D‐myo‐inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] 3‐kinase, the enzyme responsible for production of D‐myo‐inositol 1,3,4,5‐tetrakisphosphate, was activated 3‐ to 5‐fold in homogenates of rat brain cortical slices after incubation with carbachol. The effect was reproduced in response to UTP in Chinese hamster ovary (CHO) cells overexpressing Ins(1,4,5)P3 3‐kinase A, the major isoform present in rat and human neuronal cells. In ortho‐32P‐labelled cells, the phosphorylated 53 kDa enzyme could be identified after receptor activation by immunoprecipitation. The time course of phosphorylation was very similar to that observed for carbachol (or UTP)‐induced enzyme activation. Enzyme phosphorylation was prevented in the presence of okadaic acid. Calmodulin (CaM) kinase II inhibitors (i.e. KN‐93 and KN‐62) prevented phosphorylation of Ins(1,4,5)P3 3‐kinase. Identification of the phosphorylation site in transfected CHO cells indicated that the phosphorylated residue was Thr311. This residue of the human brain sequence lies in an active site peptide segment corresponding to a CaM kinase II‐mediated phosphorylation consensus site, i.e. Arg‐Ala‐Val‐Thr. The same residue in Ins(1,4,5)P3 3‐kinase A was also phosphorylated in vitro by CaM kinase II. Phosphorylation resulted in 8‐ to 10‐fold enzyme activation and a 25‐fold increase in sensitivity to the Ca2+:CaM complex. In this study, direct evidence is provided for a novel regulation mechanism for Ins(1,4,5)P3 3‐kinase (isoform A) in vitro and in intact cells.
d‐myo‐inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] is generated by hydrolysis of plasma membrane phosphatidylinositol 4,5‐bisphosphate as a consequence of activation of a wide variety of cell surface receptors (Berridge, 1993). Ins(1,4,5)P3 3‐kinase occupies a central position in inositol phosphate metabolism by terminating the Ca2+‐mobilizing effect of Ins(1,4,5)P3 and by generating d‐myo‐inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4]. This molecule is further metabolized to many highly phosphorylated inositol phosphates, some of which show specific biological functions. Ins(3,4,5,6)P4 has been shown to selectively block epithelial Ca2+‐activated chloride channels (Vajanaphanich et al., 1994; Ismailov et al., 1996). Rapid production of Ins(1,3,4,5)P4 in response to receptor activation has been observed in various cell types, such as rat brain cortical slices (Batty et al., 1985; Challiss and Nahorski, 1990) and human platelets (King and Rittenhouse, 1989). There is experimental evidence for a number of possible second messenger roles for Ins(1,3,4,5)P4, e.g. in Ca2+ homeostasis (Changya et al., 1989; Irvine, 1991). Moreover, the recent cloning and characterization of a specific high affinity Ins(1,3,4,5)P4 binding protein from pig platelets demonstrated that it corresponds to a member of the GTPase activating protein (GAP) 1 family (Ras‐GAP1IP4BP). This protein showed GAP activity towards Ras and was specifically stimulated by Ins(1,3,4,5)P4 (Cullen et al., 1995).
cDNAs encoding rat and human brain Ins(1,4,5)P3 3‐kinase A (50–53 kDa) have been isolated (Choi et al., 1990; Takazawa et al., 1990b, 1991a). Evidence has been provided to show high expression of isoform A in neuronal cells of the cortex, hippocampus and cerebellum in both rat and human (Mailleux et al., 1991, 1992). Rat and human Ins(1,4,5)P3 3‐kinases A show 93% amino acid sequence identity and polyclonal antibodies to the purified rat brain isoenzyme A also recognize the human isoform A (Takazawa et al., 1991a). cDNAs encoding an isoenzyme, i.e. Ins(1,4,5)P3 3‐kinase B, have been isolated from human and rat cDNA libraries (Takazawa et al., 1991b; Thomas et al., 1994; Vanweyenberg et al., 1995).
Both indirect and direct evidence suggests that Ins(1,4,5)P3 3‐kinase is controlled by various mechanisms. Ca2+ regulates Ins(1,3,4,5)P4 production in lysed thymocytes and in intact cells stimulated with concanavalin A (Zilberman et al., 1987). In the lacrimal acinar cells, acetylcholine activates Ca2+‐dependent K+ channels even when Ins(1,4,5)P3 perfused into the same cells does not (Morris et al., 1987). It was suggested that acetylcholine promotes the production of Ins(1,3,4,5)P4 and that possibly Ins(1,4,5)P3 3‐kinase is stimulated by receptor activation (Irvine et al., 1988). Purified Ins(1,4,5)P3 3‐kinase appeared to be sensitive to the Ca2+:calmodulin (CaM) complex, 2‐fold in rat and human brain (Lee et al., 1990; Takazawa et al., 1990a, b, 1991a) to 17‐fold in human platelets (Communi et al., 1994). Additionally, several potential phosphorylation sites based on consensus phosphorylation site sequences for Ca2+:CaM‐dependent protein kinase II (CaM kinase II), as well as for protein kinase C (PKC) and cAMP‐dependent protein kinase (PKA), are present in the primary structure of rat and human Ins(1,4,5)P3 3–kinases A and B (Takazawa et al., 1990b, 1991a, b).
At least three reports have suggested that Ins(1,4,5)P3 3‐kinase could be a substrate of protein kinase(s): in Jurkat cells (Imboden and Pattison, 1987); in rat hepatocytes (Biden et al., 1988); in human platelets (King and Rittenhouse, 1989). A small increase (180% maximum) in Ins(1,4,5)P3 3‐kinase activity in cells exposed to a given agent was observed in the three reports. For example, in Jurkat cells, stimulation of intact cells through the antigen receptor led to a 1.8‐fold increase in the Vmax of Ins(1,4,5)P3 3‐kinase. The effect was reproduced when cells were treated with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) (Imboden and Pattison, 1987). The mechanism involved has not yet been described: no direct evidence for protein phosphorylation has been shown for Ins(1,4,5)P3 3‐kinase regulation and these studies are difficult to interprete, since the isoform expressed in these cells has not yet been identified. Transformation of Rat‐1 fibroblasts with the v‐src oncogene led to a 6‐ to 8‐fold increase in Ins(1,4,5)P3 3‐kinase activity (Johnson et al., 1989). It was recently shown that this could be achieved by increasing the level of isoenzyme A expression without any tyrosine phosphorylation (Woodring and Garrison, 1996).
In vitro experiments have demonstrated that rat brain Ins(1,4,5)P3 3‐kinase A is a substrate for PKC‐ and PKA‐mediated phosphorylation (Sim et al., 1990). PKC caused a strong inhibition of enzyme activity, whereas PKA provoked only a very slight stimulation of enzyme activity. Phosphorylation of the enzyme by CaM kinase II has not yet been investigated. In this paper we report for the first time that Ins(1,4,5)P3 3‐kinase A is the target of a regulatory mechanism involving CaM kinase II‐mediated phosphorylation both in vitro and in intact cells. Treatment of rat brain cortical slices with carbachol provoked an increase in phosphorylation of Ins(1,4,5)P3 3‐kinase A and a corresponding increase in enzyme activity. These events have been reproduced in Chinese hamster ovary (CHO) cells transfected with Ins(1,4,5)P3 3‐kinase A in response to the purinoreceptor agonist UTP. The phosphorylation site has been identified as Thr311 in the human sequence, which lies in one of the two putative consensus phosphorylation sites for CaM kinase II previously identified in the primary structure of the enzyme.
Okadaic acid‐sensitive transient activation of Ins(1,4,5)P3 3‐kinase A in response to Ca2+‐raising agents
Carbachol is a well‐known agonist for muscarinic cholinoreceptors in rat brain cortical slices. It mediates enhancement of PLC activity, the production of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and intracellular calcium mobilization. Incubation of rat brain cortical slices with carbachol provoked a transient increase in Ins(1,4,5)P3 3‐kinase activity, i.e. 3‐ to 5‐fold as compared with basal activity after 3 min incubation with the agonist (Figure 1A). Maximal enzyme activation was achieved at 10–50 μM carbachol (Table I). Additionally, DNA corresponding to human brain Ins(1,4,5)P3 3‐kinase A has been transfected into CHO cells to obtain a stable cell line overexpressing Ins(1,4,5)P3 3‐kinase activity, i.e. 16 ± 5 nmol/min/mg when assayed at 10 μM Ins(1,4,5)P3. Western blot analysis of the transfected CHO cells demonstrated the presence of a unique 53 kDa protein immunodetected with polyclonal antobodies to rat brain Ins(1,4,5)P3 3‐kinase A (not shown). Ca2+ mobilization resulting from increased production of Ins(1,4,5)P3 has been shown in CHO cells in response to purinoreceptor P2Y2 activation (Iredale and Hill, 1993). Activation of the P2Y2 receptor with UTP provoked a rapid increase in Ins(1,4,5)P3 3‐kinase activity, i.e. 3‐ to 5‐fold at 15–30 s (Figure 1B). Maximal activation was observed at 10–50 μM UTP (Table I). Since UTP and ATP are equipotent agonists for the P2Y2 receptor subtype, ATP reproduced the effects of UTP (Table I). Initial experiments suggested that a critical phosphatase inhibitor had to be added to our homogenization buffer (buffer A; see Materials and methods) to observe maximal stimulation of enzyme activity, in this case okadaic acid (30 nM) and not NaF (100 mM) or sodium vanadate (1 mM) (not shown). Preincubation with okadaic acid for 30 min of cerebral cortex slices and transfected CHO cells before receptor activation provided maximal and a more sustained activation of Ins(1,4,5)P3 3‐kinase (i.e. 5‐fold; Table I). Incubation with ionophore A23187 (150 nM) for up to 5 min or thapsigargin (for 3 min) up to 5 μM also provoked activation of Ins(1,4,5)P3 3‐kinase (Table II). In both cell systems activation of Ins(1,4,5)P3 3‐kinase was related to an increase in Vmax but not to a change in the apparent Km value for Ins(1,4,5)P3 (Km = 4 ± 1 μM).
Activation of Ins(1,4,5)P3 3‐kinase was specifically inhibited by CaM kinase II inhibitors
No modulation of Ins(1,4,5)P3 3‐kinase activity was observed after incubation of cortical slices or transfected CHO cells with TPA or after preincubation in the presence of calphostin C (a potent inhibitor of PKC) before agonist stimulation (Table II). Forskolin did not provoke any change in Ins(1,4,5)P3 3‐kinase activity (Table II). However, preincubation of cortical slices or transfected CHO cells with increasing concentrations (up to 2 μM) of two potent CaM kinase II inhibitors, KN‐93 and KN‐62 (for 30 and 20 min respectively), prevented agonist (carbachol or UTP respectively)‐mediated activation of Ins(1,4,5)P3 3‐kinase (Table III).
Activation of Ins(1,4,5)P3 3‐kinase A is mediated by phosphorylation
Rat cortical slices (Figure 2) and transfected CHO cells (Figure 3) were prelabelled with ortho‐32P and incubated with an agonist (carbachol or UTP respectively) to stimulate enzyme activity. Ins(1,4,5)P3 3‐kinase was immunoprecipitated and analysed by SDS–PAGE. Enzyme activation coincided with phosphate incorporation into the 53 kDa protein band (Figures 2 and 3). Maximal 32P incorporation occurred after incubation of cerebral cortex slices with 10 μM carbachol for 3 min (Figure 2A) or after incubation of CHO cells with 10 μM UTP for 30 s (Figure 3A). Preincubation with okadaic acid before receptor activation potentiated phosphate incorporation into the 53 kDa enzyme in both cell systems (Figures 2A and 3A). Thapsigargin (up to 5 μM) reproduced the effect of carbachol on Ins(1,4,5)P3 3‐kinase phosphorylation (Figure 2B). Moreover, preincubation with KN‐93 or KN‐62 before receptor activation prevented 32P incorporation into the enzyme in a dose‐dependent manner (Figures 2B and 3B). TPA, calphostin C and forskolin had no effect on enzyme phosphorylation (not shown).
Enzyme phosphorylation increased sensitivity of Ins(1,4,5)P3 3‐kinase to the Ca2+:CaM complex
Ins(1,4,5)P3 3‐kinase A was purified from transfected CHO cells (stimulated or not with 50 μM UTP for 30 s) by CaM–Sepharose chromatography (not shown). The specific activity of the enzyme purified from quiescent transfected CHO cells was 1.7 μmol/min/mg when assayed at 5 μM Ins(1,4,5)P3. A major 53 kDa protein band could be revealed by Coomassie Blue protein staining (Figure 4A) or immunodetection (not shown). The maximal stimulation factor by the Ca2+:CaM complex was identical between non‐phosphorylated and in vivo phosphorylated enzyme (i.e. 2‐ to 2.5‐fold). The CaM concentration–response curve of phosphorylated Ins(1,4,5)P3 3‐kinase A was shifted to the left (Figure 4B): in the presence of 10 μM free Ca2+, half‐maximal stimulation of Ins(1,4,5)P3 3‐kinase activity was reached at 52 nM CaM for non‐phosphorylated enzyme and at 2 nM CaM for the enzyme activated by in vivo phosphorylation. The same increase in CaM sensitivity was observed for crude in vivo phosphorylated Ins(1,4,5)P3 3‐kinase from rat cortical slices or transfected CHO cells stimulated by carbachol or UTP respectively (not shown).
In vitro phosphorylation at one unique site and activation of Ins(1,4,5)P3 3‐kinase A by CaM kinase II
Since in vivo phosphorylation of Ins(1,4,5)P3 3‐kinase A was inhibited by potent CaM kinase II inhibitors, we investigated in vitro phosphorylation by CaM kinase II of Ins(1,4,5)P3 3‐kinase A purified from unstimulated transfected CHO cells. The data have been compared with in vitro PKC‐mediated enzyme phosphorylation. Ins(1,4,5)P3 3‐kinase A is a substrate for PKC (Sim et al., 1990). In the presence of phosphatidylserine and diacylglycerol, PKC provoked a 90% loss of enzyme activity (Figure 5A; Sim et al., 1990). CaM kinase II‐catalysed phosphorylation resulted in an increase in Ins(1,4,5)P3 3‐kinase activity in the presence of 10 μM free Ca2+ and 2 μM CaM, i.e. 8‐ to 10‐fold as compared with basal activity measured after preincubation in the absence of CaM kinase II, Ca2+ and CaM. Ca2+:CaM‐mediated stimulation was 2‐ to 2.5‐fold (Figure 5A). In vitro phosphorylation of Ins(1,4,5)P3 3‐kinase A by CaM kinase II also provoked a 25‐fold increase in CaM sensitivity (not shown). Additionally, stoichiometric measurements indicated that the enzyme was phosphorylated at one unique residue, which reached a plateau after 2 min at 37°C (Figure 5B). The in vitro phosphorylated residue was identified after chymotryptic digestion of purified Ins(1,4,5)P3 3‐kinase A phosphorylated by CaM kinase II in the presence of [γ‐32P]ATP. Peptides were separated by reverse phase HPLC (not shown) and the unique radioactive peptide was sequenced. The peptide sequence (Glu‐His‐Ala‐Gln‐Arg‐Ala‐Val‐Thr‐Lys‐Pro‐Arg‐Tyr) corresponded to amino acids 304–315 of human brain Ins(1,4,5)P3 3‐kinase A (Takazawa et al., 1991a). An estimate of the radioactivity at each cycle of Edman degradation showed the phosphorylated residue to be Thr311 (Table IV).
Identification of the in vivo phosphorylated residue
Ins(1,4,5)P3 3‐kinase A purified from ortho‐32P‐labelled, UTP‐stimulated (at 50 μM for 30 s) transfected CHO cells was used for chymotryptic digestion and reverse phase HPLC to isolate a unique radioactive peptide (Figure 6). Figure 6 also shows that no major radioactive peak was observed after chymotryptic digestion and reverse phase HPLC of enzyme isolated from labelled but unstimulated CHO cells. After repurification of the 32P‐labelled peptide, an estimate of the radioactivity at each cycle of Edman degradation demonstrated that the in vivo phosphorylated residue was Thr311 (Table IV).
Data provided mainly by Irvine and co‐workers suggest that both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are necessary to control Ca2+ entry in mouse lacrimal cells (Morris et al., 1987; Changya et al., 1989). Synergistic effects of low micromolar concentrations of Ins(1,3,4,5)P4 and sub‐optimal concentrations of a non‐metabolizable Ins(1,4,5)P3 analogue in causing Ca2+ release have been reported in mouse lymphoma cells (Cullen et al., 1990; Loomis‐Husselbee et al., 1996). The data therefore suggest, at least in these cells, a second messenger function for Ins(1,3,4,5)P4. Evidence has been provided in pancreatic acinar cells for the rapid generation of first Ins(1,4,5)P3 and then Ins(1,3,4,5)P4. A maximal increase in Ins(1,3,4,5)P4 production occured within 15–30 s (Trimble et al., 1987). In rat cerebral cortex slices an Ins(1,3,4,5)P4 mass assay also allowed the observation of a 15‐ to 25‐fold increase in Ins(1,3,4,5)P4 after stimulation with carbachol (Challiss and Nahorski, 1990). It was suggested, for example in mouse lacrimal cells, that if acetylcholine causes stimulation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4, it could be that Ins(1,4,5)P3 3‐kinase is stimulated by receptor activation (Irvine et al., 1988). This could be driven by at least two types of mechanisms: direct activation by the Ca2+:CaM complex and/or, as shown here, by phosphorylation of Ins(1,4,5)P3 3‐kinase. The first mechanism is quite general and has been reported in a large number of tissues and cell types (Yamaguchi et al., 1987; Biden et al., 1988; Takazawa et al., 1988; Communi et al., 1994). Mutagenesis studies permitted the localization of a basic amphiphilic α‐helix‐like site necessary for CaM binding, which is in the N‐terminal region close to the large C‐terminal catalytic domain (Takazawa and Erneux, 1991; Erneux et al., 1993). The stimulation factor is, however, variable, depending on the expression of possible distinct isoenzymes. Isoforms A and B show a conserved C‐terminal catalytic domain but rather different N‐terminal regulatory domains (Takazawa et al., 1990b, 1991a, b). This situation is very much comparable with the various isoenzymes of the CaM‐dependent cyclic nucleotide phosphodiesterases, where the activation characteristics by the Ca2+:CaM complex and by phosphorylation are different for each isoform and splice variant (Beltman et al., 1993; Yan et al., 1996).
The present study demonstrates that Ins(1,4,5)P3 3‐kinase A is a substrate for CaM kinase II both in vitro and in intact cells. This was shown both in rat cerebral cortex slices stimulated by carbachol and in Ins(1,4,5)P3 3‐kinase A‐transfected CHO cells stimulated by UTP. The presence of Ins(1,4,5)P3 3‐kinase A (50–53 kDa protein band on a SDS gel) in rat brain as the major expressed isoform has been previously reported (Takazawa et al., 1990b; Mailleux et al., 1991; Vanweyenberg et al., 1995). When cells were preincubated with 32P, evidence was provided that CaM kinase II inhibitors (KN‐93 and KN‐62) prevented phosphate incorporation into the 53 kDa protein after immunoprecipitation. Phosphorylation in both cell systems was protected in the presence of okadaic acid, suggesting that the phosphorylated enzyme could be a substrate for protein phosphatase 1 or 2A (Cohen et al., 1990). More directly, Ins(1,4,5)P3 3‐kinase was phosphorylated in vitro by CaM kinase II at the level of one unique residue. The in vitro and in vivo unique phosphorylated residue (from transfected CHO cells) was Thr311 (in the primary structure of the human enzyme). This residue is one of the two predicted phosphorylation consensus sites for CaM kinase II previously identified in the sequence of Ins(1,4,5)P3 3‐kinase A (Arg‐Ala‐Val‐Thr311 for consensus site Arg/Lys‐X‐X‐Ser/Thr; Soderling, 1990; Takazawa et al., 1990b). This residue is conserved in the primary structure of human and rat Ins(1,4,5)P3 3‐kinase B (Takazawa et al., 1991b; Thomas et al., 1994; Vanweyenberg et al., 1995), suggesting that this phosphorylation mechanism is general for the two isoforms. The phosphorylated residue is localized in a 15 amino acid peptidic segment (Ala309–Glu320; Takazawa and Erneux, 1991), at a C‐terminal position near the CaM binding site and forming part of the ATP/Mg2+ binding domain (Communi et al., 1995). Phosphorylation of Ins(1,4,5)P3 3‐kinase A increased both enzyme activity and sensitivity to the Ca2+:CaM complex. This mechanism was also observed for CaM‐dependent cyclic nucleotide phosphodiesterase, however, for this enzyme phosphorylation of PDE1A2 by CaM kinase II on Ser120 decreased the binding affinity of CaM (Florio et al., 1994). Although rat Ins(1,4,5)P3 3‐kinase A is also a substrate for PKC and PKA (Sim et al., 1990; unpublished data), TPA, calphostin C and forskolin did not induce any change in Ins(1,4,5)P3 3‐kinase activity nor 32P incorporation in cerebral cortex slices or transfected CHO cells. Overexpressed Ins(1,4,5)P3 3‐kinase A was also not tyrosine phosphorylated, as was recently reported for Rat 1 v‐src‐transformed cells (Woodring and Garrison, 1996). CaM kinase II, a multifunctional enzyme which catalyses phosphorylation of many proteins, has a wide tissue distribution, but is particularly abundant in brain (Schulman and Hanson, 1993). Regulation of several CaM‐dependent proteins by CaM kinase II has been reported. For example, phosphorylation of calcineurin decreased phosphatase activity by decreasing the Vmax or by increasing the Km, depending on the substrate utilized (Hashimoto et al., 1988). The mammalian AMP‐activated protein kinase, a global regulator of carbon metabolism, is activated by two distinct mechanisms, as shown in our study for Ins(1,4,5)P3 3‐kinase: AMP‐activated protein kinase is activated allosterically by 5′‐AMP, which is also required for phosphorylation by AMP‐activated protein kinase kinase. This produced a >50‐fold activation on top of the 5‐fold activation due to the allosteric mechanism (Corton et al., 1994).
What could be the physiological importance of CaM kinase II‐mediated activation of Ins(1,4,5)P3 3‐kinase A? CaM kinase II is a major neuronal protein playing a significant role in the cellular process of long‐term potentiation and depression, as well as vesicular release of neurotransmitters. We previously reported high levels of Ins(1,4,5)P3 3‐kinase A in the dendrites of hippocampal CA1 pyramidal cells, in the dorsal lateral septum and in the dendrites of cerebellar Purkinje cells. This suggested the involvement of Ins(1,4,5)P3 3‐kinase in long‐term potentiation and long‐term depression of synaptic transmission (Mailleux et al., 1991). The effect of KN‐62, which suppresses CA1 region long‐term depression, could be interpreted as due to a decrease in Ins(1,3,4,5)P4 production (Ito et al., 1991). In another study, application of Ins(1,3,4,5)P4 to normal hippocampal slices mimicked deterioration of ischaemic neurons in an extracellular Ca2+‐dependent manner and application of antibodies against rat brain Ins(1,4,5)P3 3‐kinase A, which blocks the formation of Ins(1,3,4,5)P4, protected against cell deterioration (Tsubokawa et al., 1994). It was suggested that formation of Ins(1,3,4,5)P4 plays a critical role in neuronal death and that Ins(1,3,4,5)P4 acts as a signal inducing Ca2+ entry. Interestingly, Ras‐GAP1IP4BP is also expressed at the highest levels in the hippocampus and cerebellum (Baba et al., 1995). Our data suggest that in these cells, Ins(1,3,4,5)P4 levels could be controlled by CaM kinase II‐dependent phosphorylation of Ins(1,4,5)P3 3‐kinase. In one report, KN‐62 provided neuroprotection against NMDA‐ and hypoxia‐induced cell death in fetal rat cortical cultures (Hajimohammadreza et al., 1995). Ca2+ measurements in single neurons indicated that KN–62 produced a reduction in intracellular Ca2+ concentration in response to NMDA. The NMDA receptor itself, voltage‐sensitive Ca2+ channels or, as suggested here, the enzyme responsible for Ins(1,3,4,5)P4 production could be potential targets of CaM kinase II.
Multimeric CaM kinase II exhibits physical and regulatory properties that might enable it to decode the pulsatile nature of Ca2+ signals. Each kinase subunit exists in multiple functional states controlled by autophosphorylation and that differ in their activity and rate of deactivation following a brief Ca2+ spike (Schulman et al., 1992; Hanson et al., 1994). This may possibly enable stimulus frequency‐dependent activation of CaM kinase II, activation of Ins(1,4,5)P3 3‐kinase A and control of Ins(1,3,4,5)P4 production.
Regulation of Ins(1,4,5)P3 3‐kinase by CaM kinase II could be as important as regulation of cyclic nucleotide phosphodiesterase isoenzymes by phosphorylation mechanisms. This could be achieved in addition to multiple allosteric regulatory controls (Burns et al., 1996). In the reaction catalysed by Ins(1,4,5)P3 3‐kinase, phosphorylation could in turn modulate Ins(1,3,4,5)P4 levels and its further metabolism to highly phosphorylated inositol phosphates. This could be particularly relevant in different brain areas, which produce high Ins(1,3,4,5)P4 levels as compared with many other cell types. Whether this mechanism also occurs in cells expressing a different Ins(1,4,5)P3 3‐kinase isoenzyme is currently being studied in our laboratory.
Materials and methods
Preparation of rat brain cortical slices
Cerebral cortex slices (∼350×350 μM) were prepared from freshly decapitated adult male Sprague–Dawley rats and preincubated for 1 h at 37°C in 5 vol KHB medium (116 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 25 mM NaHCO3, 1.2 mM KH2PO4, 11 mM glucose, 25 mM HEPES–NaOH, pH 7.4) under slight agitation.
Stable expression of human brain Ins(1,4,5)P3 3‐kinase A in CHO cells
The human Ins(1,4,5)P3 3‐kinase A coding sequence (Takazawa et al., 1991a) was subcloned using the BamHI and EcoRI restriction sites of the pcDNA3 expression vector (Invitrogen) for transfection into CHO cells using the calcium phosphate precipitation method (Gottesman, 1987). Selection for transfected cells was by addition of fresh complete medium (Ham's F‐12 medium supplemented with 10% fetal calf serum, 1% fungizone and 2% penicillin/streptomycin) containing 400 μg/ml geneticin sulfate G418 in an atmosphere of 5% CO2 at 37°C. After death of all non‐transfected cells, 16 geneticin‐resistant clones were isolated, four of which had high Ins(1,4,5)P3 3‐kinase activity, ranging from 10‐ to 50‐fold over non‐transfected CHO cells. The positive clone overexpressing the highest enzyme activity was used in all experiments reported here. Cell culture medium, dishes and antibiotics were from Gibco.
Cortical slices, cell incubations and Ins(1,4,5)P3 3‐kinase activity assay
Aliquots (100 μl) of packed cortical slices (2–3 mg protein) were incubated at 37°C in flat‐bottomed vials with 1 ml prewarmed KHB medium containing the agent(s). Incubations were terminated by aspirating the incubation medium prior to washing the slices twice in incubation medium. Slices were homogenized in 300 μl ice‐cold buffer A (10 mM Tris–HCl, pH 7.5, 150 mM KCl, 12 mM 2‐mercaptoethanol, 0.5% Nonidet P‐40, 100 mM NaF, 30 nM okadaic acid, 1 mM sodium vanadate, 20 mM benzamidine, 0.1 mM Pefabloc, 5 μM leupeptin and 10 μg/ml calpain inhibitors I and II) using a glass homogenizer. When the CHO cells were ∼80% confluent (3–4×106 cells in 3 ml culture medium), they were washed twice with 2 ml prewarmed KRH medium (124 mM NaCl, 5 mM KCl, 1.25 mM MgSO4, 1.45 mM CaCl2, 1.25 mM KH2PO4, 8 mM glucose, 25 mM HEPES–NaOH, pH 7.4). An aliquot of 2 ml of the same prewarmed medium containing the agent(s) was pipetted into each culture dish. Cell incubations were terminated by aspirating the incubation medium prior to rapidly rinsing the cells twice with KRH medium. Cells were harvested by scraping with a rubber policeman in 300 μl buffer A. Final cell lysates were obtained by three cycles of freeze/thawing. Ins(1,4,5)P3 3‐kinase activity was measured at 5 μM Ins(1,4,5)P3 (Takazawa et al., 1988). The Km value for Ins(1,4,5)P3 was estimated by measuring initial velocities in the presence of 0–50 μM Ins(1,4,5)P3 and by a non‐linear least squares curve fitting of substrate–velocity relationships (Marquardt–Levenberg algorithm). Sensitivity of the enzyme to CaM was measured by assaying enzyme activity at 5 μM Ins(1,4,5)P3, 1 mM EGTA, 10 μM free Ca2+ and increasing concentrations of CaM (0–1 μM). The free Ca2+ concentration was calculated using the dissociation constant of the Ca2+:EGTA complex. Okadaic acid, sodium orthovanadate, NaF, ATP, UTP, carbachol, TPA, thapsigargin, Nonidet P‐40 and leupeptin were from Sigma. Pefabloc was from Pentapharm. Calpain inhibitors I and II were from Boehringer. KN‐93, KN‐62, calphostin C and forskolin were from Calbiochem. [3H]Ins(1,4,5)P3 (15–30 Ci/mmol) was from NEN Dupont. SDS–PAGE, Western blotting and immunodetection were performed as described previously (Takazawa et al., 1990a).
Cortical slices, cell labelling and enzyme immunoprecipitation
Slices (100 μl aliquots) were incubated in KHB medium containing carrier‐free [32P]orthophosphate (1 mCi/ml; Amersham) for 2 h at 37°C. Slices were then washed five times with 20 vol. prewarmed KHB and an aliquot of 1 ml of this medium containing the agent(s) was added. When CHO cells were ∼80% confluent in 6 cm diameter culture dishes, they were washed twice and incubated for 2 h in Dulbecco's MEM pyrophosphate‐free medium supplemented with carrier‐free [32P]orthophosphate (1 mCi/ml). The cells were subsequently washed in prewarmed KRH medium and an aliquot of 2 ml of this medium containing the agent(s) was pipetted into each culture dish for incubation with an agonist. Crude slice and cell extracts were prepared as described above. Ins(1,4,5)P3 3‐kinase A was immunoprecipitated using protein A–Sepharose (Pharmacia) coupled to anti‐rabbit IgG (Sigma) and rabbit polyclonal anti‐rat brain Ins(1,4,5)P3 3‐kinase A antibodies (Takazawa et al., 1990a). An aliquot of 90 μl slice or cell extract (∼650 μg protein) was immunoprecipitated in the presence of 25 μl pretreated protein A–Sepharose and 10 μl immune (or preimmune) serum. Immune complexes were separated by SDS–PAGE and detected by autoradiography using Hyperfilm‐MP (Amersham) exposed for 24–32 h.
Preparative labelling and affinity purification of Ins(1,4,5)P3 3‐kinase A from transfected CHO cells
Transfected CHO cells were cultured in monolayers in square cell culture dishes (22×22 cm) as described above, until the cells were ∼80% confluent. Then cells were labelled with carrier‐free [32P]orthophosphate (0.25 mCi/ml) before washing and incubating as described above. Labelled cells were stimulated in the presence or absence of 50 μM UTP for 30 s (eight dishes for each cell incubation condition). The crude cell extracts were applied in the presence of 1 mM CaCl2 onto 36 ml CaM–Sepharose (Pharmacia). Purified Ins(1,4,5)P3 3‐kinase A was eluted in the presence of 2 mM EGTA, 1% Triton X‐100 (Boehringer) with phosphatase and protease inhibitors as described before (Communi et al., 1994). Ins(1,4,5)P3 3‐kinase activity was assayed for each fraction, from which a 10 μl aliquot was counted for 32P radioactivity. Fractions presenting the highest Ins(1,4,5)P3 3‐kinase activity (six fractions, 50 ml) were concentrated to obtain a final 150 μl sample (∼90 μg purified enzyme).
In vitro enzyme phosphorylation by CaM kinase II and PKC
Phosphorylation of purified human brain Ins(1,4,5)P3 3‐kinase A by CaM kinase II was performed at 37°C for 5 min in HEPES–NaOH, pH 7.4, 10 mM MgCl2, 1 mM ATP, 1 mM EGTA, 12 mM 2‐mercaptoethanol, 5 μg Ins(1,4,5)P3 3‐kinase in the presence or absence of 50 ng rat brain CaM kinase II (Calbiochem), 2 μM CaM and 10 μM free Ca2+ in a final volume of 50 μl. Enzyme phosphorylation by rat brain PKC was carried out in 20 mM HEPES–NaOH, pH 7.4, 10 mM MgCl2, 1 mM ATP, 12 mM 2‐mercaptoethanol, 5 μg Ins(1,4,5)P3 3‐kinase in the presence or absence of 100 ng PKC, 10 μM free Ca2+, 0.25 mg/ml phosphatidylserine and 20 μg/ml 1,2‐diacylglycerol (Sigma) at 37°C for 5 min in a final volume of 50 μl. Phosphorylation samples were stopped at 4°C and immediately diluted with ice‐cold enzyme dilution buffer before assaying enzyme activity. In the case of radioactive phosphorylation, reactions were carried out in the presence of 100 μM [γ‐32P]ATP (final activity ∼50 μCi/ml) instead of 1 mM ATP and stopped in SDS sample buffer before SDS–PAGE. In order to measure CaM kinase II‐induced 32P incorporation into Ins(1,4,5)P3 3‐kinase A, enzyme (5 μg) was phosphorylated in the presence of 100 μM [γ‐32P]ATP (final activity ∼200 μCi/ml) for various times (0–5 min) with 2 μM CaM and 10 μM free Ca2+. After each incubation time, the sample was spotted onto P81 phosphocellulose (Whatman), precipitated and washed in the presence of 75 mM phosphoric acid before counting radioactivity (Roskoski, 1979). Preparative in vitro phosphorylation was carried out at 37°C for 15 min in 20 mM HEPES–NaOH, pH 7.5, 100 μM [γ–32P]ATP (final activity ∼250 μCi/ml), 10 mM MgCl2, 12 mM 2–mercaptoethanol, 90 μg Ins(1,4,5)P3 3‐kinase A, 150 ng rat brain CaM kinase II, 2 μM CaM and 10 μM free Ca2+ in a final volume of 200 μl. The reaction was stopped at 4°C before chymotryptic digestion.
Reverse phase HPLC purification and microsequencing of in vivo and in vitro phosphopeptide
Purified enzyme samples (90 μg) after in vivo cell labelling and stimulation and after in vitro radioactive CaM kinase II‐promoted phosphorylation respectively were digested for 16 h at 30°C with 50 mM Tris–HCl, pH 8.0 and 2 μg α‐chymotrypsin (Sigma). Chymotryptic fragments of 32P‐labelled Ins(1,4,5)P3 3‐kinase A were separated by reverse phase HPLC using an Alltech C18 column (2.1×250 mm), as reported previously (Communi et al., 1996). Each peak detected at an absorbance of 214 nm was collected separately and a 5 μl aliquot was counted to estimate the 32P radioactivity. The unique in vivo phosphorylated 90 μl peak fraction and the unique in vitro phosphorylated 70 μl peak fraction were concentrated respectively and repurified by reverse phase HPLC in the presence 0.1% trifluoroacetic acid. A 5 μl aliquot of each peak fraction was counted for radioactivity. The amino acid sequence of the two labelled peptides was determined by Edman degradation using an Applied Biosystems model 477A peptide sequencer. The amino acid phenylthiohydantoin derivatives were collected in an internal fraction collector and counted for radioactivity to identify the labelled amino acid residue.
We would like to thank Dr R.Lecocq for experimental help. We are grateful to C.Vanhoutte for providing rats. Rat brain PKC was a generous gift from Mark H.Rider (ICP, UCL, Brussels, Belgium). This research was supported by grants from Actions de Recherche Concertées, the FRSM., Boehringer Ingelheim and The Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State Prime Minister's Office, Federal Service for Science, Technology and Culture. D.C. is Chargé de Recherche at the Fonds National pour la Recherche Scientifique.
- Copyright © 1997 European Molecular Biology Organization