹³⁴ upon PKA activation and that, as expected, IP₃R3^SAS (lane 8) or IP₃R3^AAA (lane 12) were not. Surprisingly, we did not detect phosphorylation of endogenous IP₃R3 (lanes 1-2), most likely because anti-pS934 does not recognize phosphorylated chicken IP₃R3, due to minor sequence differences in the antibody epitope.

We next set out to define the basic characteristics of IP₃R3 phosphorylation in 3KO-SSS cells. Fig 3B (upper panel) shows, surprisingly, that forskolin was not a potent stimulus and that very little phosphorylation of IP₃R3 was detected at doses of forskolin (10-30 μM) considered supramaximal in many mammalian cell lines; e.g. maximal phosphorylation of exogenous IP₃R3 in HEK cells (Fig 3C) or endogenous IP₃R3 in Rat-1 fibroblasts (data not shown) occurs at 1 μM forskolin. However, inclusion of IBMX (200 μM), an inhibitor of cAMP phosphodiesterases, greatly enhanced IP₃R3 phosphorylation in 3KO-SSS cells (Fig 3B, lower panel) and reduced the concentration of forskolin required for maximal IP₃R3 phosphorylation to ∼10 μM.

The use of IBMX to enhance IP₃R3 phosphorylation proved to be essential for the studies on Ca²⁺ signaling in 3KO-derived cells, as we found that forskolin alone caused Ca²⁺ mobilization in 3KO-SSS cells at concentrations of 10 μM and above (Fig 3D). The cause of this mobilization remains unknown, but appears to reflect IP₃R-independent Ca²⁺ release from intracellular stores, as it still occurred in 3KO cells (Fig 3D), and in the absence of extracellular Ca²⁺ (data not shown). Thus, to study the effects of IP₃R3 phosphorylation on Ca²⁺ mobilization, we chose to use 1 μM forskolin plus 200 μM IBMX, as this condition causes near maximal IP₃R3 phosphorylation (Fig 3B), but did not itself cause Ca²⁺ mobilization (Figs 4-5). Further, we could demonstrate that at least 50% of exogenous IP₃R3^SSS was phosphorylated by forskolin plus IBMX under these conditions (Fig 3E).

Figure 4. Effect of PKA activation on IP₃R3-mediated Ca²⁺ mobilization in response to BCR stimulation.

(A) Traces of typical Ca²⁺ responses to 5, 1, 0.05 and 0.005 μg/ml anti-IgM (added at t = 0.5 min) in DT40 (i) or 3KO-SSS (ii) cells. Dose-response curves (iii) for DT40 and 3KO-SSS cells are expressed as a percentage of the [Ca²⁺]i increase in response to 5 μg/ml anti-IgM. Error bars may be occluded by symbols. (B) Traces of typical Ca²⁺ responses in DT40 (i-iii) or 3KO-SSS (iv-vi) cells exposed to 5 (i and iv), 1 (ii and v), or 0.05 (iii and vi) μg/ml anti-IgM (added at t = 10.5 min) in the absence (solid line) or presence (broken line) of 1 μM forskolin plus 200 μM IBMX (added at t = 0.5 min). Histogram (vii) of [Ca²⁺]i increases in DT40 or 3KO cell lines in response to 5, 1, or 0.05 μg/ml anti-IgM in the absence (clear bars) or presence (solid bars) of forskolin plus IBMX. (C) Typical traces of Ca²⁺ responses in 3KO-SSS cells (i) exposed to 5mM EGTA (added at t = 10 min) and 1 μg/ml anti-IgM (added at t = 10.5 min) in the absence (solid line) or presence (broken line) of 1μM forskolin plus 200 μM IBMX (added at t = 0.5 min). Histogram (ii) of [Ca²⁺]i increases in 3KO-SSS and 3KO-AAA cells in the absence (clear bars) or presence (solid bars) of forskolin plus IBMX.

Figure 5. Effect of PKA activation on IP₃R1-mediated Ca²⁺ mobilization in response to BCR stimulation.

(A) HEK cells transfected with empty vector (lanes 1-2), or cDNA encoding wild-type IP₃R1 (IP₃R3^SS; lanes 3-4), IP₃R1 mutated at S¹⁷⁵⁵ (IP₃R3^SA; lanes 5-6), mutated at S¹⁵⁸⁸ (IP₃R3^AS; lanes 7-8), mutated at S¹⁵⁸⁸ and S¹⁷⁵⁵ (IP₃R1^AA; lanes 9-10), or wild-type IP₃R3 (IP₃R3^SSSS ; lanes 11-12) were exposed to 10 μM forskolin for 5 min as indicated, lysates were prepared, and IP₃R1 phosphorylated at S¹⁷⁵⁵ were immunoprecipitated with anti-pS1755 and visualized in immunoblots with CT1. (B) DT40 (lanes 1-2), or IP₃R1-expressing 3KO (lanes 3-6) cells were exposed to 1 μM forskolin plus 200 μM IBMX for 10 min as indicated, cell lysates were prepared, and IP₃R1 phosphorylated at S¹⁷⁵⁵ was immunoprecipitated with anti-pS1755 and visualized in immunoblots with CT1 (lower panel). IP₃R1 levels in lysates prior to immunoprecipitation were assessed with CT1 (upper panel). (C) 3KO-SS cells were incubated with 1μM forskolin plus 200 μM IBMX for 10 min and levels of IP₃R1 in cell lysates prior to (pre) and following (post) immunoprecipitation of phosphorylated IP₃R1 with anti-pS1755 were assessed in immunoblots with CT1. The ∼50% reduction in signal intensity post immunoprecipitation indicates that at least 50% of IP₃R1 is phosphorylated. (D) Traces of typical Ca²⁺ responses to 5 μg/ml anti-IgM (added at t = 10.5) in the absence (solid line) or presence (broken line) of 1 μM forskolin plus 200 μM IBMX (added at t = 0.5 min) in 3KO-SS cells. (E) Histogram of [Ca²⁺]i increases in 3KO-SS and 3KO-AA cells in response to 5 μg/ml or 1 μg /ml anti-IgM in the absence (clear bars) or presence (solid bars) of forskolin plus IBMX.

3.4 Effects of IP₃R3 phosphorylation on BCR-mediated Ca²⁺ mobilization

Stimulation of DT40 cells with anti-IgM results in endogenous B cell receptor (BCR) activation, PLCγ-mediated generation of IP₃, and IP₃R-mediated Ca²⁺ mobilization [22, 28]. Fig 4A shows typical Ca²⁺ responses to various doses of anti-IgM in DT40 (i) and 3KO-SSS (ii) cells, and dose-response curves (iii). Clearly, exogenous IP₃R3^SSS (ii) couples BCR activation to Ca²⁺ mobilization essentially identically to endogenous IP₃Rs (i), with the exception that 3KO-SSS cells are slightly less sensitive to anti-IgM than DT40 cells (EC₅₀ = 0.70 ± 0.06 and 0.50 ± 0.04 μg/ml, respectively). This minor difference may be explained by the fact that, in addition to IP₃R3, DT40 cells express IP₃R1 and IP₃R2 [22], both of which have a higher affinity for IP₃ than IP₃R3 [6, 7, 22]. 3KO-SSS, 3KO-AAA, 3KO-SAS and 3KO-ASA cells exhibited virtually identical maximum responses to anti-IgM (544 ± 40, 570 ± 52, 533 ± 37 and 522 ± 48 nM Ca²⁺, respectively) and anti-IgM dose-response curves (EC₅₀ = 0.70 ± 0.04, 0.72 ± 0.08, 0.79 ± 0.10, and 0.72 ± 0.06 μg/ml), demonstrating that basal phosphorylation of S⁹¹⁶, S⁹³⁴ and S¹⁸³² does not affect IP₃R3-mediated Ca²⁺ mobilization.

To determine the effects of increased IP₃R3 phosphorylation on anti-IgM - induced Ca²⁺ mobilization, we stimulated cells with a high (5 μg/ml), medium (1 μg/ml) and low (0.05 μg/ml) dose of anti-IgM, 10 min after addition of either vehicle or 1 μM forskolin plus 200 μM IBMX, a condition which phosphorylates at least 50% of IP₃R3s in transfected 3KO cells (Fig 3E) and causes phosphorylation of endogenous IP₃Rs in DT40 cells (Fig 5B). Fig 4B shows that treatment with forskolin plus IBMX had no effect on anti-IgM induced Ca²⁺ mobilization in DT40 cells (i-iii), but slightly inhibited Ca²⁺ mobilization in 3KO-SSS cells (iv-vi). However, this inhibition was unrelated to IP₃R3 phosphorylation at S⁹¹⁶, S⁹³⁴ or S¹⁸³², as it occurred to a similar extent in all IP₃R3 mutant-expressing cell lines (vii). It also occurred in 3KO-SSS and 3KO-AAA cells incubated with extracellular EGTA (Fig 4C), indicating that it is due to reduced IP₃R3-mediated Ca²⁺ release from intracellular stores, rather than reduced Ca²⁺ entry. Surprisingly, therefore, the data in Fig 4 demonstrate that phosphorylation of IP₃R3 at S⁹¹⁶, S⁹³⁴ and S¹⁸³² has no effect on Ca²⁺ mobilization in response to BCR activation, but also reveals a slight inhibitory effect of forskolin plus IBMX that is mediated by an, as yet, unidentified mechanism. This inhibition appears to be a consequence of cAMP elevation and/or PKA activation, as it was not seen when cells were incubated with either 1 μM forskolin or 200 μM IBMX alone (data not shown).

3.5 IP₃R1 phosphorylation enhances BCR-mediated Ca²⁺ mobilization

Given the lack of effect of forskolin plus IBMX on Ca²⁺ signaling in DT40 cells and 3KO-SSS cells (Fig 4), we sought evidence that IP₃R phosphorylation can have measurable effects on Ca²⁺ signaling in our experimental system. In this regard, it has been shown previously that PKA-mediated phosphorylation of IP₃R1 at S¹⁵⁸⁸ and/or S¹⁷⁵⁵ enhances Ca²⁺ release in individual IP₃R1-expressing 3KO cells [15, 16]. Thus, PKA activation should also enhance IP₃R1-mediated Ca²⁺ release in our experiments with cell suspensions.

To measure IP₃R1 phosphorylation, we developed an antibody against phospho- S¹⁷⁵⁵. This antibody, termed anti-pS1755, did not recognize phosphorylated IP₃R1 in immunoblots or via immunofluorescence microscopy (not shown), but was effective in immunoprecipitations and was specific towards phospho-S¹⁷⁵⁵ of IP₃R1 (Fig 5A). Thus, we could demonstrate forskolin plus IBMX-induced phosphorylation of endogenous IP₃R1 in DT40 cells (Fig 5B, lane 2, lower panel), and of exogenous IP₃R1^SS in 3KO-SS cells (lane 4, lower panel), but not in 3KO-AA cells (lane 6 lower panel). Further, using the same approach utilized for IP₃R3 (Fig 3E), we could show that at least 50% of the receptors in 3KO-SS cells are phosphorylated in response to forskolin plus IBMX (Fig 5C).

Fig 5D shows that treatment with forskolin plus IBMX enhanced the Ca²⁺ response to 5 μg/ml anti-IgM by ∼30 % in 3KO-SS cells. This effect was also seen at 1 μg/ml anti-IgM, but was not evident in 3KO-AA cells (Fig 5E), indicating that it is a direct consequence of IP₃R1 phosphorylation at S¹⁷⁵⁵ and/or S¹⁵⁸⁸. Thus, IP₃R phosphorylation at defined sites is capable of modulating Ca²⁺ signals in our experimental system.

3.6 Effects of IP₃R phosphorylation on trypsin-mediated Ca²⁺ mobilization

To determine whether activation of Ca²⁺ mobilization via a different signaling pathway might uncover effects of IP₃R3 phosphorylation, we examined Ca²⁺ signaling in response to trypsin, which activates the PAR2 G protein-coupled receptor in DT40 cells [29] and which elevates intracellular Ca²⁺ in our experimental system (Fig 6A). However, as with anti-IgM, trypsin-induced Ca²⁺ mobilization was not affected by forskolin plus IBMX treatment in DT40 cells, and was inhibited slightly by this treatment in both 3KO-SSS and 3KO-AAA cells (Fig 6B). Thus, again, this inhibitory effect is not due to phosphorylation of S⁹¹⁶, S⁹³⁴, or S¹⁸³². Enhancement of trypsin-induced responses by forskolin plus IBMX treatment in 3KO-SS cells, but not in 3KO-AA cells, again confirms that Ca²⁺ mobilization can be regulated by IP₃R phosphorylation (Fig 6B).

Figure 6. Effect of PKA activation on trypsin-induced Ca²⁺ mobilization.

(A) Trace showing a typical Ca²⁺ response to a maximal dose of trypsin (1000 U/ml) in DT40 cells. (B) Histogram of [Ca²⁺]i increases in DT40 and 3KO cell lines in response to 1000 U/ml or 50 U/ml trypsin in the absence (clear bars) or presence (solid bars) of 1 μM forskolin plus 200 μM IBMX.

4. Discussion

The major findings presented herein are (i) that IP₃R2 is a very poor substrate for PKA and (ii) that phosphorylation of S⁹¹⁶, S⁹³⁴ and S¹⁸³² has no effect on the ability of IP₃R3 to mobilize Ca²⁺.

The inability of maximally activated PKA to substantially phosphorylate exogenous IP₃R2 expressed in HEK cells (Fig 1) is consistent with findings that endogenous IP₃R2 in AR42J cells is only very weakly phosphorylated by PKA [9], and that PKA catalytic subunit does not affect IP₃-mediated Ca²⁺ release in permeabilized cells that predominantly express IP₃R2 [11]. Taken together, these data indicate that IP₃R2 is not a physiological substrate for PKA. Therefore, the enhanced Ca²⁺ signaling that has been attributed to PKA-mediated phosphorylation of IP₃R2 in parotid acinar cells [19] is most likely the result of phosphorylation of other proteins, possibly other IP₃R types. Indeed, the evidence for IP₃R2 phosphorylation in parotid acinar cells was that IP₃R2 was immunoprecipitated from forskolin-stimulated cells by an anti-phosphoserine/threonine antibody [19]; however, as parotid cells also express IP₃R1 and IP₃R3 [30], and IP₃Rs heterotetramerize [4, 5], it is entirely plausible that non-phosphorylated IP₃R2 was immunoprecipitated because of its interaction with phosphorylated IP₃R1 and/or IP₃R3.

We have also found that phosphorylation of IP₃R3 at S⁹¹⁶, S⁹³⁴ and S¹⁸³², the only sites in IP₃R3 known to be modified by PKA [18], does not affect the receptor's ability to mobilize Ca²⁺. This conclusion was validated by the positive control that Ca²⁺ mobilization via IP₃R1^SS, but not IP₃R1^AA, was enhanced by PKA activation; thus, 3KO cells contain the requisite proteins to allow for Ca²⁺ signaling via exogenous IP₃Rs to be modified by their phosphorylation. Phosphorylation of IP₃R3 at S⁹¹⁶, S⁹³⁴ and S¹⁸³², therefore, may play a role other than regulation of Ca²⁺ channel activity, such as providing docking sites for ancillary proteins. Interestingly, similar findings have recently been reported for Akt kinase-mediated phosphorylation of IP₃R1; this kinase phosphorylates IP₃R1 at S²⁶⁸¹, but this modification does not affect the receptor's ability to mobilize Ca²⁺ [31].

Surprisingly, we also found that forskolin plus IBMX treatment inhibited Ca²⁺ release by ∼10% for each of the exogenous IP₃R3s, irrespective of whether S⁹¹⁶, S⁹³⁴ or S¹⁸³² were mutated or not. Conceivably, this inhibition is a result of phosphorylation of a previously uncharacterized site in IP₃R3 that is revealed when the receptor is expressed in 3KO cells, although this possibility is unlikely, as S⁹¹⁶, S⁹³⁴ and S¹⁸³² comprise the full complement of sites phosphorylated by PKA in intact HEK cells and in vitro [18]. Alternatively, it could be the effect of an, as yet, unidentified PKA substrate that specifically associates with and inhibits IP₃R3. Either event may go some way to explaining the PKA-mediated inhibition of Ca²⁺ signaling that has been attributed to IP₃R3 phosphorylation in pancreatic acinar cells [20, 21].

The development of anti-pS934 and anti-pS1755 allowed us, for the first time, to characterize the phosphorylation of exogenous IP₃Rs expressed in 3KO cells and endogenous IP₃Rs in DT40 cells. Interestingly, our data revealed that forskolin is a very weak stimulus in these cells and that co-incubation with IBMX is required for efficient IP₃R phosphorylation (Fig 3B), perhaps indicating that DT40-derived cell lines contain high levels of cAMP phosphodiesterase activity. Clearly, this should be taken into account when examining PKA-mediated events in DT40-derived cells. Further, and somewhat surprisingly, while endogenous IP₃Rs are phosphorylated by PKA in DT40 cells (Fig 5B), this does not affect Ca²⁺ signaling (Fig 4). As IP₃R1, IP₃R2 and IP₃R3 are all present in DT40 cells [22], this could reflect the combined effects of enhanced IP₃R1 activity and inhibited IP₃R3 activity. Alternatively, as DT40 cell IP₃R complexes are likely to be heterotetrameric [5], and as nothing is currently known about how phosphorylation affects IP₃R heterotetramers, it is possible that the IP₃R complexes in DT40 cells are unaffected by phosphorylation, even though they contain IP₃R1 and/or IP₃R3.

In summary, we demonstrate the differential phosphorylation of IP₃R types by PKA, with IP₃R2 being only very weakly phosphorylated compared to IP₃R1 and IP₃R3. Further, phosphorylation of IP₃R3 at the sites known to be targeted by PKA (S⁹¹⁶, S⁹³⁴ and S¹⁸³²) does not affect its ability to mobilize Ca²⁺, suggesting that modification of Ca²⁺ flux may not be the primary effect of IP₃R3 phosphorylation.