Amersham Corp., and Immobilon-P was from Millipore (Bedford, MA). cADPR was prepared as described (4). The 30-kDa chymotryptic frag- ment of CaM kinase ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 51, Issue of December 22, pp. 30257–30259, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Communication Requirement of Calmodulindependent Protein Kinase II in Cyclic ADP-ribose-mediated Intracellular Ca21 Mobilization* (Received for publication, September 13, 1995, and in revised form, October 18, 1995) Shin Takasawa‡, Atsuhiko Ishida§, Koji Nata‡, Kei Nakagawa‡, Naoya Noguchi‡, Akira Tohgo‡¶, Ichiro Kato‡, Hideto Yonekura‡, Hitoshi Fujisawa§, and Hiroshi Okamoto‡i From the ‡Department of Biochemistry, Tohoku University School of Medicine, Sendai 980-77, Miyagi, Japan and the §Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Hokkaido, Japan
Cyclic ADP-ribose (cADPR) is generated in pancreatic islets by glucose stimulation, serving as a second messenger for Ca21 mobilization from the endoplasmic reticulum for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370 – 373). In the present study, we observed that the addition of calmodulin (CaM) to rat islet microsomes sensitized and activated the cADPR-mediated Ca21 release. Inhibitors for CaM-dependent protein kinase II (CaM kinase II) completely abolished the glucose-induced insulin secretion as well as the cADPR-mediated and CaM-activated Ca21 mobilization. Western blot analysis revealed that the microsomes contain the a isoform of CaM kinase II but do not contain CaM. When the active 30-kDa chymotryptic fragment of CaM kinase II was added to the microsomes, fully activated cADPR-mediated Ca21 release was observed in the absence of CaM. These results along with available evidence strongly suggest that CaM kinase II is required to phosphorylate and activate the ryanodine-like receptor, a Ca21 channel for cADPR as an endogenous activator, for the cADPR-mediated Ca21 release.
Glucose is the primary stimulus of insulin secretion and synthesis in the pancreatic islets of Langerhans (1–3). Cyclic ADP-ribose (cADPR)1 is generated by glucose stimulation, serving as a second messenger for Ca21 mobilization from the endoplasmic reticulum to secrete insulin (4 – 6). cADPR has been thought to activate or enhance the ryanodine-like receptor * This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan. 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. ¶ Recipient of a fellowship from the Japan Society for the Promotion of Science. i To whom correspondence should be addressed: Dept. of Biochemistry, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-77, Miyagi, Japan. Tel.: 81-22-274-1111 (ext. 2211); Fax: 81-22-272-8101. 1 The abbreviations used are: cADPR, cyclic ADP-ribose; RyR, ryanodine-like receptor; CaM, calmodulin; CaM kinase II, calmodulin-dependent protein kinase II; RIA, radioimmunoassay; AIP, autocamtide2-related inhibitory peptide; KRB, Krebs-Ringer’s bicarbonate buffer.
(RyR) of a variety of cells to release Ca21 from the intracellular stores (4 –13). In sea urchin eggs, it was recently suggested that calmodulin (CaM) directly interacts with RyR to enhance the cADPR-mediated Ca21 release (14 –16). In the present study, we showed that CaM-dependent protein kinase II (CaM kinase II) is essential for the cADPR-mediated Ca21 mobilization for insulin secretion in islets. A possible mode of action of cADPR and CaM kinase II on RyR is discussed. EXPERIMENTAL PROCEDURES
Materials—CaM was purchased from Calbiochem. Monoclonal antibody against CaM (clone 3402 2D1.15.282) (17), W-5, W-7, KN-04, KN-64, KN-92, and KN-93 were obtained from Seikagaku Corp. (Tokyo, Japan). Monoclonal antibodies against rat a isoform and b isoform of CaM kinase II (CBa-2 and CBb-1, respectively) were from Life Technologies, Inc. The rat insulin radioimmunoassay (RIA) kit was from Amersham Corp., and Immobilon-P was from Millipore (Bedford, MA). cADPR was prepared as described (4). The 30-kDa chymotryptic fragment of CaM kinase II from rat brain (18) and autocamtide-2-related inhibitory peptide (AIP: KKALRRQEAVDAL) (19) were prepared as described previously. Calcium Release Assay—Microsomes were prepared as described previously (4) with some modifications. In brief, 2,000 islets from Wistar male rats (240 –280 g) were homogenized with a Pellet mixer (Treff, Degersheim, Switzerland) in 0.2 ml of acetate intracellular medium composed of 250 mM potassium acetate, 250 mM N-methylglucamine, 1 mM MgCl2, and 20 mM Hepes (pH 7.2) supplemented with 0.5 mM ATP, 4 mM phosphocreatine, creatine phosphokinase (2 units/ml), 2.5 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride. After the homogenates had been centrifuged for 45 s at 13,000 3 g, microsomes were prepared by two cycles of Percoll density gradient centrifugation at 20,000 3 g for 40 min at 10 °C (4). For cerebellar microsomes, rat cerebellum was homogenized with a Dounce-type glass tissue homogenizer, and microsomes were prepared as described above. Release of Ca21 was monitored in 0.6 ml of gluconate intracellular medium composed of 250 mM potassium gluconate, 250 mM N-methylglucamine, 1 mM MgCl2, and 20 mM Hepes (pH 7.2) supplemented with 1 mM ATP, 4 mM phosphocreatine, creatine phosphokinase (2 units/ml), 2.5 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 3 mM Fluo-3 (Molecular Probes), and 30 ml of islet microsomes (10 mg of protein) or cerebellar microsomes (50 mg of protein). Fluorescence was measured at 490 nm excitation and 535 nm emission with a Jasco CAF-110 intracellular ion analyzer (Tokyo, Japan) at 37 °C. At the end of each experiment, calibration was performed by adding 1 mM CaCl2, followed by 10 mM MnCl2 to obtain Fmax and Fmin, respectively (15, 20). The ambient-free Ca21 concentration ([Ca21]) was calculated using the following equation: [Ca21] 5 Kd 3 (F 2 Fmin)/(Fmax 2 F), where Kd 5 400 nM (20). The estimated free [Ca21] was 130.1 6 30.4 nM when CaM was added. Western Blot Analysis—Islets were homogenized as described (25% homogenate) (4) and diluted with acetate intracellular medium as 1% homogenate. The protein concentration of the 1% homogenate was 0.92 mg/ml. Western blot analysis was carried out as described previously (21, 22). Insulin Secretion from Isolated Islets—Islets (20/tube) were preincubated at 37 °C for 30 min in 1 ml of Krebs-Ringer’s bicarbonate buffer (KRB) containing 0.2% bovine serum albumin (3, 4) with vehicle or inhibitors under an atmosphere of 95% O2, 5% CO2. Following the preincubation, the medium was discarded, and islets were incubated in 0.1 ml of fresh KRB buffer containing 20 mM glucose in the presence of inhibitors or vehicle. After 30 min of incubation, the medium was removed from the islets, and the insulin content was determined by RIA using a rat insulin RIA kit and rat insulin standard. RESULTS AND DISCUSSION
We measured the Ca21 release by cADPR from rat islet microsomes in the presence or absence of CaM (Fig. 1A). In the absence of CaM, the cADPR responsiveness for Ca21 release was restored only at high concentrations of cADPR. On the other hand, in the presence of CaM, islet microsomes were sensitized to cADPR at
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Activation of cADPR-mediated Ca21 Release by CaM Kinase II
FIG. 1. cADPR sensitivity-conferring activity of CaM. A, islet microsomes were incubated with or without 7 mg/ml CaM and challenged with the indicated concentrations of cADPR. The Ca21 release measurement was a peak value estimated from Fluo-3 fluorescence. B, CaM content was estimated in islet homogenate and in islet microsomes by immunoblot analysis. Islet homogenate (1 mg of protein, lane 1), islet microsome (1 mg of protein, lane 2), and bovine CaM (0.01, 0.1, 1 mg; lanes 3–5, respectively) were chromatographed on 15% SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P. The membrane was incubated at room temperature for 1 h with a monoclonal antibody against CaM. The antibody was diluted at 1 mg/ml with 5% milk powder solution. After rinsing, the membrane was further incubated at room temperature for 1 h with a secondary antibody labeled with horseradish peroxidase and developed using an ECL detection system (Amersham Corp.).
much lower concentrations (0.1– 0.3 mM), and the Ca21 release by cADPR was greatly increased. The maximal response was achieved at 3–10 mg/ml CaM. CaM was not detected in islet microsomes by immunoblot analysis (Fig. 1B, lane 2), but CaM was present in islet homogenates, and the CaM concentration in islets was estimated to be about 3–10 mg/ml (Fig. 1B). Therefore, Ca21 release from the microsomes in the presence of 3–10 mg/ml CaM rather than in its absence is physiological. The activation of cADPR-mediated Ca21 release by CaM was also observed with rat cerebellar microsomes (data not shown). These results suggest that CaM is a positive modulator for cADPR-mediated Ca21 release not only in sea urchin egg microsomes (14 –16) but also in mammalian microsomes. We then examined the effects of CaM and CaM kinase II inhibitors on the cADPR/CaM-induced Ca21 release from islet microsomes. A CaM inhibitor, W-7 (50 mM), and CaM kinase II inhibitors such as KN-62 (10 mM) and KN-93 (10 mM) completely inhibited the cADPR/CaM-induced Ca21 release, but non-inhibitory analogues, W-5 (50 mM), KN-04 (10 mM), and KN-92 (10 mM), did not (Fig. 2A). In addition, AIP (18, 19), which is a more
FIG. 2. A, effects of the cell-permeable CaM antagonist and CaM kinase II antagonists on the cADPR-mediated Ca21 release and on the glucose-induced insulin secretion. Ca21 release was induced by the addition of 100 nM cADPR in the presence of 7 mg/ml CaM and measured as in Fig. 1A. The concentrations of CaM antagonists (W-7 and W-5) and CaM kinase II antagonists (KN-62, KN-04, KM-93, and KN92) were 50 and 10 mM, respectively. Values are expressed by percent (mean 6 S.E., n 5 3– 4) of control without antagonist. The average Ca21 release of control was 0.327 6 0.018 mM. For insulin secretion, the antagonists were used at the same concentrations in KRB as for the Ca21 release. Values are expressed by percent (mean 6 S.E., n 5 5– 6) of control without antagonist. The average insulin secretion of control was 40.9 6 11.3 ng/islet/h. B, inhibition of cADPR/CaM-mediated Ca21 release by AIP. cADPR/CaM-mediated Ca21 release was measured in the presence of the indicated concentrations of AIP. Ca21 release was induced by the addition of 100 nM cADPR in the presence of 7 mg/ml CaM and measured as in Fig. 1A.
specific CaM kinase II peptide inhibitor, also inhibited the activation of cADPR-mediated Ca21 mobilization by CaM, and the dose-inhibition curve of Ca21 mobilization by AIP (Fig. 2B) was well fitted to that of CaM kinase II activity by AIP (19). These results strongly suggest that the activation of cADPRmediated Ca21 mobilization by CaM is achieved by activation of CaM kinase II but not by the direct interaction of CaM with the Ca21 release machinery. We therefore examined the existence of CaM kinase II in the microsomes by immunoblot analysis and revealed that the a isoform of CaM kinase II but not the b isoform actually exists in islet microsomes (Fig. 3A). To confirm the essential requirement of CaM kinase II activity for the cADPR-mediated Ca21 mobilization, we then added the active 30-kDa chymotryptic fragment of the CaM kinase II (18), which lacks the autoinhibitory domain and is therefore activated in the absence of CaM, to islet microsomes and measured the cADPR-mediated Ca21 release. As shown in Fig. 3B, the cADPR-mediated Ca21 release was as fully and dose dependently activated by the 30-
Activation of cADPR-mediated Ca21 Release by CaM Kinase II
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FIG. 4. Model for cADPR-induced Ca21 mobilization via RyR for the glucose-induced insulin secretion. Glucose stimulation induces cADPR accumulation in islets, and cADPR acts on RyR as a second messenger for intracellular Ca21 mobilization (4 – 6, 21, 23, 24). On the other hand, CaM kinase II is activated by glucose stimulation (28, 29), and the activated kinase phosphorylates RyR to sensitize the Ca21 channel for the cADPR signal. Pi, phosphorylation of RyR by CaM kinase II. FIG. 3. Evidence that CaM kinase II is the cADPR sensitivityconferring factor. A, cerebral homogenate (50 mg of protein, lanes 1, 3, and 5) and islet microsome (50 mg of protein, lanes 2, 4, and 6; 100 mg of protein, lane 7) were chromatographed on 10% SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P. The membrane was incubated at room temperature for 1 h with the monoclonal antibody against the a isoform (lanes 1 and 2) or b isoform (lanes 3 and 4) of CaM kinase II and with a 1:1 mixture of the antibodies (lanes 5–7). The concentration of antibody was 1 mg/ml each with 5% milk powder solution. After rinsing, the membrane was further incubated with a secondary antibody at room temperature for 1 h and developed as described in Fig. 1B. B, concentration dependence of CaM kinase II in cADPR-mediated Ca21 release from islet microsomes. For the Ca21 release assay, the active 30-kDa chymotryptic fragment of CaM kinase II at the indicated concentrations was used in the absence of CaM. The same amounts of the 30-kDa CaM kinase II fragment previously inactivated by 5-min boiling were used as controls. Ca21 release was induced by the addition of 100 nM cADPR and measured as in Fig. 1A.
kDa fragment of CaM kinase II as by the addition of CaM to microsomes. We have proposed a model for insulin secretion by glucose via cADPR-mediated Ca21 mobilization. In the process of glucose metabolism, millimolar concentrations of ATP are generated, inducing cADPR accumulation by inhibiting the cADPR hydrolase activity of CD38, and cADPR acts as a second messenger for intracellular Ca21 mobilization from microsomes for insulin secretion (4 – 6, 21, 23, 24). Recently, in sea urchin eggs, CaM has been reported to act on the microsomes and sensitize the cADPR-mediated Ca21 release without the involvement of CaM-dependent enzymes such as CaM kinase II (14 –16). The results in this study showed that CaM sensitized and activated the cADPR-mediated Ca21 release from islet microsomes, which contained the a isoform of CaM kinase II, and that the effect of CaM on cADPR-mediated Ca21 mobilization as well as the glucose-induced insulin secretion were abolished by specific inhibitors for CaM kinase II. Furthermore, the addition of the 30-kDa active CaM kinase II fragment to the microsomes fully activated the cADPR-mediated Ca21 release in the absence of CaM. These results suggest that the cADPR-mediated Ca21 mobilization for insulin secretion is achieved by the CaM-activated a isoform of CaM kinase II. RyR is thought to be a Ca21 channel for cADPR as an endogenous activator, and phosphorylation and activation of RyR by CaM kinase II were reported (25–27). Furthermore, in islets, CaM kinase II was observed to be activated by glucose stimulation (28, 29). Therefore, as de-
picted in Fig. 4, when islets are stimulated by glucose, RyR can be synergistically activated not only by cADPR but also by phosphorylation of the Ca21 channel by CaM kinase II. Acknowledgment—We are grateful to Brent Bell for valuable assistance in preparing the manuscript for publication. REFERENCES 1. 2. 3. 4. 5.
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