Biol Res 37: 593-602, 2004

ARTICLE

Modulation of cytosolic calcium signaling by protein kinase A-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors

STEPHEN V. STRAUB, LARRY E. WAGNER II, JASON I.E. BRUCE and DAVID I. YULE

University of Rochester, Department of Pharmacology and Physiology, Rochester, New York 14642 USA

Dirección para Correspondencia

ABSTRACT

Calcium release via intracellular Ca²⁺ release channels is a central event underpinning the generation of numerous, often divergent physiological processes. In electrically non-excitable cells, this Ca²⁺ release is brought about primarily through activation of inositol 1,4,5-trisphosphate receptors and typically takes the form of calcium oscillations. It is widely believed that information is carried in the temporal and spatial characteristics of these signals. Furthermore, stimulation of individual cells with different agonists can generate Ca²⁺ oscillations with dramatically different spatial and temporal characteristics. Thus, mechanisms must exist for the acute regulation of Ca²⁺ release such that agonist-specific Ca²⁺ signals can be generated. One such mechanism by which Ca²⁺ signals can be modulated is through simultaneous activation of multiple second messenger pathways. For example, activation of both the InsP₃ and cAMP pathways leads to the modulation of Ca²⁺ release through protein kinase A mediated phosphoregulation of the InsP₃R. Indeed, each InsP₃R subtype is a potential substrate for PKA, although the functional consequences of this phosphorylation are not clear. This review will focus on recent advances in our understanding of phosphoregulation of InsP₃R, as well as the functional consequences of this modulation in terms of eliciting specific cellular events.

Key words: Inositol 1,4,5-trisphosphate receptor, protein kinase A, calcium signaling, pancreatic acinar cells, parotid acinar cells.

SPECIFIC ACTIVATION OF DISTINCT CELLULAR PROCESSES IS ENCODED IN THE SPATIAL AND TEMPORAL CHARACTERISTICS OF Ca²⁺ SIGNALS

Cytosolic calcium is the most versatile signaling molecule in biology. Changes in the concentration of cytosolic Ca²⁺ ([Ca²⁺]_c) have been found to exert control over a myriad of different cellular processes including, but certainly not limited to, increases in gene transcription (Dolmetsch et al., 1997; Dolmetsch et al., 2001), muscle contraction (Jaggar et al., 2000), and exocytosis (Augustine, 2001). In addition, [Ca²⁺]_c changes are involved in exerting control over a number of opposing physiological processes, including smooth muscle contraction and relaxation (Jaggar et al., 2000), as well as mitochondrial metabolism and apoptotic cell death (Gunter and Gunter, 2001). The control of such a diverse array of cellular functions by a single messenger suggests that specificity to exert control upon an individual effector must exist within the signal transduction system. Indeed, the Ca²⁺ signal itself appears uniquely suited to encode specific information, as diverse Ca²⁺ signals, displaying a wide range of temporal and spatial characteristics, can be generated in a given cell (Cuthbertson et al., 1981; Cuthbertson and Cobbold, 1985; Woods et al., 1986; Woods et al., 1987). Thus, Ca²⁺ signals with a particular spatial and temporal "signature" could potentially underlie the generation of a specific, singular cellular process. Indeed, this assertion has been confirmed in numerous studies to date. For example, it has been shown that Ca²⁺ oscillations of a particular frequency are capable of selectively eliciting the activation Ca²⁺-calmodulin-dependent protein kinase II (De Koninck and Schulman, 1998), while spatially localized Ca²⁺ signals, such as Ca²⁺ influx through L-type Ca²⁺ channels in neurons, are effective at activating transcription factors such as CREB and MEF-2 due to spatial targeting of Ca²⁺-sensitive signal transduction components in the vicinity of the L-channels (Dolmetsch et al., 2001). Therefore, the ability to generate a variety of Ca²⁺ signals allows for the selective modulation of a myriad of cellular effects by a singular signaling molecule (Berridge et al., 2000). Thus, the mechanisms by which Ca²⁺ release events are modulated, such that diverse signaling patterns can be generated, are integral to the universality and specificity inherent in Ca²⁺ signaling.

PHOSPHOREGULATION OF Ca²⁺ RELEASE FROM InsP₃R

In most electrically non-excitable cells, such as hepatocytes and the exocrine cells of the pancreas and parotid salivary gland, Ca²⁺ release from InsP₃R is the major mechanism by which [Ca²⁺]_c changes are brought about (Thomas et al., 1996; Bird et al., 1998). Therefore, regulation of Ca²⁺ release at the level of the InsP₃R is potentially of importance in allowing the cell to generate a multitude of Ca²⁺ signaling patterns. Three subtypes of InsP₃R are expressed in mammalian cells, type I, II, and III (Furuichi et al., 1989a; Furuichi et al., 1989b; Sudhof et al., 1991; Thomas et al., 1996; Patel et al., 1999). In addition, multiple splice variants of the receptors exist. For example, the type I InsP₃R can be spliced at three distinct regions, denoted as SI-SIII, with five unique variants resulting from splicing at the SII site. In terms of expression, two splice variants of the type I receptor appear to predominate. The SII⁺ isoform, also known as the long or neuronal form, is expressed primarily in neuronal tissues, while the SII^- isoform (short form) is expressed primarily in the periphery. Each functional receptor exists as a tetrameric assembly of similar or dissimilar subunits, thus allowing for the formation of homo- or hetero-tetramers. The N-terminal 600 amino acids of each subunit comprise a binding region for InsP₃. Giving the critical requirement for a number of amino acid residues in different regions of this N-terminus, it is believed that the three-dimensional structure of the N-terminus forms a binding pocket for InsP₃. The C-terminal region of the protein comprises the channel domain and, along with the N-terminus, shows high sequence homology across subtypes and species (Patel et al., 1999).

A primary distinction between receptor subtypes is the differential sensitivity to activation by InsP₃ and Ca²⁺. Activation of each subtype requires the binding of InsP₃, but Ca²⁺ itself acts as a co-agonist of the receptor (Kaftan et al., 1997; see also Foskett and Mak, 2004, this issue). Indeed, the binding of calcium to an activation site is thought to be required for channel activation under physiological conditions (Mak et al., 2001). Thus, the distinct InsP₃ and Ca²⁺ sensitivities may result in each subtype being uniquely suited to generate Ca²⁺ signals in different cellular environments. For example, the type III InsP₃R has been proposed to act as a trigger for Ca²⁺ release events, given a less significant effect of Ca²⁺ inhibition on this subtype (Hagar et al., 1998), as well as an enhanced sensitivity to activation by near resting Ca²⁺ levels. While each receptor subtype possesses an inherently different sensitivity to InsP₃ and Ca²⁺, the primary difference between receptor subtypes at the amino acid level, and perhaps functionally as well, exists in the large regulatory domain, which spans the region between the N- and C-termini, and comprises the majority of the protein, being approximately 1600 amino acids in length (Patel et al., 1999). This region contains sites for potential interactions with a range of regulatory factors, including Ca²⁺, ATP, regulatory proteins such as CaM and FKBP, as well as sites for phosphorylation by a range of protein kinases, including PKA, PKG, CaM-kinase, and PKC. Each subtype appears to have a unique susceptibility to modulation, and similar modulatory events appear to have the capacity to evoke different effects dependent upon the receptor subtype involved.

Phosphorylation of InsP₃R has been extensively examined in the type I InsP₃R, particularly in terms of the effects of PKA phosphorylation on Ca²⁺ release from this subtype (Supattapone et al., 1988; Ferris et al., 1991; Wojcikiewicz and Luo, 1998; Tang et al., 2003). Despite this intensive investigation, a consensus does not exist as to the effects of this phosphorylation, although recent findings have enhanced our understanding of this phenomenon. Initial experiments investigating PKA-mediated phosphorylation of type I InsP₃R performed by Ross and colleagues suggested that two PKA phosphorylation sites exist in the type I receptor, at serines 1589 and 1755 (Ferris et al., 1991). Interestingly, serine 1589 was suggested to be phosphorylated in the SII^- (short, peripheral) splice variant, while serine 1755 was phosphorylated in the SII⁺ (long, neuronal) form of the receptor. In a subsequent study, Snyder and colleagues found that PKA phosphorylation of InsP₃R purified from cerebellum (SII⁺) resulted in 10-fold decrease in the sensitivity of the receptor to InsP₃, measured as a decrease in ⁴⁵Ca²⁺ flux from microsomal vesicles (Supattapone et al., 1988). In contrast, studies from Wojcikiewicz and Luo suggested that PKA phosphorylation of the SII⁺ splice variant, presumably at serine 1755, resulted in a modest 20% increase in the sensitivity of the receptor to InsP₃, as measured in permeabilized SH-SY5Y neuroblastoma cells (Wojcikiewicz and Luo, 1998). Compared to the neuronal form of the receptor, there has been much less investigation of effects of phosphorylation on the SII^- splice variant. One elegant study performed by Tertyshnikova and Fein suggested that PKA phosphorylation of this InsP₃R resulted in decreased Ca²⁺ release in response to photorelease of caged InsP₃ in megakaryocytes. This effect on Ca²⁺ release was not due to effects of PKA on Ca²⁺ clearance (Tertyshnikova and Fein, 1998).

A major advance in the study of InsP₃R regulation was the generation of a triple InsP₃R knockout cell line by Kurosaki and colleagues, which functions as the only known null background for InsP₃R (Sugawara et al., 1997). Our lab has utilized this DT40 chicken B-cell line to dissect the molecular loci and functional consequences of PKA phosphorylation on both major splice variants of the type I InsP₃R. Transient transfection of the InsP₃R of interest allows for the study of this receptor in the complete absence of all other InsP₃R subtypes, thus allowing for the study of individual subtypes as well as receptors containing mutations of interest. In cells transfected with the SII⁺ type I InsP₃R, as well as M3 muscarinic receptor and the fluorescent indicator protein HcRed, stimulation with low (nM) concentrations of carbachol (CCh) elicited increases in [Ca²⁺]_c, as measured by fura-2 fluorescence (Fig. 1A) (Wagner et al., 2003). Treatment of cells with forskolin, to raise cellular levels of cAMP and activate PKA, resulted in an enhanced [Ca²⁺]_c increase upon subsequent exposure to agonist. Consistent with previous studies which indicated that serine 1755 was the residue phosphorylated in the neuronal form of the receptor, our studies found that this increased sensitivity to agonist was dependent upon phosphorylation of serine 1755, as [Ca²⁺]_c increases in cells expressing a receptor containing an S1755A point mutation were not affected by forskolin treatment (Fig. 1B). In addition, expression of receptors containing an S1589A mutation still showed the same level of potentiation of the [Ca²⁺]_c signal following forskolin treatment as did wild type SII⁺ receptors (Fig. 1E), suggesting that in the long form of the type I receptor, phosphorylation of serine 1589 is functionally insignificant in terms of effects on Ca²⁺ release. In contrast, in the peripheral (SII^-) form of the type I receptor, phosphorylation of either S1589 or S1755 resulted in enhanced Ca²⁺ release (Fig. 1 C-D,F), suggesting that in this form of the receptor, both potential phosphorylation sites are functionally important. Thus, utilization of the triple InsP₃R knockout DT40 cell line allows for the effects of potential modulatory events occurring at the level of the InsP₃R to be studied with InsP₃R populations of unambiguous composition, as well as InsP₃R of altered molecular composition.

Figure 1. Investigation of PKA-mediated phosphorylation of type I InsP3R expressed in DT40 cells. A) Fura-2 loaded triple InsP3R knockout DT40 cells expressing the SII+ (neuronal) form of the type I InsP3R and M3 muscarinic receptor were stimulated with CCh to elicit an elevation of [Ca2+]c. Only cells positively identified as transfected, as detected by HcRed expression, responded to agonist (black trace). Exposure of cells to forskolin to activate PKA resulted in a potentiated [Ca2+]c increase compared to the control response. B) Mutation of serine 1755 to alanine in the SII+ receptor prevented the effect of PKA activation on the [Ca2+]c increase, suggesting that phosphorylation of this residue is critical in mediating the effects of PKA phosphorylation on this form of the type I receptor. C) [Ca2+]c increases elicited by CCh stimulation prior to and following exposure to forskolin in DT40 expressing the SII- (peripheral) form of type I InsP3R. D) Mutation of both serine residues (1589 and 1755) in the SII- receptor was required in order to inhibit potentiation of the [Ca2+]c increase following PKA activation, suggesting both residues are functionally important for altering Ca2+ release in this form of the receptor. E) Pooled data showing the fold increase in [Ca2+]c (normalized to the control response) following exposure to forskolin for wild type SII+, S1589A and S1755A point mutants of the type I InsP3R. F) Pooled data showing fold increase in [Ca2+]c elicited following treatment with forskolin and CCh (normalized to control) for cells expressing wild type SII-, S1589A or S1755A single point mutants and double (S1589A, S1755A) mutant InsP3R. Adapted from Wagner et al., 2003, with permission. © (2003) The American Society for Biochemistry and Molecular Biology.

SPECIFIC PHYSIOLOGICAL OUTCOMES GENERATED BY InsP₃R PHOSPHORYLATION IN NON-EXCITABLE CELLS

While the use of heterologous expression systems is helpful in determining the effect of InsP₃R phosphorylation on Ca²⁺ release from the channel, receptor modulation in the context of a biological system is likely to have consequences beyond simply altering the rate or magnitude of Ca²⁺ release. Consistent with this assertion, we have found that InsP₃R phosphorylation may be involved in modulating physiological processes in exocrine acinar cells of the pancreas and parotid salivary gland (Bruce et al., 2002; Straub et al., 2002). In parotid acinar cells, where the major physiological function is fluid secretion leading to the generation of the primary saliva, it is known that maximal secretion occurs following stimulation of both sympathetic and parasympathetic pathways (Bobyock and Chernick, 1989; Larsson and Olgart, 1989). Elevation of cAMP was found to result in the generation of a [Ca²⁺]_c signal in response to a previously sub-threshold concentration of agonist, and the conversion of an oscillatory [Ca²⁺]_c signal into a peak-and-plateau type response (Fig. 2 A) (Bruce et al., 2002), suggesting an increased sensitivity of the signal transduction machinery to agonist. This increased sensitivity was not due to a change in the activity of Ca²⁺ clearance mechanisms, or effects on InsP₃ production, Ca²⁺ influx or release from ryanodine receptors, but rather due to a direct effect on InsP₃R, as Ca²⁺ release evoked by InsP₃ was enhanced following elevation of cAMP in permeabilized parotid cells (Bruce et al., 2002).

Similar to most cell types, parotid cells express all three InsP₃R subtypes with the types II and III comprising the majority of receptors and the type I accounting for only 5% of the receptor population (Zhang et al., 1999; Giovannucci et al., 2002). Phosphorylation appears to occur at the level of the type II receptor in this system, as robust phosphorylation was found in immunoprecipitated type II InsP₃R following treatment of cells with forskolin (Bruce et al., 2002). This finding of potentiated Ca²⁺ release following phosphorylation of the type II InsP₃R is consistent with early literature from hepatocytes showing similar effects of type II phosphorylation by PKA (Hajnoczky et al., 1993). In parotid, the effector for the enhanced Ca²⁺ release generated during peak secretion is likely to be one or more plasma membrane resident Ca²⁺-activated ion channels involved in the generation of a transcellular osmotic gradient responsible for fluid movement (Iwatsuki et al., 1985; Kasai and Augustine, 1990). Following a [Ca²⁺]_c increase, Ca²⁺-activated Cl^- channels present on the apical membrane become activated due to the close association between these channels and the InsP₃R, the majority of which are present in the apical region of the cell (Kasai and Augustine, 1990). Activation of these channels leads to Cl^- efflux from the cell into the lumen, establishing an osmotic gradient, which results in the paracellular movement of Na⁺ and water. The potentiated Ca²⁺ signal is also likely to enhance the activity of Ca²⁺-activated K⁺ channels, present on the basolateral membrane, which function to reverse membrane depolarization resulting from Cl^- efflux. Thus, the PKA-mediated potentiation of InsP₃-evoked Ca²⁺ release is able to enhance fluid secretion by altering the activity of the Ca²⁺ sensitive secretory machinery.

Figure 2. Functional effects of InsP3R phosphorylation in exocrine cells. A) Fura-2 loaded parotid acinar cells were stimulated with sub-threshold (30 nM) and oscillatory (100 nM) concentrations of CCh to elicit an increase in [Ca2+]c. [Ca2+]c increases in the apical region of the cell are noted with an arrow while [Ca2+]c changes in the basal region are shown in the unlabeled trace. Exposure to forskolin was found to enhance the CCh-evoked [Ca2+]c increase, such that previously sub-threshold concentrations of agonist now evoked oscillations while previously oscillatory agonist concentrations now evoked a peak-and-plateau type response. B) Stimulation of pancreatic acinar cells with CCh or CCK evokes dramatically different patterns of [Ca2+]c oscillations in fura-2-loaded cells. One characteristic difference between the [Ca2+]c increases elicited by these agonists is the kinetics of the rising phase of the [Ca2+]c oscillations. This difference in the [Ca2+]c responses was found to be due to the PKA-mediated phosphorylation of type III InsP3R produced during stimulation with CCK. C) Whole cell patch clamped, fura-2 loaded pancreatic acinar cells were stimulated with CCK, evoking the typical pattern of baseline [Ca2+]c oscillations associated with this agonist. D) Inclusion of HT31 in the patch pipette, to disrupt the targeting of PKA to AKAP and inhibit the effects of a targeted PKA-mediated phosphorylation event, resulted in a disruption in the normal pattern of oscillations evoked by CCK stimulation. Adapted from Bruce et al., 2002, and Straub et al., 2002, with permission. © (2002) The American Society for Biochemistry and Molecular Biology.

In pancreatic acinar cells, there exists a situation similar to that in parotid acinar cells: the type II and III InsP₃R comprise the majority of the InsP₃R population, while type I receptors make up less than 3% of the receptor complement (Wojcikiewicz, 1995). However, in the pancreas the physiologically relevant phosphorylation event appears to occur at the level of the type III receptor (Straub et al., 2002). In these cells, Ca²⁺ signals are generated in response to a wide range of agonists, and it is well documented that stimulation with different agonists results in the generation of agonist-specific [Ca²⁺]_c signaling patterns (Fig. 2B) (Yule et al.; see also Petersen, this issue). In particular, stimulation with acetylcholine (ACh) results in the generation of frequent [Ca²⁺]_c oscillations (4-6/min) which are superimposed upon an elevated [Ca²⁺]_c plateau, while stimulation with the hormone cholecystokinin (CCK) results in the generation of infrequent oscillations (1/min) which are generated from the baseline [Ca²⁺]_c level, and are thus termed baseline [Ca²⁺]_c oscillations. The agonists appear to utilize similar signal transduction machinery to generate [Ca²⁺]_c signals, as inhibition of the G_q family of heterotrimeric G proteins (Yule et al., 1999) or of Ca²⁺ release from InsP₃R (Thorn et al., 1994; Cancela et al., 1999) prevents [Ca²⁺]_c increases from these agonists. Thus, an agonist specific modulation of a common signal transduction component appeared to be involved in the generation of [Ca²⁺]_c signals to one or both of these agonists. Previous studies had shown that CCK receptors were capable of coupling to G_q as well as G_s family G proteins (Schnefel et al., 1990) and that stimulation with supraphysiological CCK concentrations resulted in the formation of cAMP and activation of PKA (Marino et al., 1993). Thus, we hypothesized that CCK stimulation could result in the PKA-mediated phosphorylation of InsP₃R, leading to an alteration of Ca²⁺ release compared to that which occurs during stimulation with ACh. Consistent with this, it was found that stimulation of acinar cells with physiological concentrations of CCK, which result in baseline [Ca²⁺]_c oscillations, evoked phosphorylation of type III InsP₃R which was dependent upon activation of PKA (LeBeau et al., 1999; Straub et al., 2002). Interestingly, stimulation with ACh did not evoke receptor phosphorylation at agonist concentrations which generate [Ca²⁺]_c oscillations. In terms of functional effects on Ca²⁺ release and the characteristics of the evoked [Ca²⁺]_c signal, phosphorylation of the type III receptor was found to slow the rising phase of [Ca²⁺]_c increases evoked by ACh stimulation or photorelease of caged InsP₃, and this slowing in the kinetics of Ca²⁺ release was found to mimic the difference in the rising phase of [Ca²⁺]_c increases evoked by CCK and ACh (Fig 2B) (Straub et al., 2002). Furthermore, activation of PKA during a train of Ach-evoked oscillations was found to decrease the frequency and maximal amplitude of the oscillations (Giovannucci et al., 2000).

Since all three InsP₃R subtypes are expressed in parotid and pancreatic acinar cells, and all three subtypes have been shown to be substrates for InsP₃R phosphorylation, how is it that a particular subtype is preferentially phosphorylated by PKA? In addition to a cytosolic localization, PKA can also be localized to its substrate through binding to a family of scaffolding proteins known as A kinase anchoring proteins, or AKAPs (Colledge and Scott, 1999). These proteins comprise a growing family of functionally similar but structurally diverse proteins, which, in addition to binding PKA, can form macromolecular complexes containing PKA, several protein phosphatases such as PP1 and PP2A, as well as the substrate for PKA phosphorylation. Each AKAP contains a targeting domain, which directs the protein to a particular subcellular region, allowing PKA to function as a spatially targeted kinase. Thus, targeting of PKA to a particular InsP₃R subtype through binding of an AKAP directly to the channel, or to a docking site in the vicinity of the channel, would allow for the selective phosphorylation of a particular InsP₃R subtype. The binding of PKA to an AKAP occurs through multiple physical interactions between amphipathic helices present in both the RII regulatory subunits of PKA and the AKAP and can be selectively disrupted by peptide known as HT31, which contains the RII binding site from an AKAP expressed in human thyroid (Colledge and Scott, 1999). This peptide can be utilized to dissect the effects of PKA which are due to a targeted kinase versus those effects due to cytosolic PKA. When HT31 was introduced into pancreatic acinar cells via diffusion through a patch clamp pipette, it was found that the typical pattern of baseline oscillations normally associated with CCK stimulation was severely disrupted, such that the oscillations were now elicited upon an elevated [Ca²⁺]_c plateau (Fig. 2C, D) (Straub et al., 2002). Interestingly, HT31 was without effect on oscillations evoked by ACh stimulation, suggesting that a targeted, PKA-mediated phosphorylation event, presumably occurring at the level of the type III InsP₃R, is involved in generating the physiological pattern of [Ca²⁺]_c oscillations associated with CCK stimulation in pancreatic acinar cells. Furthermore, it was found that stimulation with another agonist, bombesin, which activates neuromedin-type receptors and evokes a baseline pattern of [Ca²⁺]_c oscillations similar to those evoked by CCK, also elicited phosphorylation of type III InsP ₃R which was again dependent upon activation of PKA (Straub et al., 2002). These findings suggest that PKA-mediated phosphorylation of InsP₃R may be part of a general mechanism by which a baseline pattern of [Ca²⁺]_c oscillations is evoked in pancreatic acinar cells.

CONCLUSIONS

Overall, these findings highlight one potential mechanism by which InsP₃-evoked Ca²⁺ signals can be shaped such that an array of physiological outcomes can be achieved with a high degree of specificity. Utilization of the DT40 cell line will allow for the dissection of the molecular loci at which phosphorylation occurs in type II and III InsP₃R, as well as for a determination of the functional consequences of these phosphorylation events. These findings will be utilized to enhance our understanding of the mechanisms by which crosstalk between the Ca²⁺ signaling and cAMP pathways contributes to the generation of specific physiological processes in primary tissues.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Trevor J. Shuttleworth for helpful comments on the manuscript. D.I.Y. is supported by grants from NIH (RO1-DK54568, RO1-DE14756, and PO1-DE13539). S.V.S. is supported by an NIH training grant through NIDCR (T32-DE007202).

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Corresponding Author: David I. Yule, Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Rochester, New York 14642 USA. Phone: (1-585) 273-2154, Fax (1-585) 273-2652, E-mail: David_Yule@urmc.rochester.edu

Received: April 5, 2004. Accepted: May 6, 2004.