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J. Biol. Chem., Vol. 277, Issue 18, 15813-15818, May 3, 2002

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STO-609, a Specific Inhibitor of the Ca²⁺/Calmodulin-dependent Protein Kinase Kinase^*

Hiroshi Tokumitsu^§, Hiroyuki Inuzuka, Yumi Ishikawa, Masahiko Ikeda^¶, Ikutaro Saji^¶, and Ryoji Kobayashi

From the  Department of Chemistry, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan and the ^¶ Exploratory Research Group, Research Division, Sumitomo Pharmaceuticals Co., Ltd., 4-2-1 Takatukasa, Takarazuka, Hyogo 665, Japan

Received for publication, February 1, 2002, and in revised form, February 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

STO-609, a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase kinase (CaM-KK) was synthesized, and its inhibitory properties were investigated both in vitro and in vivo. STO-609 inhibits the activities of recombinant CaM-KK and CaM-KK isoforms, with K_i values of 80 and 15 ng/ml, respectively, and also inhibits their autophosphorylation activities. Comparison of the inhibitory potency of the compound against various protein kinases revealed that STO-609 is highly selective for CaM-KK without any significant effect on the downstream CaM kinases (CaM-KI and -IV), and the IC₅₀ value of the compound against CaM-KII is ~10 µg/ml. STO-609 inhibits constitutively active CaM-KK (glutathione S-transferase (GST)-CaM-KK-(84-434)) as well as the wild-type enzyme. Kinetic analysis indicates that the compound is a competitive inhibitor of ATP. In transfected HeLa cells, STO-609 suppresses the Ca²⁺-induced activation of CaM-KIV in a dose-dependent manner. In agreement with this observation, the inhibitor significantly reduces the endogenous activity of CaM-KK in SH-SY5Y neuroblastoma cells at a concentration of 1 µg/ml (~80% inhibitory rate). Taken together, these results indicate that STO-609 is a selective and cell-permeable inhibitor of CaM-KK and that it may be a useful tool for evaluating the physiological significance of the CaM-KK-mediated pathway in vivo as well as in vitro.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Ca²⁺/calmodulin-dependent protein kinases (CaM-Ks)¹ constitute a diverse group of enzymes, which are involved in many cellular responses mediated by an increase in the concentration of intracellular calcium. Previous studies have demonstrated that two multifunctional CaM kinases, CaM-KI and -IV, are activated by phosphorylation of an activation loop Thr residue by an upstream CaM kinase kinase (CaM-KK) resulting in a large increase in catalytic efficiency (reviewed in Refs. 1 and 2). In mammals, two CaM-KK genes (CaM-KK and CaM-KK) have been cloned, both of which are highly expressed in the brain, and the  isoform is also expressed in various peripheral tissues such as thymus and spleen (3-5). The CaM-KK gene has been found in Caenorhabditis elegans and Aspergillus nidulans, and the proteins they encode are components of the CaM kinase cascade of these organisms (6-9). Interestingly, both mammalian CaM-KK isoforms bind to Ca²⁺/CaM complexes indicating that CaM-KK is a member of the CaM kinase family (3, 5). Indeed, Ca²⁺/CaM binding is absolutely required for the relief of CaM-KK autoinhibition, which results in its activation. In contrast, CaM-KK exhibits an enhanced Ca²⁺/CaM-independent activity, due to suppression of autoinhibition by the second regulatory segment (residues 129-151) located at the N terminus of the catalytic domain (10-13). Recent structural and functional studies of CaM-KK have revealed that it binds Ca²⁺/CaM in a manner different from other CaM kinases such as CaM-KII and MLCK, as confirmed for the C. elegans CaM-KK by 1.8-Å resolution crystal structure analysis of Ca²⁺/CaM, complexed with its CaM-binding peptide fragment (14, 15). The unique feature of the CaM-binding segment in CaM-KK is required for the autoinhibitory mechanism through Ile-441 in CaM-KK (16).

A functional CaM kinase cascade leading to the activation of CaM-KIV in response to Ca²⁺ mobilization has been demonstrated for using transfected COS-7 cells (10), Jurkat cells (17), and cultured hippocampal neurons (18); the cascade has also been shown to be required for the activation of CaM-KI in PC-12 cells upon membrane depolarization (19). The CaM kinase cascade has been reported to engage in cross-talk with other signaling pathways such as those that lead to the activation of MAP kinases (20) and to be subject to down-regulation by PKA (21, 22). An important role has been demonstrated for the CaM-KIV cascade in the regulation of Ca²⁺-dependent gene expression by the phosphorylation of transcription factors such as CREB (23-26). A recent study of transgenic mice carrying dominant negative CaM-KIV alleles that confer a defect in the phosphorylation of CREB indicates that these animals exhibit a disruption of late phase long term potentiation and that they are impaired in the consolidation/retention phase of hippocampus-dependent memory (27). Analysis of mice deficient in CaM-KIV revealed that the CaM-KIV-mediated pathway plays an important role in the function and development of the cerebellum and is critical for male and female fertility (28-30). In addition, the physiological role has been predicted for CaM-KK, with the suggestion that it may act as a regulatory protein kinase in the CaM kinase cascade, but this has not been demonstrated in vivo. Therefore, to evaluate the physiological functions of CaM-KK and of the CaM kinase cascade, we attempted to synthesize a potent and specific inhibitor of CaM-KK. In this study, we characterize the effects of the inhibitor STO-609 on CaM-KK activity both in vitro and in intact cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- CaM-KK cDNA (GenBank^TM accession number L42810) (3) was obtained from a rat brain cDNA library. Recombinant CaM-KK and - were expressed in Escherichia coli and purified as described previously (13). A constitutively active CaM-KK (GST-CaM-KK-(84-434)) was expressed in E. coli JM109 and purified by glutathione-Sepharose column chromatography (16). Recombinant rat CaM was expressed in the E. coli strain BL-21 (DE3) using pET-CaM (31) (kindly provided by Dr. Nobuhiro Hayashi, Fujita Health University, Toyoake, Japan) and purified by phenyl-Sepharose column chromatography. Rat CaM-KI was expressed in E. coli JM-109 as a GST fusion protein and purified by glutathione-Sepharose column chromatography (7). Recombinant CaM-KIV was expressed in Sf9 cells using a baculovirus system and purified by CaM-Sepharose, which was kindly provided by Dr. Tom Soderling (Vollum Institute, Oregon Health Sciences University) (32). Rat CaM-KII holoenzyme was purified from rat forebrain as previously described (33). Purified myosin light chain kinase from chicken gizzard was kindly provided by Dr. Hiroshi Hosoya (Hiroshima University, Higashihiroshima, Japan). cAMP-dependent protein kinase and protein kinase C were obtained from CLONTECH. p42 MAP kinase was obtained from Upstate Biotechnology.

Synthesis of STO-609-- 1,8-Naphthoylene benzimidazole-3-carboxylic acid (STO-609, M_r = 314.29) was synthesized as follows. 3-Bromo-1,8-naphthoylene benzimidazole was prepared by the method of Nakaya et al. (37). Reaction of 3-bromo-1,8-naphthoylene benzimidazole with cuprous cyanide in pyridine, followed by acid hydrolysis of 3-cyano-1,8-naphthoylene benzimidazole with H₂SO₄ in aqueous acetic acid, yielded STO-609 as a yellow solid. Recrystallization from acetic acid yielded pure STO-609 as an acetic acid adduct in a molar ratio of 1:1. The structure and purity of the synthetic compound were confirmed by ¹H NMR, electrospray ionization mass spectroscopy, and elemental analysis.

In Vitro Assay for CaM-KK Activity-- Purified recombinant CaM-KKs (CaM-KK, 0.28 µg/ml; CaM-KK, 0.52 µg/ml; constitutively active CaM-KK, 0.3 µg/ml) were incubated with 10 µg of GST-CaM-KI-(1-293)-K49E at 30 °C for 5 min in a solution (25 µl) containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)₂, 1 mM DTT, various concentrations (50-400 µM) of [-³²P]ATP (650-6500 cpm/pmol) with various concentrations of STO-609 (0-10 µg/ml in Me₂SO at a final concentration of 4%) in the presence of either 1 mM EGTA (for constitutively active CaM-KK) or 1 mM CaCl₂, 2 µM CaM. The reaction was initiated by the addition of [-³²P]ATP and terminated by spotting aliquots (15 µl) onto phosphocellulose paper (Whatman P-81) followed by several washes with 75 mM phosphoric acid (34). Phosphate incorporation into GST-CaM-KI-(1-293)-K49E was determined by liquid scintillation counting of the filters. A 5-min reaction was chosen to determine CaM-KK activity based on the time course experiment described recently (13). Specific activities of CaM-KK, CaM-KK, and constitutively active CaM-KK in the absence of STO-609 were calculated to be 723 ± 7 µmol/min/mg, 338 ± 18 µmol/min/mg, and 927 ± 40 µmol/min/mg, respectively.

Autophosphorylation of CaM-KK and --- Purified recombinant CaM-KK and - (0.8 µg) were assayed at 30 °C for 5 min in a solution (25 µl) containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)₂, 1 mM DTT, 50 µM [-³²P]ATP (6500 cpm/pmol) with various concentrations of STO-609 (0-10 µg/ml in Me₂SO at a final concentration of 4%) in the presence of either 1 mM EGTA (for CaM-KK and CaM-KK) or 1 mM CaCl₂, 2 µM CaM (for CaM-KK). The reaction was initiated by the addition of [-³²P]ATP and terminated by the addition of SDS-PAGE sample buffer. The samples were subjected to SDS-10% PAGE followed by autoradiography. ³²P incorporation into CaM-KK was estimated by densitometric scanning of the x-ray film.

In Vitro Assay for CaM-KI, -II, and -IV and MLCK Activities-- CaM-KI (2.5 µg/ml), CaM-KII (0.75 µg/ml), CaM-KIV (9 µg/ml), and MLCK (0.6 µg/ml) were incubated with 40 µM syntide-2 or 50 µM MLC peptide (for MLCK) at 30 °C for 5 min in a solution (25 µl) containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)₂, 1 mM DTT, 50 µM [-³²P]ATP (4500 cpm/pmol) with various concentrations of STO-609 (0-10 µg/ml in Me₂SO at a final concentration of 4%) in the presence of 1 mM CaCl₂, 2 µM CaM. Protein kinase activity was measured by the phosphocellulose filter method as described above. Specific activities of CaM-KI, CaM-KII, CaM-KIV, and MLCK in the absence of STO-609 were calculated to be 24 ± 1 µmol/min/mg, 122 ± 3 µmol/min/mg, 48 ± 1 µmol/min/mg, and 178 ± 6 µmol/min/mg, respectively.

In Vitro Assay for PKA, PKC, and p42 MAP Kinase Activities-- PKA (8 µg/ml), PKC (25 ng/ml), and p42 MAP kinase (2 µg/ml) were incubated with 100 µM kemptamide (for PKA), 100 µM neurogranin peptide (for PKC, Promega), or 0.4 mg/ml myelin basic protein (for p42 MAP kinase) at 30 °C for 5 min in a solution (25 µl) containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)₂, 1 mM DTT, 50 µM [-³²P]ATP (4500 cpm/pmol) with various concentrations of STO-609 (0-10 µg/ml in Me₂SO at a final concentration of 4%) in the absence (for PKA and p42 MAP kinase) or presence of 1 mM CaCl₂, 0.4 mg/ml phosphatidylserine, and 0.1 mg/ml bovine serum albumin. Protein kinase activity was measured by the phosphocellulose filter method as described above. Specific activities of PKA, PKC and p42 MAP kinase in the absence of STO-609 were calculated to be 22 ± 1 µmol/min/mg, 7.522 ± 0.062 mmol/min/mg, and 181 ± 3 µmol/min/mg, respectively. Protein kinase activity was measured under linear conditions based on the results obtained from titration experiments for each enzyme.

Transient Expression and Immunoprecipitation of HA-CaM-KIV-- HeLa cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were subcultured in 6-cm dishes 12 h before transfection. The cells were then transferred to serum-free medium and treated with a mixture of either 3 µg of pME18s plasmid DNA (DNAX Research Institute, Inc) or 3 µg of HA (hemagglutinin-tagged)-CaM-KIV and 20 µg of LipofectAMINE Reagent (Invitrogen) in 2.5 ml of medium. After 20 h of incubation, the cells were further cultured in serum-free medium for 6 h in either the absence or presence of various concentrations of STO-609 (0.01-10 µg/ml in Me₂SO at a final concentration of 0.5%) and then treated with or without 1 µM ionomycin for 5 min. Stimulation was terminated by the addition of 1 ml of lysis buffer (150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 2 mM EGTA, 1% Nonidet P-40, 10% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/liter leupeptin, 10 mg/liter trypsin inhibitor, and 1 µM microcystin LR), and the cells were lysed for 30 min on ice. The cell extract was collected and centrifuged at 15,000 × g for 15 min, the supernatant was precleared with 40 µl of Protein G-Sepharose (50% slurry, Amersham Biosciences) for 2 h at 4 °C, and the supernatant was mixed with 4 µg of anti-HA antibody (clone 12CA5, Roche Molecular Biochemicals) for 3 h. 40 µl of Protein G-Sepharose was then applied to the extract and incubated overnight. The immunoprecipitated resin was washed three times with 1 ml of the lysis buffer as described above and then washed with 1 ml of kinase buffer (50 mM HEPES (pH 7.5), 10 mM Mg(Ac)₂, 1 mM DTT, 1 mM EGTA, and 1 µM microcystin LR). Protein G-Sepharose with immunoprecipitated HA-CaM-KIV was subjected to the protein kinase assay (50 µl reaction volume) in the presence of 1 mM EGTA using syntide-2 as a substrate as described above. To estimate the amount of immunoprecipitated HA-CaM-KIV, SDS-PAGE sample buffer (50 µl) was added to immunoprecipitated samples and then heated at 95 °C for 10 min. After centrifugation, 10 µl of the sample was subjected to SDS-10% PAGE followed by Western blotting using anti-CaM-KIV antibody (1:2000, Transduction Laboratories).

Expression of Ca²⁺/CaM-independent CaM-KIV in SH-SY5Y Neuroblastoma Cells by Infection with Recombinant Adenovirus-- Recombinant adenoviruses carrying cDNAs encoding Ca²⁺/CaM-independent CaM-KIV (³⁰⁵HMDT-DEDD) (32), a kinase-deficient mutant (³⁰⁵HMDT-DEDD, K71E), or a constitutively active CaM-KK-(1-434) (3) were constructed as follows. Briefly, CaM-KIV mutants and constitutively active CaM-KK cDNAs in pME18s plasmid were digested, blunt-ended, and then ligated into pShuttle (CLONTECH). Recombinant viruses were obtained from HEK293 cells using the Adeno-X Expression System (CLONTECH) according to the manufacturer's protocol. For virus infection, confluent SY5Y cells in 6-well culture plates were infected with viruses at a multiplicity of infection of 10 plaque-forming units/cell at 37 °C for 1 h. After infection, virus was aspirated, and the cells were further cultured in RPMI medium containing 10% fetal bovine serum for 12 h. The cells were then serum-starved for 6 h in either the absence or presence of various concentrations of STO-609 (0.01-10 µg/ml in Me₂SO at a final concentration of 0.5%). The cells were stimulated with 1 µM ionomycin for 10 min (or not subjected to ionomycin treatment) in the absence or presence of various concentrations of STO-609 and then lysed, and the extract was subjected to SDS-7.5% PAGE followed by Western blotting using anti-CaM-KIV antibody. The intensity of the immunoreactive band was measured by densitometric scanning of the x-ray film.

Others-- Western blotting was performed as described previously (13) using horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham Pharmacia Biotech) as a secondary antibody and chemiluminescence reagent (PerkinElmer Life Sciences) for detection. Protein concentration was estimated by Coomassie dye binding (Bio-Rad) using bovine serum albumin as a standard.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We have synthesized 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609, Fig. 1A) to test its effects on CaM-KK kinase activity using GST-CaM-KI-(1-293)-K49E as a substrate and on the autophosphorylation activities of the CaM-KK and - isoforms. Fig. 1B shows that the CaM-KK and - kinase activities are inhibited by more than 80% in the presence of 1 and 0.1 µg/ml STO-609, respectively. It has been shown that CaM-KK undergoes autophosphorylation in a Ca²⁺/CaM-dependent manner whereas the CaM-KK and autophosphorylation activities of CaM-KK are highly independent on Ca²⁺/CaM (5, 13, 16). We thus tested the inhibitory effect of STO-609 on the Ca²⁺/CaM-dependent autophosphorylation of CaM-KK and the Ca²⁺/CaM-independent autophosphorylation of the  isoform. As shown in Fig. 1C, the autophosphorylation activities of both CaM-KK isoforms are inhibited by more than 80% in the presence of 1 µg/ml STO-609 with an IC₅₀ value of ~100 ng/ml, which is similar to the inhibitory effect of STO-609 on CaM-KK kinase activities, as shown in Fig. 1B. We have investigated the specificity of STO-609 for various protein kinases in the presence of 50 µM [-³²P]ATP. The activities of multifunctional CaM kinases including the downstream kinases CaM-KI, -IV, and -KII, are unaffected or only slightly affected by the presence of 1 µg/ml STO-609 (CaM-KI, 6%; CaM-KII, 18% of inhibitory rate; CaM-KIV, not detected) (Fig. 2). We further tested the inhibitory effects of STO-609 on other protein kinases such as protein kinase C, cAMP-dependent protein kinase, and p42 MAP kinase in the presence of 50 µM [-³²P]ATP (Table I) and found that those protein kinases were only slightly affected by the presence of 10 µg/ml STO-609 (~20% inhibitory rate). CaM-KII and MLCK are significantly inhibited (~50% inhibitory rate) only by concentrations as high as 10 µg/ml STO-609, which represents an ~100-fold or much lower inhibitory potency of the compound against these kinases than against the two CaM-KK isoforms (CaM-KK, IC₅₀ = 120 ng/ml; CaM-KK, IC₅₀ = 40 ng/ml). These results indicate that STO-609 more significantly inhibits CaM-KKs than other protein kinases we tested. Thus, the newly synthesized compound STO-609 is a selective and potent inhibitor of CaM-KK.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Inhibition of CaM-KK activity by STO-609. A, chemical structure of STO-609. B, purified recombinant CaM-KK (open circle) and - (closed circle) were incubated with 10 µg of GST-CaM-KI-(1-293)-K49E at 30 °C for 5 min in a solution (25 µl) containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM DTT, 50 µM [-32P]ATP with various concentrations of STO-609 (0-10 µg/ml in Me2SO at a final concentration of 4%) in the presence of 1 mM CaCl2, 2 µM CaM. CaM-KK activity was measured as described under "Experimental Procedures" and is expressed as a percentage of the value in the absence of STO-609. Results represent the mean ± S.E. of three experiments. C, autophosphorylation activities of recombinant CaM-KK and - were measured at 30 °C for 5 min in a solution containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM DTT, 50 µM [-32P]ATP with various concentrations (0-10 µg/ml in Me2SO at a final concentration of 4%) of STO-609 in the presence of either 1 mM EGTA without STO-609 (EGTA, lane EGTA in inset) or 1 mM CaCl2, 2 µM CaM for CaM-KK (open bar) or in the presence of 1 mM EGTA for CaM-KK (closed bar) as described under "Experimental Procedures." After termination of the reaction by the addition of the SDS-PAGE sample buffer, the samples were subjected to SDS-10% PAGE followed by autoradiography (insets). Autophosphorylation activities of both CaM-KK isoforms were estimated by densitometric scanning of the autoradiograms (insets, lane a and lane EGTA for CaM-KK; Me2SO control, lane b; 0.001 µg/ml, lane c; 0.01 µg/ml, lane d; 0.1 µg/ml, lane e; 1 µg/ml, lane f; 10 µg/ml STO-609) for CaM-KK (upper inset) and CaM-KK (lower inset) and are expressed as a percentage of the value in the absence of STO-609 (lane a).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of STO-609 on the activities of multifunctional Ca2+/CaM-dependent protein kinases. Protein kinase activities of CaM-KI (open circle), CaM-KII (open triangle), CaM-KIV (open square), and CaM-KK (closed circle) were measured at 30 °C for 5 min in the presence of 1 mM CaCl2, 2 µM CaM and 50 µM [-32P]ATP with various concentrations of STO-609 (0-10 µg/ml in Me2SO at a final concentration of 4%) as described under "Experimental Procedures" and are expressed as a percentage of the value of the activity in the absence of STO-609. Results represent the mean ± S.E. of three experiments.

To clarify the mechanisms involved in the inhibition of CaM-KK activity, STO-609 was tested for its ability to inhibit a constitutively active mutant form of CaM-KK (GST-CaM-KK-(84-434)), which lacks residues required for both autoinhibition and CaM binding (16). As shown in Fig. 3A, in the absence of Ca²⁺/CaM the activity of GST-CaM-KK-(84-434) is suppressed by the addition of STO-609 at exactly the same concentration that inhibits the wild type enzyme (IC₅₀ ~100 ng/ml). This result indicates that STO-609 targets the catalytic domain of CaM-KK and that it is not a CaM antagonist, like trifluoperazine, the interpretations that are also consistent with the inhibition of Ca²⁺/CaM-independent autophosphorylation of CaM-KK by STO-609 as shown in Fig. 1C. Next we performed a kinetic analysis of the inhibition of CaM-KK isoforms by STO-609. Fig. 3B shows the degree of inhibition observed with varying concentrations of ATP (50-400 µM) in the absence or presence (0.1 or 1.0 µg/ml) of STO-609 for CaM-KK (left panel) and in the absence or presence (0.01 or 0.1 µg/ml) of STO-609 for CaM-KK (right panel). As there is no change in the V_max value for the two CaM-KK isoforms, the apparent K_m value for ATP increases with increasing concentrations of STO-609, indicating that the inhibition is competitive with respect to ATP. Based on kinetic data, the K_i values of STO-609 were calculated to be 80 ng/ml for CaM-KK and 15 ng/ml for CaM-KK.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Inhibitory mechanism of STO-609. A, recombinant CaM-KK (closed circle) and constitutively active CaM-KK-(84-434) (open circle) were incubated with 10 µg of GST-CaM-KI-(1-293)-K49E at 30 °C for 5 min in a solution (25 µl) containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM DTT, 50 µM [-32P]ATP with various concentrations of STO-609 (0-10 µg/ml in Me2SO at a final concentration of 4%) in the presence of either 1 mM EGTA (for constitutively active CaM-KK) or 1 mM CaCl2, 2 µM CaM (for CaM-KK) as described under "Experimental Procedures." Activities are expressed as a percentage of the value in the absence of STO-609. Results represent mean ± S.E. of three experiments. B, protein kinase activities of purified recombinant CaM-KK (left panel) and - (right panel) were measured with various concentrations (50-400 µM) of [-32P]ATP as described in Fig. 1 in either the absence (open circle) or presence of 0.1 µg/ml (open triangle) or 1.0 µg/ml (closed triangle) STO-609 for CaM-KK and in either the absence (open circle) or presence of 0.01 µg/ml (open triangle) or 0.1 µg/ml (closed triangle) STO-609 for CaM-KK. The results represent the mean ± S.E. of three experiments and are presented as double reciprocal plots (Lineweaver-Burk).

To examine the effect of STO-609 on the CaM kinase cascade in intact cells, we transfected into HeLa cells an expression vector carrying an HA-tagged CaM-KIV cDNA. Transfected HeLa cells were either untreated or treated with various concentration of STO-609 for 6 h prior to stimulation with 1 µM ionomycin for 5 min. HA-CaM-KIV was then immunoprecipitated, and its Ca²⁺/CaM-independent activity was measured (Fig. 4). The Ca²⁺/CaM-independent activity of CaM-KIV has been shown to be generated by phosphorylation of Thr-196 by CaM-KK as well as induction of Ca²⁺/CaM-dependent activity (3, 32, 35). The Ca²⁺/CaM-independent activity of immunoprecipitated HA-CaM-KIV was found to be greatly enhanced by stimulation with ionomycin, indicative of CaM-KK activity in HeLa cells. Ca²⁺-induced HA-CaM-KIV activation is suppressed by STO-609 in a dose-dependent manner. CaM-KIV activation in HeLa cells is inhibited by ~90% by treatment with 10 µg/ml STO-609 whereas the level of expression of the HA-CaM-KIV protein is not altered by STO-609 treatment (Fig. 4, inset). This result suggests that STO-609 inhibits the endogenous activity of CaM-KK in HeLa cells resulting in a reduction of Ca²⁺-induced CaM-KIV activation.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Suppression of Ca2+-dependent CaM-KIV activation by STO-609 in transfected HeLa cells. After transfection of either HA-CaM-KIV cDNA (3 µg) or empty vector (Mock) into HeLa cells, the cells were either untreated or treated with various concentrations of STO-609 (0.01-10 µg/ml) as indicated for 6 h in serum-free medium and then stimulated with 1 µM ionomycin for 5 min (+) or not stimulated (). HA-CaM-KIV was immunoprecipitated, and its Ca2+/CaM-independent activity was measured at 30 °C for 10 min in the presence of 1 mM EGTA as described under "Experimental Procedures." Results represent mean ± S.E. of three independent transfections. Immunoprecipitated samples ( volume) including a sample prepared from cells transfected with the empty vector (Mock) were subjected to Western blot analysis with an anti-CaM-KIV antibody (inset) as described under "Experimental Procedures." Upper arrow indicates HA-CaM-KIV. Lower arrow indicates the anti-HA antibody (IgG).

To confirm the cell permeability of STO-609 and its inhibitory effect on CaM-KK activity in intact cells as shown in Fig. 4, we assayed endogenous CaM-KK activity in SH-SY5Y neuroblastoma cells treated with STO-609 by using an adenovirus infection system. When Ca²⁺/CaM-independent CaM-KIV (³⁰⁵HMDT-DEDD) is overexpressed from recombinant adenoviruses in SH-SY5Y cells with a constitutively active CaM-KK-(1-434) by infection, the mobility of an immunoreactive band corresponding to CaM-KIV on the SDS-7.5% PAGE gel is decreased compared with that observed for the CaM-KIV mutant alone (Fig. 5A). The mobility shift of the CaM-KIV band is likely due to hyper-autophosphorylation subsequent to activation/phosphorylation of the Thr-196 residue by CaM-KK because the mobility shift is not detected for a corresponding CaM-KIV kinase-deficient mutant (³⁰⁵HMDT-DEDD, K71E) co-expressed with constitutively active CaM-KK. This is consistent with previous reports that the autophosphorylation of multiple Ser residues at the N terminus of CaM-KIV is highly induced by phosphorylation of Thr-196 by CaM-KK (10, 36). We have observed that the protein kinase activity of the Ca²⁺/CaM-independent CaM-KIV is 8-10-fold induced by co-infection of adenovirus bearing a constitutively active CaM-KK (data not shown) indicating that the mobility shift of the CaM-KIV band on SDS-PAGE gel reflects an activated form of CaM-KIV produced by CaM-KK phosphorylation. Therefore, we quantified the intensity of the mobility-shifted CaM-KIV band to confirm endogenous CaM-KK activity, and also we examined the effect of the inhibitor on endogenous CaM-KK activity in SH-SY5Y cells (Fig. 5B). Overexpression of Ca²⁺/CaM-independent CaM-KIV after serum withdrawal consistently results in the appearance of a small amount of the mobility-shifted band, indicating the presence of Ca²⁺/CaM-independent CaM-KK activity in these cells. This is consistent with previous observations demonstrating that CaM-KK, unlike CaM-KK, is highly Ca²⁺/CaM-independent (5, 13). Treatment of infected SH-SY5Y cells with 1 µM ionomycin for 10 min significantly induces the activation of CaM-KIV, which is dramatically suppressed by treatment with 1 µg/ml STO-609 (~80% inhibition). Concentrations of ~0.1 µg/ml STO-609 cause a 50-60% inhibition of the CaM-KIV activation. This result correlates well with the inhibitory effect of STO-609 on CaM-KK in vitro as shown in Fig. 1. SH-SY5Y neuroblastoma cells are apparently more sensitive to STO-609 than are HeLa cells, as shown in Fig. 4 with respect to the inhibition of endogenous CaM-KK activity, suggesting that the permeability of cells to the compound is cell type-dependent. It is noteworthy that concentrations of STO-609 up to 10 µg/ml do not affect the viability of treated cells including transfected HeLa cells and SH-SY5Y cells.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effect of STO-609 on endogenous CaM-KK activity in SH-SY5Y neuroblastoma cells. A, SH-SY5Y cells were infected with recombinant adenovirus carrying either a Ca2+/CaM-independent CaM-KIV (305HMDT-DEDD, CaM-KIVc) or a kinase-deficient mutant (305HMDT-DEDD, K71E, CaM-KIVd), with or without constitutively active CaM-KK-(1-434) (CaM-KKc) as indicated, and cell extracts (20 µg of protein) were subjected to SDS-7.5% PAGE followed by Western blot analysis with an anti-CaM-KIV antibody. B, after infection of recombinant adenovirus carrying Ca2+/CaM-independent CaM-KIV (305HMDT-DEDD, CaM-KIVc), the cells were treated with various concentrations of STO-609 (0.01-10 µg/ml) as indicated for 6 h or untreated with the inhibitor and then stimulated with 1 µM ionomycin for 10 min (+) or not stimulated (), as described under "Experimental Procedures." Cell extracts were analyzed by Western blotting analysis with an anti-CaM-KIV antibody as shown in A. The intensity of the mobility-shifted band (upper arrow) was estimated by densitometric scanning of the x-ray film, and CaM-KK activity is expressed as a percent inhibition of the value in the absence of STO-609 with ionomycin stimulation.

In summary, we have recently developed a potent and relatively selective inhibitor of CaM-KK, STO-609, which can permeate cells and which is a competitive inhibitor of ATP. Recent studies demonstrate that CaM-KK is a regulatory protein kinase for CaM-KI and CaM-KIV in vitro and in transfected cells; however, this property has not been demonstrated directly in vivo. We have shown in this report that STO-609 suppresses CaM-KK activity resulting in the inhibition of downstream CaM-KIV activity in intact cells, although it cannot inhibit downstream CaM kinase activities in vitro. Thus STO-609 could be a useful tool for evaluating the regulatory roles of CaM-KK for various physiological functions of the CaM kinase cascade such as the regulation of gene expression mediated by the CaM-KIV pathway. Furthermore, STO-609 could be used to distinguish between the functions of the two CaM-KK isoforms because the sensitivity of CaM-KK to the compound is ~5-fold higher than that of the  isoform. The mechanism of differential sensitivity of CaM-KK isoforms to STO-609 is not clear, but it is not likely due to their differences in affinity for ATP because the apparent K_m values of CaM-KK and CaM-KK for ATP are indistinguishable (~33 µM, Fig. 3B). Furthermore, physiological function(s) controlled by the CaM-KK/CaM-KI cascade have not been well studied, in contrast to what is known for CaM-KIV, and this question may be addressed with the use of STO-609.

	ACKNOWLEDGEMENTS

We thank Sachi Tanaka and Nahoko Ishikawa (Kagawa Medical University) for excellent technical assistance.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The 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. Tel./Fax: 81-87-891-2368; E-mail: tokumit@kms.ac.jp.

Published, JBC Papers in Press, February 26, 2002, DOI 10.1074/jbc.M201075200

	ABBREVIATIONS

The abbreviations used are: CaM-K, Ca²⁺/CaM-dependent protein kinase; CaM, calmodulin; MLCK, myosin light chain kinase; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; MAP kinase, mitogen-activated protein kinase; CREB, cAMP-response element binding protein; HA, hemagglutinin; GST, glutathione S-transferase; DTT, dithiothreitol.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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