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J. Biol. Chem., Vol. 275, Issue 26, 20090-20095, June 30, 2000

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Regulatory Mechanism of Ca²⁺/Calmodulin-dependent Protein Kinase Kinase^*

Hiroshi Tokumitsu^§, Masa-aki Muramatsu^¶, Mitsuhiko Ikura, and Ryoji Kobayashi

From the  Department of Chemistry, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-93, Japan, ^¶ Helix Research Institute, Inc., 1532-3 Yana, Kisarazu-shi, Chiba 292-0812, Japan, and  Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Ontario M5G 2M9, Canada

Received for publication, March 16, 2000, and in revised form, April 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Ca²⁺/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM-binding peptide of CaM-KK (residues 438-463), which suppressed the activity of constitutively active CaM-KK (84-434) in the absence of Ca²⁺/CaM but competitively with ATP. Truncation and site-directed mutagenesis of the CaM-binding region in CaM-KK reveal that Ile⁴⁴¹ is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu⁴⁴⁰, exhibited enhanced Ca²⁺/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, M. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol. 6, 819-824), the chimeric CaM-KKs containing Ile⁴⁴¹ remained Ca²⁺/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile⁴⁴¹ for autoinhibition, which is located at the 3 position from the N-terminal anchoring residue (Trp⁴⁴⁴) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Ca²⁺/calmodulin-dependent protein kinase kinase (CaM-KK)¹ has been identified and cloned as an activator for two multifunctional CaM kinases, CaM-KI and IV. The identification of this new CaM kinase regulating other CaM kinases revealed a novel intracellular Ca²⁺-mediated signal transduction pathway, the CaM kinase cascade (1-6). Phosphorylation by CaM-KK of Thr, located in the activation loop of the catalytic domain of CaM-KI and CaM-KIV, results in a large increase in the catalytic efficiency of each CaM kinase (5-7). Two CaM-KK genes ( and ) have been cloned in mammal (4, 8, 9), whereas only one gene was characterized in Caenorhabditis elegans (10, 11). In mammals, the highest expression of the  and  isoforms of CaM-KK is observed in the brain, although the  isoform is also expressed in various peripheral tissues such as thymus and spleen (4).

Numerous studies demonstrated that the CaM-KK/CaM-KIV cascade is present and functional in various cell types such as Jurkat cells (12), cultured hippocampal neurons (13), and transfected COS-7 cells (7). An important role for the CaM-KK/CaM-KIV cascade in the regulation of Ca²⁺-dependent gene expression through phosphorylation of transcription factors such as cAMP-response element-binding protein has been demonstrated (13-17). The CaM-KK/CaM-KI cascade is activated in PC12 cells upon membrane depolarization, although its physiological role remains to be determined (18). Recently a kinase homologous to mammalian CaM-KI (CeCaM-KI) has been cloned in C. elegans (19). Unlike mammalian CaM-KI, overexpressed CeCaM-KI is predominantly localized in the nucleus of transfected cells because of the presence of an N-terminal nuclear localization signal. In transfected cells, CeCaM-KK and CeCaM-KI reconstitute a signaling pathway that mediates Ca²⁺-dependent phosphorylation of cAMP-response element-binding protein and cAMP-response element-dependent transcription, similarly to the mammalian CaM-KK/CaM-KIV pathway. In addition to CaM-KI and IV, protein kinase B has been identified as a target for CaM-KK. The phosphorylation and activation of protein kinase B by CaM-KK is responsible for the anti-apoptotic effect upon modest elevation of intracellular Ca²⁺ in NG108 cells (20).

CaM-KK has an N-terminal catalytic domain and a regulatory domain containing autoinhibitory and CaM-binding segments at its C terminus in a similar manner to other CaM kinases. The catalytic domain of CaM-KK contains a unique Arg-Pro-rich 22 residues insert, which plays an important role for recognition of downstream CaM kinases (11). The binding of Ca²⁺/CaM to CaM-KK is absolutely required for its activation and efficient phosphorylation of target protein kinases (7, 27). Previous studies using site-directed mutagenesis and synthetic peptides identified the CaM-binding region of CaM-KK as spanning residues 438-463 (21). The three-dimensional structure of the Ca²⁺-CaM complex with the CaM-KK peptide (residues 438-463) has been resolved by NMR spectroscopy. The CaM-KK peptide forms a fold comprising an -helix (residues 444-454) and a hairpin-like loop (residues 455-459) whose C terminus folds back onto the helix (22). This unique loop structure in CaM-KK peptide is stabilized by intramolecular hydrophobic interaction between Met⁴⁵³ and Phe⁴⁵⁹, which anchor to the C-terminal domain of CaM, as well as interactions involving Phe⁴⁶³, Ile⁴⁴⁸, and Leu⁴⁴⁹. This was confirmed by mutation of either Phe⁴⁵⁹ or Phe⁴⁶³ to Asp, which resulted in a significant reduction of CaM-binding ability of CaM-KK. Trp⁴⁴⁴ was also identified as another anchoring residue to the N-terminal domain of CaM. Therefore, the orientation of the CaM-KK peptide with respect to the two CaM domains is opposite to that of the MLCKs and CaM-KII peptides (35-37). These observations raised the question why CaM-KK requires an opposite orientation of CaM binding to that of other CaM kinases for the regulation of its protein kinase activity. In this report, we present evidences using CaM-KK mutants and chimeras to assume the critical role of the unique regulatory domain of CaM-KK and the orientation of CaM binding for the kinase autoinhibition.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- CaM-KK cDNA (GenBank^TM accession number L42810) was from a rat brain cDNA library (4). GST-CaM-KK-(84-434) was constructed by the insertion of a XbaI-digested fragment from pME-CaM-KK-(1-434, 21) into pGEX-KG vector, and the recombinant enzyme was expressed in Escherichia coli JM109 and purified on glutathione-Sepharose. GST-CaM-KI-(1-293, K49E) into pGEX-4T3 was constructed by polymerase chain reaction followed by mutagenesis using a mutagenic oligonucleotide (5'-AAACTGGTGGCCATCGAATGCATTGCCAAGAAG-3') and a GeneEditor in vitro site-directed mutagenesis system (Promega Co.). Expressed GST-CaM-KI-(1-293, K49E) in E. coli JM109 was purified on glutathione-Sepharose. Recombinant rat CaM was expressed in E. coli BL-21 (DE3) using pET-CaM (23), which was kindly provided from Dr. Nobuhiro Hayashi (Fujita Health University, Toyoake, Japan), and purified by phenyl-Sepharose column chromatography. Nucleotide sequences of those constructs were confirmed. Twenty six-residue synthetic peptides corresponding to the calmodulin-binding domain of both rat CaM-KK (residues 438-463) (21) and C. elegans CaM-KK (residues 331-356) (11) were synthesized by the Peptide Institute Inc. (Osaka, Japan). Purity of either peptide was estimated at higher than 95% judging by high pressure liquid chromatography. All other chemicals were from standard commercial sources.

Construction of CaM-KK Mutants-- Mutagenesis of CaM-KK using pME-CaM-KK (wild-type, 4) plasmid as a template was carried out by a GeneEditor in vitro site-directed mutagenesis system (Promega Co.) and mutagenic oligonucleotides as follows; I441A, 5'-AACTCAGTCAAGCTTGCCCCCAGCTGGACC-3'; I441R, 5'-AACTCAGTCAAGCTTCGCCCCAGCTGGACC-3'; I441D, 5'-AACTCAGTCAAGCTTGACCCCAGCTGGACC-3'.

For CaM-KK truncation and chimera mutants, pME-CaM-KK-(1-463, 21) was mutated at Ile⁴⁴¹ by a mutagenic oligonucleotide (5'-AAGAACTCAGTCAAGCTTAAGCCCAGCTGGACCACT-3') to introduce the AflII site and then digested with AflII followed by treating with mung bean nuclease to create a blunt end at the first codon (C) of Leu⁴⁴⁰. After digestion with NotI, the linearized plasmid ligated with pre-annealed double-stranded oligonucleotides containing the NotI site as follow: pME-CaM-KK-(1-440), 5'-TCTGACATATGCGCTCTAGAACTAGTGC-3' and 5'-GGCCGCACTAGTTCTAGAGCGCATATGTCAGA-3'; pME-CaM-KK-(1-441), 5'-TCATCTGACATATGCGCTCTAGAACTAGTGC-3' and 5'-GGCCGCACTAGTTCTAGAGCGCATATGTCAGATGA-3'; pME-CaM-KK-(1-443), 5'-TCATCCCCAGCTGACATATGCGCTCTAGAACTAGTGC-3' and 5'-GGCCGCACTAGTTCTAGAGCGCATATGTCAGCTGGGGATGA-3'; pME-CaM-KK-(1-444), 5'-TCATCCCCAGCTGGTGACATATGCGCTCTAGAACTAGTGC-3' and 5'-GGCCGCACTAGTTCTAGAGCGCATATGTCACCAGCTGGGGATGA-3'; pME-CaM-KK-440/smMLCK (chicken gizzard, residues 797-816), 5'-TCAGAAGAAAATGGCAGAAAACAGGCCATGCTGTCCGAGCAATAGGAAGACTGTCATCCATGTGAGC-3' and 5'-GGCCGCTCACATGGATGACAGTCTTCCTATTGCTCGGACAGCATGGCCTGTTTTCTGCCATTTTCTTCTGA-3'; pME-CaM-KK-440/skMLCK (rabbit skeletal muscle, residues 577-596), 5'-TCAAGAGGCGCTGGAAGAAAAACTTCATTGCCGTCAGCGCTGCCAACCGCTTCAAGAAGATCTGAGC-3' and 5'-GGCCGCTCAGATCTTCTTGAAGCGGTTGGCAGCGCTGACGGCAATGAAGTTTTTCTTCCAGCGCCTCTTGA-3'; pME-CaM-KK-440/CaM-KII (rat, residues 296-313), 5'-TCCGACGGAAATTGAAGGGTGCCATCTTGACAACTATGCTGGCTACGAGAAATTTTTGAGC-3' and 5'-GGCCGCTCAAAAATTTCTCGTAGCCAGCATAGTTGTCAAGATGGCACCCTTCAATTTCCGTCGGA-3'. pME-CaMKK-(1-448) and (1-434) were constructed as described previously (21).

For construction of Ile⁴⁴¹-inserted CaM-KK chimera mutants, each chimeric mutant described above was digested with EcoRI and NotI and re-inserted into a newly digested pME18s plasmid, which was used as a template for mutagenesis. Mutagenic oligonucleotides are described as follows: pME-CaM-KK-441/skMLCK, 5'-AAGAACTCAGTCAAGCTTATCAAGAGGCGCTGGAAGAA; pME-CaM-KK-441/smMLCK, 5'-AAGAACTCAGTCAAGCTTATCAGAAGAAAATGGCAGAA-3'; pME- CaM-KK-441/CaM-KII, 5'-AAGAACTCAGTCAAGCTTATCCGACGGAAATTGAAGGG. The nucleotide sequence of each mutant was confirmed by automated sequencing using an Applied Biosystems 377 DNA sequencer.

Transient Expression and Partial Purification of CaM-KK Mutants-- COS-7 cells were maintained in Dulbeco's modified Eagle's medium containing 10% fetal calf serum. Cells were subcultured in 10-cm dishes 12 h before transfection. The cells were then transferred to serum-free medium and treated with a mixture of either 10 µg of pME18s plasmid DNA (DNAX Research Institute, Inc.) or CaM-KK cDNA containing plasmid DNAs and 60 µg of LipofectAMINE Reagent (Life Technologies, Inc.) in 6.4 ml of medium. After a 32-48-h incubation, the cells (3 plates) were collected and homogenized with 1 ml of lysis buffer (150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/liter leupeptin, 10 mg/liter pepstatin A, 10 mg/liter trypsin inhibitor) at 4 °C. After centrifugation at 15,000 × g for 15 min, the supernatant was diluted by the addition of twice the volume of 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/liter leupeptin, 10 mg/liter pepstatin A, and 10 mg/liter trypsin inhibitor (buffer A) and then applied to a Q-Sepahrose column (0.5-ml bed volume), which had been pre-equilibrated with 50 mM NaCl containing buffer A. After washing the column with 10 ml of the equilibration buffer, CaM-KK was eluted by the addition of 0.5 ml of 0.6 M NaCl containing buffer A and stored at 80 °C.

In Vitro Assay of CaM-KK Mutants-- Either partially purified CaM-KK (1 µl) from transfected COS-7 cells or E. coli expressed GST-CaM-KK-(84-434, 0.7 µg/ml) was 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 dithiothreitol, 100 µM [-³²P]ATP (1000-2000 cpm/pmol) in the presence of either 2 mM EGTA or 2 mM CaCl₂, 3 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 washing in 75 mM phosphoric acid (24). Phosphate incorporation into GST-CaM-KI-(1-293, K49E) was quantitated by liquid scintillation counting of the filters. For each experiment, ~10-fold volume of partially purified CaM-KKs used in the assay was analyzed by Western blotting to check the amount of the enzymes. CaM-KK activity was measured for 5 min, which was under the linear condition.

Others-- Western blotting was carried out using antiserum (1/1000 dilution) against a peptide corresponding to a conserved protein kinase motif (residues 132-146 of CaM-KII), and the biotinylated-CaM overlay was done as described previously (4). Detection was performed by using chemiluminescence reagent (NEN Life Science Products). Protein concentration was estimated by Coomassie Blue dye binding (Bio-Rad) using bovine serum albumin as a standard (25).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We have previously identified the CaM-binding domain at the C-terminal of rat CaM-KK (residues 438-463) by using mutagenesis and a synthetic peptide (Fig. 1A) (21). Recently, a study using NMR spectroscopy has shown that the CaM-binding peptide (residues 438-463) of CaM-KK bound to the N- and C-terminal domains of CaM in an opposite direction to all other known CaM kinases such as MLCKs and CaM-KII (22). This result raised the possibility that CaM-KK is regulated by a mechanism distinct from that controlling other CaM kinases. Indeed, we have found that the CaM-binding peptide (residues 438-463) of CaM-KK is a weak inhibitor of the enzyme, when CaM-KIV activation assay was used to measure CaM-KK activity (21). In this study, we performed a direct phosphorylation assay toward the GST-CaM-KI-(1-293, K49E) mutant as a substrate to determine CaM-KK activity. The use of this CaM-KI mutant has numerous advantages. First, CaM-KI lacking a regulatory domain (including an autoinhibitory and CaM-binding domains) is more suitable for measuring the Ca²⁺/CaM sensitivity of CaM-KK. Second, substitution of Lys⁴⁹ by Glu abolishes the binding of ATP and allows us to rule out the possibility of a feedback phosphorylation of CaM-KK by activated CaM-KI, which would indirectly affect CaM-KK activity (26). Finally, a direct phosphorylation assay allows us to perform kinetic analysis of CaM-KK activity. Thus we examined the inhibitory effect of the CaM-KK peptide (residues 438-463) on the constitutively active mutant (84-434) of CaM-KK lacking both an autoinhibitory and CaM-binding domains (7). As shown in Fig. 1B, the peptide inhibits CaM-KK 84-434 activity with an IC₅₀ of ~15 µM in the absence of Ca²⁺/CaM. This inhibition is comparable to that obtained with the corresponding peptide from C. elegans CaM-KK (residues 331-356). Kinetic analysis indicated that the peptide inhibition of CaM-KK 84-434 was competitive with respect to ATP (Fig. 1B, inset) but not to its protein substrate (data not shown). When Ca²⁺/CaM was added into the inhibitory reaction using 24 µM CaM-KK peptide, the activity of CaM-KK 84-434 was restored up to 80-90% of its original activity in a dose-dependent manner (Fig. 1C). These results indicated that the CaM-binding peptide could also interact with the catalytic domain of CaM-KK and function as an autoinhibitory peptide. Previous mutagenesis study revealed that block mutation of either Lys⁴³⁵-Asn⁴³⁶-Ser⁴³⁷ or Val⁴³⁸-Lys⁴³⁹-Leu⁴⁴⁰ by triple Asp resulted in increased Ca²⁺/CaM-independent activity (10-20% of total activity), suggesting that these residues are involved in autoinhibitory function either directly or indirectly (21). Because constitutive activity of those mutants is low compared with that of fully constitutive active mutant (1-434), we tested the possibility that other regions of CaM-KK, further C-terminal from residue 440, could be important for autoinhibition. When we truncated at residue 448 in CaM-KK, the mutant was inactive (21). A recent study demonstrated that the truncation mutant of CaM-KK at Trp⁴⁴⁴, which turns out to be the anchoring residue to N-terminal domain of CaM (22), was also inactive (27). Based on these results, we postulated that the important residue(s) for the autoinhibition are located between residue 438 and 444. 

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Inhibition of CaM-KK (84-434) activity by CaM-KK peptide (residues 438-463). A, amino acid sequence alignment of the Ca2+/CaM-binding region of various CaM-KKs (rat  and  isoforms and C. elegans) (4, 8-11). CeCaM-KK, C. elegans CaM-KK. B, GST-CaM-KK-(84-434, 0.7 µg/ml) was incubated with GST-CaM-KI-(1-293, K49E, 0.4 mg/ml) without Ca2+/CaM in the absence or presence of either CaM-KK peptide (residue 438-463, 1-100 µM, closed circle) or C. elegans CaM-KK peptide (residue 331-356, 1-100 µM, open circle) for 5 min at 30 °C as described under "Experimental Procedures." Inset, GST-CaM-KK-(84-434) activity was measured as described in A in the either absence (open circle) or presence of 20 µM CaM-KK peptide (residue 438-463, closed circle) using 50-400 µM [-32P]ATP. The results are presented as double reciprocal plots (Lineweaver-Burk). C, GST-CaM-KK-(84-434) activity was measured as described in A using 20 µM of [-32P]ATP in the either absence () or presence of 24 µM CaM-KK peptide (residue 438-463, +) with various concentrations of calmodulin (0-26.8 µM) as indicated. The experiments were performed in triplicate for each point and the results are presented as the mean and S.E. of three experiments.

We, therefore, constructed other truncated CaM-KK mutants (Fig. 2 A) and overexpressed them in COS-7. The expression level of each mutant was analyzed by immunoblotting after partial purification on Q-Sepharose (Fig. 2B). Direct phosphorylation assay toward GST-CaM-KI-(1-293, K49E) by each mutant was performed in the absence of Ca²⁺/CaM (Fig. 2C). Consistent with previous observation (21), a truncated version of CaM-KK at Phe⁴⁶³ did not exhibit significant Ca²⁺/CaM-independent activity compared with the wild-type enzyme. Further truncation from residue 448 to 441 generated detectable but weak Ca²⁺/CaM-independent activity, which is ~20-25% of fully constitutively active mutant (1-434). Interestingly, truncation at Ile⁴⁴¹ resulted in complete constitutive activity of CaM-KK similar to that of 1-434 mutant. This result suggests that Ile⁴⁴¹ is essential but residue 435-440 may not be absolutely critical for CaM-KK autoinhibition. Our previous result demonstrating a 10-20% constitutive activity of CaM-KK after triple Asp substitution between residues 435-440 was likely because of the indirect effect of the mutation on the function of residue 441. It is noteworthy that Ile⁴⁴¹ is conserved in -isoform (8, 9) and C. elegans CaM-KK (10, 11) (Fig. 1A).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Ca2+/CaM-independent activity of truncated CaM-KK mutants. A, schematic representation of wild-type and truncated mutants of CaM-KK. A series of truncation mutant of CaM-KK were constructed and expressed in COS-7 cells followed by partial purification as described under "Experimental Procedures." B, partially purified truncation mutants of CaM-KK described in A (~1 µg of protein) including Mock were subjected to SDS/10% polyacrylamide gel electrophoresis followed by Western blotting using antiserum (1/1000 dilution) against a peptide corresponding to a conserved protein kinase motif (residues 132-146 of CaM-KII). C, partially purified truncated CaM-KKs (~0.1 µg of protein) were incubated with GST-CaM-KI-(1-293, K49E, 0.4 mg/ml) in the absence of Ca2+/CaM for 5 min at 30 °C as described under "Experimental Procedures." The experiment was performed in triplicate for each point, and the results are presented as the mean and S.E. of three experiments. WT, wild type.

To confirm the involvement of Ile⁴⁴¹ in the regulatory mechanism of CaM-KK, we mutated Ile⁴⁴¹ by Ala, Arg, or Asp in the wild-type CaM-KK. Each mutant exhibited 40-60% of Ca²⁺/CaM-independent activity and was further activated by Ca²⁺/CaM (Fig. 3B). A similar result was obtained when these mutations were introduced into the CaM-KK 1-463 mutant (data not shown). The mutation of Ile⁴⁴¹ by Asp significantly reduced the binding ability of CaM compared with either wild type or other Ile⁴⁴¹ mutants (Fig. 3A, lower panel). This result is in accordance with the previous observation that the mutation of Val⁴³⁸-Lys⁴³⁹-Leu⁴⁴⁰ by triple Asp reduced the binding ability of Ca²⁺-CaM complex (21). This is probably because of an intermolecular electrostatic repulsion, because there are many acidic residues in the region of CaM, which binds to the N terminus of the CaM-KK peptide (residues 438-463) (22). These results demonstrated that the CaM binding and autoinhibitory regions in CaM-KK are overlapped at Ile⁴⁴¹.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Point mutation of Ile441 in CaM-KK. A, wild-type CaM-KK was mutated at Ile441 by Ala (I441A), Arg (I441R), or Asp (I441D) and expressed in COS-7 cells followed by partial purification as described under "Experimental Procedures." Each mutant (~2 µg of protein) was analyzed by either Western blotting (upper panel) as shown in Fig. 2B or the CaM overlay method (lower panel). B, each CaM-KK mutant (~0.3 µg of protein) including Mock was incubated with GST-CaM-KI-(1-293, K49E, 0.4 mg/ml) in the either absence (open bar) or presence (closed bar) of Ca2+/CaM for 5 min at 30 °C as described under "Experimental Procedures." The experiment was performed in triplicate for each point, and the results are presented as the mean and S.E. of three experiments. WT, wild type.

Extensive mutagenesis studies indicated that most of the elements required for CaM binding and autoinhibition of MLCK and CaM-KII were located within the combined segment but clearly separated because mutation of the CaM recognition region has little effect on autoinhibition of the kinases (28-33). Moreover, the CaM recognition sequence among MLCKs and CaM-KII can be interchangeable (28, 34). This may be guaranteed by the fact that MLCKs and CaM-KII contain basic amino acid clusters located at N-terminal of the first hydrophobic anchoring residue resulting in the same direction of CaM-binding (35-37). In contrast, the three-dimensional structure of the Ca²⁺-CaM complex with the CaM-KK peptide (residues 438-463) has shown that the orientation of CaM binding to the peptide was opposite of that observed for MLCKs and CaM-KII. This difference can be due to the presence of a basic amino acid cluster (Lys⁴⁵¹, Arg⁴⁵⁵-Lys⁴⁵⁶-Arg⁴⁵⁷) in the C-terminal region, which interacts with the negatively charged C-terminal channel outlet of CaM (22).

To determine whether the proper orientation of CaM binding to CaM-KK was required for the regulation of the enzyme, we constructed chimera CaM-KK mutants that contained CaM-binding segment of either chicken smMLCK (residues 797-816) (38), rabbit skMLCK (residues 577-596) (39), or rat CaM-KII (residues 296-313) (40, 41) based on the position of an N-terminal anchoring residue to CaM (Trp⁴⁴⁴ in CaM-KK, Trp⁸⁰⁰ in smMLCK, Trp⁵⁸⁰ in skMLCK, and Leu²⁹⁹ in CaM-KII) (Fig. 4A). The clusters of basic amino acids, which are critical for the orientation of the binding of CaM to the peptide, are located at the N terminus of the CaM-binding sequence of each CaM kinase. Therefore, we fused each CaM-binding segment with the catalytic portion of CaM-KK at Leu⁴⁴⁰. These chimera enzymes were expressed in COS-7 cells and partially purified to analyze their enzymatic activities and Ca²⁺/CaM binding by CaM overlay method (Fig. 4B). All of the chimeric enzymes retained the ability to bind Ca²⁺/CaM as expected (Fig. 4B, lower panel). Unlike the wild-type and 1-463 mutant of CaM-KK, which are Ca²⁺/CaM-dependent enzymes, all of the chimeric mutants expressed ~60% of constitutive activity (Fig. 4C). It is not surprising because Ile⁴⁴¹, which is critical for autoinhibition described above, was replaced by a basic residue (Arg or Lys) in all of the chimera mutants. In the presence of Ca²⁺/CaM, all chimeric enzymes slightly enhanced the CaM-KK activity suggesting that the CaM-binding region of each CaM kinase slightly blocks the catalytic activity. This is consistent with that Ile⁴⁴¹ mutants of CaM-KK, which exhibited 40-60% constitutive activities and were further activated by the addition of Ca²⁺/CaM (Fig. 3B).

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Characterization of CaM-KK chimera mutants. A, schematic representation of wild-type, 1-463 mutant, and the chimera mutant of CaM-KK containing the CaM-binding region of rabbit skeletal muscle MLCK (residues 577-596, 440-skMLCK) (39), chicken smooth muscle MLCK (residues 797-816, 440-smMLCK), (38) or rat CaM kinase II (residues 296-313, 440-CaM-KII) (40, 41). Chimera CaM-KKs were constructed and expressed in COS-7 cells followed by partial purification as described under "Experimental Procedures." The binding direction of CaM to the CaM-binding sequence of each chimera mutant is indicated in the right lane based on previous structural analysis using NMR and crystallography (22, 35-37). Hydrophobic anchoring residues to N- and C-terminal domains of CaM are indicated by asterisks. B, partially purified CaM-KK chimera mutants (~2 µg of protein) including wild-type, 1-463 mutant, and Mock were analyzed by either Western blotting (upper panel) as shown in Fig. 2B or the CaM overlay method (lower panel). C, partially purified CaM-KK chimera mutants (~0.2 µg of protein) including wild-type, 1-463 mutant, and Mock were incubated with GST-CaM-KI-(1-293, K49E, 0.4 mg/ml) in the either absence (open bar) or presence (closed bar) of Ca2+/CaM for 5 min at 30 °C as described under "Experimental Procedures." The experiment was performed in triplicate for each point, and the results are presented as the mean and S.E. of three experiments.

Finally we inserted Ile⁴⁴¹ into the boundary between the catalytic domain and CaM-binding region of each chimeric CaM-KK to examine Ca²⁺/CaM dependence of the mutants (Fig. 5A). As shown in Fig. 5B, all of the chimera CaM-KKs containing Ile⁴⁴¹ are virtually inactive in the absence of Ca²⁺/CaM and are activated by Ca²⁺/CaM to the same extent to the wild-type CaM-KK. These results demonstrate that the orientation of Ca²⁺/CaM binding to the CaM-binding region is not critical for the relief of CaM-KK autoinhibition.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effect of Ile441 insertion on the activities of CaM-KK chimera mutants. A, schematic representation of wild-type and chimera mutant of CaM-KK contained CaM-binding region of skeletal muscle MLCK (441-skMLCK), smooth muscle MLCK (441-smMLCK), or CaM kinase II (441-CaM-KII) in which Ile441 is inserted. Chimera CaM-KKs containing Ile441 were constructed and expressed in COS-7 cells followed by partial purification as described under "Experimental Procedures." Ile441 in each chimera mutant is indicated by a box. Hydrophobic anchoring residues to N- and C-terminal domains of CaM are indicated by asterisks. B, partially purified CaM-KK chimera mutants (~0.4 µg of protein) including wild-type and Mock were incubated with GST-CaM-KI-(1-293, K49E, 0.4 mg/ml) in the either the absence (open bar) or presence (closed bar) of Ca2+/CaM for 5 min at 30 °C as described under "Experimental Procedures." The experiment was performed in triplicate for each point, and the results are presented as the mean and S.E. of three experiments. Inset, partially purified CaM-KK chimera mutants (~3 µg pf protein) including wild-type and Mock were analyzed by Western blotting (indicated by arrows) as shown in Fig. 2B. Lane 1, mock; lane 2, wild-type CaM-KK; lane 3, 441-skMLCK; lane 4, 441-smMLCK; lane 5, 441-CaM-KII.

Conclusion-- The present mutagenesis identified Ile⁴⁴¹ as a critical residue for autoinhibition of CaM-KK located at 3 position from Trp⁴⁴⁴, which is an anchoring residue to the N-terminal hydrophobic pocket of CaM. Three-dimensional structural analysis of Ca²⁺/CaM binding (35-37) and sequence alignment of MLCKs and CaM-KII show that the residues at the 1 to 3 position from the N-terminal anchoring residue to CaM (Fig. 4A) are basic in all of them. These residues are critical for the determination of the direction in which CaM is binding, as they interact with more negatively charged C-terminal domain than N-terminal domain of CaM. In the case of CaM-KK, substitution of Ile⁴⁴¹ by either Ala, Arg, or Asp resulted in increased constitutive activity (Fig. 3B), suggesting that a residue with a lower hydrophobicity at 3 position from Trp⁴⁴⁴ caused suppression of the autoinhibitory function. Thus, a basic residue should not be located at a key position for the autoinhibition in CaM-KK. In agreement with this, the cluster of basic amino acid is located at the C-terminal portion in CaM-binding segment of CaM-KK (Lys⁴⁵¹, Arg⁴⁵⁵-Lys⁴⁵⁶-Arg⁴⁵⁷), resulting in an opposite direction of Ca²⁺/CaM binding compared with other CaM kinases such as MLCKs and CaM-KII (22), although the direction of Ca²⁺/CaM binding itself is not critical for relief of the autoinhibition. This is supported by the result that all of the chimeric CaM-KKs lacking Ile⁴⁴¹ exhibited enhanced Ca²⁺/CaM-independent activity (Fig. 4). Kinetic analysis of CaM-KK peptide inhibition raises the possibility that Ile⁴⁴¹ interacts with the ATP-binding domain directly and/or distort the ATP-binding site allosterically by interacting with the catalytic domain of CaM-KK. This might be analogous to Phe³⁰⁷ in the CaM-binding domain of CaM-KI that interacts with Phe³¹ in its glycine-rich ATP loop resulted in obstructing the nucleotide binding pocket (42). We propose that the Ca²⁺/CaM-binding segment of CaM-KK with an opposite orientation of CaM binding to other CaM kinases plays a key role for its autoinhibitory function through Ile⁴⁴¹. Ile⁴⁴¹ is likely the residue released from the catalytic domain upon binding of Ca²⁺/CaM to the regulatory region. Future studies including structural determination will be necessary to establish the molecular mechanism of interaction between Ile⁴⁴¹ and the catalytic domain of CaM-KK and to determine how this residue contributes to the autoinhibitory function of CaM-KK.

	ACKNOWLEDGEMENTS

We thank Naomi Takahashi (Helix Research Institute), Masato Iwabu, Yumi Ishikawa, and Miki Okada (Kagawa Medical University) for excellent technical assistance and Dr. Hérve Enslen for critical reading of the manuscript.

	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, April 18, 2000, DOI 10.1074/jbc.M002193200

	ABBREVIATIONS

The abbreviations used are: CaM-KK, Ca²⁺/CaM-dependent protein kinase kinase; CaM, calmodulin; MLCK, myosin light chain kinase; GST, glutathione S-transferase; sk, skeletal muscle; sm, smooth muscle.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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