ology, University of Texas Southwestern Medical Center, 5323 Harry. Hines Blvd. ..... Supattapone, S., Worley, P. E., Baraban, L. M., and Snyder, S. H. (1988). 1.
Vol. 268, No. 15,Issue of May 25, pp. 10997-11001,1993
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S. A.
Ca2+-dependentKinase and PhosphataseControl Inositol 1,4,5Trisphosphate-mediated Ca2+ Release MODIFICATION BY AGONISTSTIMULATION* (Received for publication, February 16, 1993, and in revised form, March 5, 1993)
Bin-Xian Zhang, Hong Zhao,and Shmuel Muallem$ From the Department of Physiology, University of Texas SouthwesternMedical Center, Dallas, Texas 75235-9040
regulation of IP3-mediated Ca2+ release (IMCR) by Ca2+itself. A biphasic (activation/inhibition) [Ca"] dependence of IMCR was demonstrated in permeabilized smooth muscle fibers (4) and cerebellarmicrosomes ( 5 ) and for theCa2+ channel reconstituted into bilayers (6). The mechanism of channel activation by Caz+is not known. The inhibitionof IMCR by Ca2+ has been observedin intact (7, 8) and permeabilized cells (4, 91, in isolated microsomes (IO),and in channels reconstituted into bilayers (3, 6). How Ca2+ inhibits the channel and whether channel activation stimulated by agonists is also regulated by Ca2+ is not known. This problem is particularly significant, sincemodification of Ca2+ channel activity is likely to control [Ca2+]i oscillations (11-13). Indeed, we have recently shown that feedback inhibition of Ca2+release by [Ca2+], isrequired for [Caz+]i oscillations in agonist-stimulated pancreatic acini (14). Ca2+canactivateandinhibitIMCReither directly by interacting with an activatory or inhibitory site on the channel or indirectly through activation or inhibition of other channel regulating enzymes. If the dual regulation of IMCR by Ca2+ is essential for [Ca2+];oscillation, then it should be modified by agonist stimulation. To address these questions,we used a permeable cell system in which Ca2+release can be evoked either by IP3 or agonist stimulation. Weprovide evidence in support of the regulation of IMCR by two Ca2+-dependent kinase I1 enzymes, Ca'+/calmodulin-dependent protein (CaMKII) and Ca2+-dependent phosphatase 2B (PrP-2B). An active kinase appears tobe required for Ca2+release, whereas activation of the phosphatase by Ca2+ inhibitsrelease. Agoof Ca2+ release by Ca2+. nists act to prevent the inhibition Agonists mobilize Ca2+ from intracellular stores by stimu- This appears to be the result of modification of the Ca2+ lating the formationof IP31 (I),which activates Ca2+channels dependence for activation and inhibitionof Caz+release. located in the intracellular stores membrane (2,3). Regulation MATERIALS AND METHODS of Ca2+ release evokedby IP3 or agonist is poorly understood. A form of regulation that attracted attention recently is the Solutions and Adjustment of [Ca2+l-The basic solution used in
A permeable cell system in which Ca2+release can be evoked by inositol 1,4,5-trisphosphate (IPa) or agonist stimulation was used to study the regulation of Ca2+ release by Ca2+itself. At low concentrations, Ca2+activated IP3-mediated Ca2+ release (IMCR) with halfmaximal effect at about 15 nM. At high concentrations, Ca2+inhibited IMCR giving rise to a biphasic [Ca"'] dependence of IMCR. The activation of IMCR by Ca2+ appears to be mediated by a kinase, probably the Ca"+and calmodulin-dependent protein kinase (CaMKII). Thus, the activation required MgATP, completely blocked at 0 "C, required Ca"+,and was inhibited by the CaMKII inhibitors KT5926 and KN62. The inhibition of IMCR seems to be mediated by a protein phosphatase, probably the Ca2+-dependent protein phosphatase 2B. Hence, the inhibition required Ca"+, was prevented by the general protein phosphatase inhibitor pyrophosphateand by the immunosuppressants cyclosporin A and FK506, but not by okadaic acid or V O f - , and was modified by chelating agents such as EGTA. Stimulation with agonists modified the activities of the kinase and phosphatase to make the release independent of [Ca"']. This appears to be due to an increase in the apparent affinity for Ca2+in stimulating IMCR and inhibition of the phosphatase. We suggest that agonist-dependent modification of the kinase/ phosphatase activity ratiocan be the biochemical pathway responsible for regulation of Ca2+release and in turn [Ca2+Iioscillations.
* This work was supported by National Institutes of Health Grant DK9893 and by an Established Investigator award from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, T X 75235-9040. Tel.: 214-688-2593; Fax: 214688-8685. The abbreviations used are: IPS, inositol 1,4,5-trisphosphate; [Ca"], free Ca2+concentration; [Caz+Ii,intracellular free Ca2+concentration; IMCR, IPS-mediated Ca2+ release; AMCR, agonist-mediated Ca2+ release; CaMKII, calcium- and calmodulin-dependent protein kinase 11; PrP, proteinphosphatase; IC, inactive channel (when dephosphorylated); ACP, active channel (when phosphorylated); ATPyS, adenosine 5'-0-(thiotriphosphate); GDPPS, guanyl5"yl thiodiphosphate; GTPrS, guanosine 5'-3-O-(thio)triphosphate.
the present studies is termed 5HK and contained (in mM) KCl, 120; Hepes, 10 (pH 7.2 with NaOH); MgClz, 3; CaC12, 0.035; EGTA, 0.05 to maintain [Ca"] at 5 pM. Cell permeabilization and Ca2+uptake were achieved by incubating the cells in 5HK solution containing 1 mM ATP, 5 mM creatine phosphate, 15 units/ml creatine phosphokinase, 45Ca(lo6 cpm/ml) 10 p~ antimycin A, 5 pg/ml oligomycin, and 22.5 pg/ml p-escin (uptake medium). Efflux medium was similar to 5HK except that CaCIZand EGTA concentrations were varied to adjust [Ca"'] as desired. When included in efflux medium, IP3 con. term agonist is used to indicate that centration was always 5 p ~ The efflux medium contained 2.5 p~ bradykinin and 10 ,,LM GTPyS. [Ca"] in all solutions was verified by measurement with Fura 2 as described (9) except that a sample of 5HK, uptake medium, or efflux medium supplemented with 1 p~ of the tetra K+ form of Fura 2 was maintained at the required temperature (37 or 0 "c).The Kd values for Fura 2and EGTA at 37 "C were those published in the literature (16, 17). In some experiments (Fig. 2C), the incubation temperature during efflux was 0 "C. The Kd values of Fura 2 and EGTA are sensitive to temperature and thus were adjusted to 0 "C from the
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Ca2+ KinaselPhosphatase Release and
calculated enthalpy using the Kd values a t 37 "C and 23 "C (16, 17). The KC. for EGTA a t 37 and 0 "C used were 2.29 X and 3.98 x 10"' M, and the KC. for Fura 2 at 37 "C and 0 "C used were 220 and 69 nM, respectively. In solutions containing PPi, Fura 2 could not be used since its fluorescence was quenched by PPI. In these solutions, [Ca"] was measured and adjusted with the aid of Flu0 3 using a Kc. at 37 "cof 0.37 pM. Measurement of Ca2+ Uptake and Rekase"ST6 fibroblasts were grown in 24 multi-well plates as described (15). The cells were used after 4-6 days in culture. Prior topermeabilization, the cell layer was washed twice with 5HK. Cell permeabilization and loading the internal stores with CaZ+labeled with '5Ca were achieved by incubating the cells with uptake medium (0.2 ml/well) for 30 min at 37 "C. [Ca"] of the uptake medium was always 5 p~ except in the experiments in Fig. 2C (squares) where it was reduced to 0.1 p ~At. the end of this period, between 85 and 95% of the cells were permeable to molecules the size of IPS, but not to heparin (4-6 kDa). To initiate the efflux, the uptake medium was removed by aspiration and replaced with 0.5 ml of efflux mediumwith or without IP3or the agonist and containing the desired [CaZ+].After a 20-s incubation, efflux medium was removed and the cell layer was washed twice with a 5HK solution containing 1 mM Lac13 to stop the reaction. '5Ca content of the cells and protein were determined after solubilization in 0.5 ml of 0.2 M NaOH (9). Mass Measurement of ZP3-The cells were permeabilized, and internal stores were loaded with Ca2+exactly as described for Ca2+ release measurements, except that the uptake medium in 12 wells (one half plate) did not contain "Ca. After a 30-min incubation at 37 "C, uptake medium was removedand replaced with 0.2 ml of efflux medium containing 2 mM EGTA, sufficient CaCIZ toset [Ca"] between approximately 2 and 60 nM, and the agonist. After a 20-s incubation at 37 "C, '5Ca-containing cells were processed as described above for efflux measurements, whereas the reaction in the remaining wells was terminated with 25 p1 of a solution containing 45% perchloric acid and 2 mM ATP. Following a 20-min incubation at 0 'C, the supernatantwas collected, centrifuged a t 1000 X g for 5 min, and transferred to a clean tube. Perchloric acid was removed, and IP3 in the sample was extracted by the addition of 0.2 ml of Freon and 0.2 ml of tri-n-octylamine. After mixing and centrifugation, the upper layer was collected. Samples of authentic IP3 were processed in the same manner to obtain a standard curve. The IP3 content in each sample was determined from displacement of [3H]IP3bound to cerebellar microsomes exactly as described (9).
A possible explanation for the difference between the behavior of AMCR and IMCR is that the localized high concentration of IPS due to agonist (15) antagonized the inhibition by Ca'+. Alternatively, the agonist may have mobilized Caz+ from more than one pool or from an IP3-insensitive pool(s). These are considered unlikely for the following reasons. (a) Increasing [IP3] to 100 ~ L Mdid not prevent the inhibition of IMCR by Ca2+. ( b ) Bradykinin or GTP+alonedidnot release any Ca2+, and 100 p~ GDP(3S inhibited the effect of bradykinin and GTPyS. (c) AMCR was completely blocked by reducing the temperature to 0 "C after the uptake and before initiation of the release. ( d ) There was a tight correlation between agonist-induced increase in the level of IP3 and Ca2+ release.Thus, only stimulation with bradykinin and GTP-yS together resulted in rapid increase in IP3 levels and rapid Ca2+ release; the time course of the two activities was identical (not shown), and the two activities had identical [Ca"] dependence (see Fig. 3A, circles). Together, these observations suggest that all the AMCR is mediated by IPS. Therefore,theresults in Fig. 1 indicatethattheagonist prevented the inhibitionof IMCR by Ca2+. To study how the agonist may have modified the [Ca2+] dependence of IMCR, it is necessary to understand how Ca2+ regulates IMCR. Although no explicit mechanism has been proposed, it is assumedthat Ca2+ inhibitsthe release by directly interacting with the Ca2+ channelitself. We propose here and test a possible alternativemechanism based on activation and inhibitionof the channelby a Ca2+ dependent kinaseandphosphatase, respectively(see Scheme 1). To obtain evidence in support of this mechanism,we determined whether an active kinase is required for Ca2+ release and whether inhibition of P r P activity can modify the release. The resultsin Fig. 2 (A-C) test for the possible involvement of a protein kinase-mediated phosphorylation in Ca2+ release. The first requirement of a kinase-mediated process is that ATP must be present during Ca2+ release. Fig. 2A shows that when ATPwas removed prior toefflux, both IPS- and agonist-
RESULTS AND DISCUSSION
Fig. 1 shows the effect of [Ca'+] on Ca2+ release evoked by IP3 or agonist stimulation in permeabilized 3T6 fibroblasts. Maximal IMCR was measured a t 0.06 p~ Ca2+. Increasing [Ca'+] beyond 0.4 p~ completely inhibited IMCR. We have reported the same behavior in other cell types (9). To study the effect of theagonist, Ca2+release was triggered with bradykinin and GTP$% At low [Ca"], the agonist released about 1.8-fold moreCa2+ thandid IPS.Amore dramatic difference is that agonist-mediatedCa2+ release(AMCR) was not inhibited at thehigher [Ca2+].
C Temperature Senslfwty
Uptake
- 100 0
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-80
E
IP3+Mg2*
Uptake at 0.1 ( m . 0 ) or 5pM ( A , A)CaZ' 60 Keep 37'C (m, A ) ACoolO'C(0.A) Measure efflux
IP3-MgZ*
-20
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-
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FIG. 2. Dependence of Ca2+release on the activity of a protein kinase. Cells were permeabilized, and stores were loaded with CaZ+labeled with 'Ta as described under "Materials and Methods." Panel A , uptake medium was removed, and the cells were washed once with 5HK to remove ATP. Subsequently, efflux was initiated with medium containing different [Ca"] andeither agonist (O), agonist and 1 mM ATP (O),5 p~ IP3 (A),or IP3 and ATP (A). Panel B , efflux was initiated by washing the cells with a MP-free 5HK solution that contained 1 mM ATP, followed by addition of M$+-free efflux medium of different [Ca"] also containing agonist (O),agonist 0.5 and 3 mM MgC12 (0),IP3 (A),or IP3 and M P (A).Panel C, the Ca" Concentratlon (pM) channel was stabilized at the active (ACP) or inactive (IC) states FIG. 1. CaZ+dependence of IPS-or agonist-mediated Ca2+ prior to efflux by setting [Ca"] in uptake medium at either 0.1 p M release. After loading the internal storeswith '6Ca by incubating the (0,W) to stabilize ACP or 5 p~ (A, A) to stabilize IC. At the end of cells in uptake medium containing 5 p~ Ca2+,efflux was measured the permeabilization and uptake period at 37 "C, some of the cells by removal of uptake medium and addition of efflux medium contain- were kept at 37 "C and the Ca" dependence of IMCR was measured (W, A). A second portion was transferred to an ice-cold water bath ing different [CaZ+]without '5Ca and with either IP3 or bradykinin andGTPyS.The fraction ofIPB- or agonist-mediated release is for 2 min. Then the Ca" dependence of IMCR was measured as plotted against [Ca"] present during efflux. The figure shows the above except for the use of ice-cold efflux medium (,A). The results in panels A-C represent the average of at least eight experiments. mean +- S.E. of eight experiments.
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Ca" Kinase/Phosphatase Release and
mediated Ca2+release were largely inhibited. The small A. amount of release is probably due t o incomplete removal of ATP. When ATPwas added back, agonist- and IPS-mediated 100 Ca2+release were restored.Proteinkinase-mediatedphos0 phorylation requires MgATP as substrate.Fig. 2B shows that a 80 n removing Mg2+ before initiation of the release reduced the m effect of the agonist and IPS, whichcould berestored by m 60 adding back the Mg2+. Another commonly used test to dems 0onstrate the involvementof a protein kinase is toshow that " ATPyS can substitute for ATP and stably thiophosphorylate rn + ~ p o n , r t - 30 0 40 5 -0the target enzyme. It was not possible to successfully employ 20 this test, sinceATP-yS only slowly thiophosphorylates target 0 enzymes and is not a substrate for the Ca2+ pump. Nonetheless, in a protocol similar to that in Fig. 2 A , after Ca2+ uptake, 10 20 30 40 60 Release by Can+ in nM the ATP was washed and the cells exposed for 4 min to a Agonist Ip3 medium containing 5 p M Ca2+ and0.5 mM ATPyS. Although FIG. 3. Ca2+dependence and blocker sensitivity of the kiabout 70%of the stored Ca2+ leaked during the incubation nase. Panel A, cell permeabilization and Caz+uptake were performed with ATPyS, both IP3 and the agonist released Ca2+ without by incubating the cells in uptake medium containing 5 p M Ca2+and '5Ca.After 30 min at 37 "C, uptake further addition of ATP and at [Ca"] of 1 and 5 p~ (not with (0,A, W) or without (0) 20- medium was removed and the cells were exposed for 20 s to efflux shown). On the other hand, including ATPyS during the medium containing 2 mM EGTA and sufficient CaC12to adjust [Ca'+] s efflux period a t 37 "C did not substitute for ATP. These to between
