©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stabilization of Calmodulin-dependent Protein Kinase II through the Autoinhibitory Domain (*)

(Received for publication, August 15, 1994; and in revised form, November 14, 1994)

Atsuhiko Ishida Hitoshi Fujisawa

From the Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The active 30-kDa chymotryptic fragment of calmodulin-dependent protein kinase II (CaM kinase II), devoid of the autoinhibitory domain, and the enzyme, autothiophosphorylated at Thr/Thr, were much more labile than was the original native enzyme. They were markedly stabilized by synthetic peptides, designed after the sequence around the autophosphorylation site in the autoinhibitory domain, such as autocamtide-2 and CaMK-(281-309), but such marked stabilizations were not observed with the ordinary exogenous substrates, such as syntide-2. These results suggest that the autoinhibitory domain of CaM kinase II plays a crucial role in stabilizing the enzyme. A nonphosphorylatable analog of autocamtide-2, AIP, strongly inhibited the activity of the 30-kDa fragment. Kinetic analysis revealed that the inhibition by AIP was competitive with respect to autocamtide-2 and CaMK-(281-289) and noncompetitive with respect to syntide-2 and ATP/Mg, suggesting that CaM kinase II possesses at least two distinct substrate-binding sites; one for ordinary exogenous substrates such as syntide-2 and the other for an endogenous substrate, the autophosphorylation site (Thr/Thr) in the autoinhibitory domain. Fluorescence analysis of the binding of 7-nitrobenz-2-oxa-1,3-diazole-4-yl labeled AIP to the 30-kDa fragment also supported this contention. Thus, the autoinhibitory domain appears to play a crucial role in keeping the enzyme stable by binding to the substrate-binding site for the autophosphorylation site.


INTRODUCTION

Calmodulin-dependent protein kinase II (CaM kinase II) (^1)is known to be a second-messenger-responsive multifunctional protein kinase which plays important roles in controlling a variety of cellular functions in response to an increase in intracellular Ca (reviewed in (1, 2, 3) ). CaM kinase II is widely distributed in many tissues and organisms, but most abundantly in brain. CaM kinase II is thought to play important roles in the central nervous system, and possible involvements of CaM kinase II in the regulation of neuronal functions such as neurotransmitter synthesis(4, 5, 6) , neurotransmitter release(7, 8) , long-term potentiation(9, 10, 11) , and formation of spatial learning (12) have so far been suggested.

Rat brain CaM kinase II is an oligomeric enzyme with a molecular weight of about 540,000 and is composed of subunits ranging in molecular weight from about 55,000 to 60,000(1, 2, 3) . The primary structure of four rat brain CaM kinase II isoforms, alpha(13) , beta(14, 15) , (16) , and (17) , have been determined from the cDNA clones. Each subunit has the consensus ATP-binding domain near the amino terminus, the catalytic domain in the amino-terminal region, and the regulatory domain consisting of the autoinhibitory and calmodulin-binding domains in the central region of the protein(1, 2, 3) . Upon binding Ca/calmodulin in the presence of ATP/Mg, CaM kinase II undergoes a rapid autophosphorylation on Thr/Thr (in alpha/beta, , and isoforms) located within the autoinhibitory domain, leading to the full activation of the enzyme (18, 19) as well as the generation of the Ca/calmodulin-independent activity(20, 21, 22, 23, 24) . This autophosphorylation has recently been reported to cause a dramatic increase in the affinity of the enzyme for Ca/ calmodulin(25) .

Further autophosphorylation of the enzyme following the initial rapid autophosphorylation on Thr results in a gradual loss of the enzymatic activity(26, 27, 28) . Lai et al.(29) have suggested that the inactivation may be a consequence of a decrease in the thermal stability of the enzyme. Colbran (30) has reported that basal autophosphorylation at Thr in the calmodulin-binding site blocks calmodulin binding, resulting in inactivation of the enzyme.

We have recently reported that incubation of the enzyme with Ca/calmodulin causes a marked inactivation of the enzyme and that the inactivation was prevented by autocamtide-2, a synthetic substrate peptide modeled after the sequence around the autophosphorylation site (Thr) within the autoinhibitory domain, but not prevented by syntide-2, a synthetic substrate peptide modeled after a phosphorylation site of glycogen synthase(31) . These observations led us to a postulation that there are two distinct substrate-binding sites in CaM kinase II, one for ordinary exogenous substrates and the other for the endogenous substrate, the autophosphorylation site (Thr) in the autoinhibitory domain, and that the latter may be involved in stabilizing the enzyme. The present paper describes several experimental data supporting this contention. Thus, the autoinhibitory domain of CaM kinase II appears to play a crucial role in stabilizing the enzyme as well as in inhibiting the catalytic activity of the enzyme, keeping the enzyme inactive and stable. Similar stabilizing and inhibitory effects have been observed with the pseudosubstrate domain of smooth muscle myosin light chain kinase(32) .


EXPERIMENTAL PROCEDURES

Materials

ATP was purchased from Sigma. ATPS was from Boehringer Mannheim. N-tosyl-L-lysyl chloromethyl ketone-treated chymotrypsin and 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD-F) were from Sigma. N-Tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin was obtained from Cooper Biomedical. [-P]ATP (5000 Ci/mmol) was from Amersham International. Tween 20 was purchased from Pierce. Synthetic peptide substrates syntide-2 (PLARTLSVAGLPGKK) (33) and autocamtide-2 (KKALRRQETVDAL) (34) were synthesized by American Peptide Company, Inc. Autocamtide-2-related inhibitory peptide, AIP (KKALRRQEAVDAL), was synthesized by Accord Co. Ltd. CaMK-(281-309) (MHRQETVDCLKKFNARRKLKGAILTTMLA)(35) , CaMK-(281-289) (MHRQETVDC)(35) , and synapsin I site 3 peptide (YRQGPPQLPPGPAGPTRQASQAGP-NH(2)) (36) were synthesized by a Shimadzu PSSM-8 automated peptide synthesizer. All these peptides were purified by reversed-phase HPLC. Concentrations of the peptides were determined by amino acid analysis.

Protein Preparations

Microtubule-associated protein 2 (MAP2) was purified from porcine brain as described previously(37) . Myosin light chain was prepared from chicken gizzard myosin by the method of Perrie and Perry(38) . Chicken gizzard myosin was prepared by the method of Ebashi(39) . Calmodulin was prepared from rat testis essentially according to Gopalakrishna and Anderson(40) . CaM kinase II was purified from rat cerebral cortex as described previously(31) , except that H-9-Sepharose chromatography was omitted. An active 30-kDa proteolytic fragment of CaM kinase II was prepared according to the method of Yamagata et al.(41) with slight modifications as described before(31) , except that 1 mM phenylmethanesulfonyl fluoride in the dialysis buffer was omitted. The fragment thus obtained gives a single polypeptide band, corresponding to 30 kDa, on SDS-polyacrylamide gel electrophoresis and exhibits a completely Ca/calmodulin-independent protein kinase activity, and it should be the fragment of amino acid residues 1-271 of CaM kinase II(31, 41) .

Inactivation of the Active 30-kDa Fragment of CaM Kinase II

The inactivation reaction was carried out in a mixture containing 85-556 nM CaM kinase II fragment, 40 mM Hepes-NaOH (pH 8.0), 0.01% Tween 20, and 0.1 mM EGTA, with the indicated additions. Polypropylene tubes (Eppendorf) were used, because the enzyme protein was adsorbed on glass tubes. Unless otherwise stated, the reaction was carried out at 30 °C. The reaction was started by adding the enzyme to the mixture, and aliquots were withdrawn at the indicated times, followed by immediate dilution with 19 volumes of ice-cold 40 mM Hepes-NaOH (pH 8.0) containing 1 mM dithiothreitol, 0.01% Tween 20, and 1 mM EGTA (stop buffer). For determination of the original activity, the enzyme was added to the incubation mixture on ice and aliquots were withdrawn, followed by immediate dilution with the ice-cold stop buffer. The activity of the diluted enzyme was immediately measured as described below. Unless otherwise specified, syntide-2 was used for substrate. The remaining activity was expressed as a percentage of the original activity.

Inactivation of Autothiophosphorylated CaM Kinase II

Approximately 0.22-0.24 µM CaM kinase II (the concentration as holoenzyme) was incubated at 5 °C for 10 min in the reaction mixture containing 40 mM Hepes-NaOH (pH 8.0), 50 µM ATPS, 5 mM magnesium acetate, 0.3 mM CaCl(2), 0.1 mM EGTA, 0.01% Tween 20, and 5 µM calmodulin. Under the conditions used, autothiophosphorylation should occur only at Thr(24) . The autothiophosphorylation reaction was stopped by the addition of 10 mM EDTA, 1 mM EGTA. A control experiment was done by adding EDTA/EGTA before starting the reaction by the addition of the enzyme. After reaction for autothiophosphorylation, 3-µl aliquots of the incubation mixture were withdrawn and incubated in order to observe inactivation at 30 °C in the mixture containing 40 mM Hepes-NaOH (pH 8.0), 5 mM EDTA, 1 mM EGTA, and 0.01% Tween 20, with the indicated additions, in a total volume of 30 µl. At the indicated times, aliquots were withdrawn, diluted 20-fold with the ice-cold stop buffer, and immediately assayed for CaM kinase II activity as described below, with syntide-2 as substrate. The remaining activity was expressed as a percentage of the original activity.

Assay of Autothiophosphorylated CaM Kinase II and the Active 30-kDa Fragment of CaM Kinase II

The activities of autothiophosphorylated CaM kinase II and the active 30-kDa fragment of CaM kinase II were measured by the phosphocellulose paper method of Roskoski(42) , as described previously(31) . Autothiophosphorylated CaM kinase II was assayed in the presence of Ca/calmodulin, and the CaM kinase II fragment was assayed in the absence of Ca/calmodulin.

Preparation of NBD-labeled AIP (NBD-AIP)

Labeling of AIP was achieved essentially as described by Rapaport and Shai(43) . The reaction was carried out at 24 °C for 66 h, followed by precipitation with diethyl ether. The precipitate was washed once with diethyl ether and dried under a stream of nitrogen. The yellow powder thus obtained was dissolved into 0.1% trifluoroacetic acid and purified by reversed-phase HPLC on a C(18) column. Positive-ion fast atom bombardment mass spectrum of the purified peptide, measured with a JEOL JMS-SX102 mass spectrometer, showed a prominent peak at m/e 1661, indicating that 1 mol of NBD was introduced into 1 mol of AIP. The concentration of the labeled peptide was determined spectrophotometrically using an absorption coefficient, A, of 26,650 M cm as described by Allegrini et al.(44) .

Fluorescence Assay for Binding of NBD-AIP to the Active 30-kDa Fragment of CaM Kinase II

Fluorescence spectra of 0.2 µM NBD-AIP were measured in the mixture consisting of 40 mM Hepes-NaOH (pH 8.0) and 0.01% Tween 20, with the indicated additions, before and after the addition of 0.23 µM of the active 30-kDa fragment of CaM kinase II, with a Hitachi F-3000 fluorescence spectrophotometer at 30 °C, with an excitation wavelength of 472 nm. Changes in relative fluorescence intensity at an emission wavelength of 521.6 nm (DeltaF) were calculated according to ,

where F and F are the relative fluorescence intensities at 521.6 nm before and after the addition of the 30-kDa fragment, respectively. The values were corrected for dilution.

Other Analytical Procedures

SDS-polyacrylamide gel electrophoresis was carried out according to the method of Laemmli (45) . The concentrations of CaM kinase II and calmodulin were determined spectrophotometrically using absorption coefficients, A(1 mg/ml), of 1.30 and 0.21, respectively, as described previously(37) . Other proteins were determined by the method of Lowry et al.(46) , as modified by Peterson (47) with bovine serum albumin as a standard. Fluorography was carried out by the method of Chamberlain(48) . The molecular weights for CaM kinase II, the 30-kDa fragment of CaM kinase II, calmodulin, and MAP2 were taken as 540,000, 30,000, 17,000, and 300,000, respectively.


RESULTS

Thermal Stability of the Active 30-kDa Fragment and Autothiophosphorylated CaM Kinase II

Fig. 1shows thermal stabilities of the active 30-kDa fragment of CaM kinase II and the autothiophosphorylated CaM kinase II, compared with the original native enzyme. Incubation of the native enzyme for 3 min caused no significant change in the activity over the incubation temperature range from 0 to 40 °C. In contrast, incubation of the 30-kDa fragment resulted in a progressive decrease in the activity with increasing the incubation temperature at above 20 °C, and more than 90% of the original activity was lost at 40 °C, as shown in Fig. 1A. When the effect of the concentration of the fragment on the rate of the inactivation was examined at two different concentrations of the fragment, 85 nM and 556 nM, no significant difference between the former and the latter was observed (data not shown), suggesting that the inactivation was not affected by concentrations of the fragment ranging from 85 nM to 556 nM. No molecular size difference was observed between the active and the inactivated 30-kDa fragments, as judged by SDS-polyacrylamide gel electrophoresis (Fig. 2, A and B), suggesting that the inactivation was not due to proteolysis. Thus, the removal of the region corresponding to the C-terminal side of the residue 271(41) , in which autoinhibitory domain was included, led to a dramatic decrease in the thermal stability of CaM kinase II. The susceptibility of the inactivated 30-kDa fragment to tryptic digestion was significantly higher than that of the active fragment (Fig. 2C), indicating that a conformational change occurred during the inactivation process.


Figure 1: Effect of temperature on thermal stability of the 30-kDa fragment of CaM kinase II and autothiophosphorylated CaM kinase II. A, the 30-kDa fragment (163 nM) of CaM kinase II (bullet) or native CaM kinase II (16.7 nM as holoenzyme) (circle) was incubated at the indicated temperatures for 3 min, and then aliquots were assayed for the kinase activity. The CaM kinase II fragment was assayed in the absence of Ca/calmodulin, and the native CaM kinase II was assayed in the presence of Ca/calmodulin. B, CaM kinase II autothiophosphorylated as described under ``Experimental Procedures'' (bullet) or the nonthiophosphorylated enzyme, which was prepared by adding EDTA/EGTA before starting the autothiophosphorylation reaction as described under ``Experimental Procedures'' (circle), was incubated at the indicated temperatures for 3 min, and aliquots were assayed for the kinase activity in the presence of Ca/calmodulin.




Figure 2: Analyses of the inactivated 30-kDa fragment of CaM kinase II and its susceptibility to tryptic digestion on SDS-polyacrylamide gel electrophoresis. A, the 30-kDa fragment (407 nM) of CaM kinase II was incubated at 30 °C for inactivation, and after incubation for 0, 2, 5, and 10 min (lanes 1-4, respectively), aliquots were subjected to SDS-polyacrylamide gel electrophoresis on a 15% polyacrylamide gel, followed by silver staining. B, the 30-kDa fragment (167 nM) was incubated at 0, 10, 20, 30, and 40 °C (lanes 1-5, respectively) for 3 min, and aliquots were subjected to SDS-polyacrylamide gel electrophoresis on a 15% polyacrylamide gel, followed by silver staining. C, approximately 980 ng of the active 30-kDa fragment (lanes 1-5) or the inactivated 30-kDa fragment (lanes 6-10), obtained by incubation for 10 min at 30 °C as described under ``Experimental Procedures,'' was incubated with 21 ng of N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin at 0 °C. After incubation for 0 (lanes 1 and 6), 5 (lanes 2 and 7), 30 (lanes 3 and 8), 60 (lanes 4 and 9), and 120 min (lanes 5 and 10), aliquots were mixed with phenylmethanesulfonyl fluoride (10 mM) and subjected to SDS-polyacrylamide gel electrophoresis on a 15% polyacrylamide gel, followed by silver staining.



Fig. 1B shows that autothiophosphorylation of CaM kinase II resulted in a decrease in the thermal stability of the enzyme. Under the experimental conditions used, autothiophosphorylation should occur only at Thr(24) , which is located in the autoinhibitory domain of the enzyme. Thus, not only the removal of the autoinhibitory domain but also the thiophosphorylation of Thr in the autoinhibitory domain led to a large decrease in the thermal stability of CaM kinase II.

Stabilization of the Active 30-kDa Fragment and the Autothiophosphorylated CaM Kinase II by Autocamtide-2 and CaMK-(281-309)

Fig. 3shows protective effects of the two synthetic peptide substrates, syntide-2 and autocamtide-2, on the inactivation of the active 30-kDa fragment and the autothiophosphorylated enzyme. Although both peptides have so far been commonly used as substrate for CaM kinase II, there are marked differences in their structural designs; syntide-2 was designed after the sequence of the phosphorylation site of glycogen synthase(33) , and autocamtide-2 was modeled after the sequence of the autophosphorylation site (Thr) in the autoinhibitory domain of CaM kinase II (34) . The addition of 1 µM and 5 µM autocamtide-2 markedly stabilized the 30-kDa fragment (Fig. 3A) and the autothiophosphorylated enzyme (Fig. 3B), respectively. In contrast, syntide-2, even at 0.3 mM, only slightly stabilized the 30-kDa fragment and did not stabilize the autothiophosphorylated enzyme at all. These results suggest that the autoinhibitory domain of CaM kinase II stabilizes the enzyme. In order to confirm the involvement of the autoinhibitory domain in stabilizing the enzyme, effects of various compounds, including CaMK-(281-309), a synthetic peptide corresponding to the amino acid sequences of the regulatory domain of the enzyme, and several ordinary substrates such as MAP2, myosin light chain, syntide-2, and synapsin I site 3 peptide, on the thermal stabilities of the 30-kDa fragment and the autothiophosphorylated enzyme were studied, as summarized in Table 1. Among a number of compounds tested, CaMK-(281-309) and autocamtide-2 were most effective in stabilizing both the 30-kDa fragment and the autothiophosphorylated enzyme. ATP/Mg was also highly effective in stabilizing the 30-kDa fragment, but its stabilizing effect on the autothiophosphorylated enzyme could not be examined, because the reaction was carried out in the presence of 6 mM EDTA to prevent further autothiophosphorylation. CaMK-(281-289), a synthetic peptide corresponding to the short segment of the sequence around the autophosphorylation site, also significantly stabilized the enzyme. In contrast to CaMK-(281-309) and autocamtide-2, ordinary exogenous substrates for the enzyme, such as MAP2, myosin light chain, syntide-2, and synapsin I site 3 peptide, showed no or little, if any, effect on stabilizing the enzyme.


Figure 3: Effect of syntide-2 or autocamtide-2 on thermal stability of the 30-kDa fragment of CaM kinase II and autothiophosphorylated CaM kinase II. A, the 30-kDa fragment (163 nM) of CaM kinase II (, bullet, times) or native CaM kinase II (17 nM as holoenzyme) (circle) was incubated in the inactivation mixture with additions of none (, circle), 1 µM autocamtide-2 (bullet), or 0.3 mM syntide-2 (times) at 30 °C for the indicated times, and then aliquots were assayed for the kinase activity. B, CaM kinase II autothiophosphorylated as described under ``Experimental Procedures'' (, bullet, times) or the nonthiophosphorylated enzyme, which was prepared as described in the legend for Fig. 1(circle), was incubated at 30 °C for the indicated times with the addition of none (, circle), 5 µM autocamtide-2 (bullet), or 0.3 mM syntide-2 (times), and aliquots were assayed for the kinase activity.





In order to estimate affinities of CaMK-(281-309) and autocamtide-2 for the 30-kDa fragment, the effect of varying their concentrations on the thermal stability of the fragment was examined as shown in Fig. 4. Under our experimental conditions, the concentration of the 30-kDa fragment required for accurate determination of the remaining activity was at least 85 nM. Both CaMK-(281-309) and autocamtide-2 almost completely protected 85 nM enzyme against inactivation at concentrations as low as about 100 nM, indicating that the two peptides have very high affinities for the enzyme.


Figure 4: Effect of varying the concentration of autocamtide-2 and CaMK-(281-309) on thermal stability of the 30-kDa fragment of CaM kinase II. A, the 30-kDa fragment (85 nM) of CaM kinase II was incubated at 30 °C for 10 min with the indicated concentrations of autocamtide-2, and then aliquots were assayed for the kinase activity. B, the 30-kDa fragment (85 nM) of CaM kinase II was incubated at 30 °C for 10 min with the indicated concentrations of CaMK-(281-309), and aliquots were assayed for the kinase activity.



Two Distinct Binding Sites on CaM Kinase II for Peptide Substrates

The observation that synthetic peptide substrates designed after the sequence around the autophosphorylation site in the autoinhibitory domain of CaM kinase II, such as autocamtide-2, CaMK-(281-309), and CaMK-(281-289), stabilized both the 30-kDa fragment and the autothiophosphorylated enzyme, but that ordinary exogenous substrates, such as syntide-2, MAP2, myosin light chain, and synapsin I site 3 peptide did not, led us to the notion that the binding site for the autophosphorylation site in the autoinhibitory domain (the endogenous substrate) is distinct from that for ordinary exogenous substrates. In order to prove this hypothesis, a kinetic analysis of the activity of the active 30-kDa fragment was carried out using a novel synthetic peptide inhibitor, AIP (KKALRRQEAVDAL), which was made by the substitution of Ala for Thr^9 of autocamtide-2 (KKALRRQETVDAL), as shown in Fig. 5. As expected, AIP was a potent inhibitor of the 30-kDa fragment of the enzyme with a K(i) value of about 2-8 nM, the inhibition being competitive with respect to autocamtide-2 (Fig. 5B) and CaMK-(281-289) (Fig. 5C) and noncompetitive with respect to syntide-2 (Fig. 5A) and ATP (Fig. 5D). Thus, the binding site for AIP may be the same as those for autocamtide-2 and CaMK-(281-289) and distinct from those for syntide-2 and ATP. Kinetic studies of the inhibition of the activity of the 30-kDa fragment by CaMK-(281-309), a synthetic peptide corresponding to the regulatory domain of the enzyme, indicated that CaMK-(281-309), in contrast with AIP, inhibited the enzyme, competitively with respect to both autocamtide-2 and ATP but noncompetitively with respect to syntide-2, as shown in Fig. 6, in agreement with earlier observations (49) that CaMK-(281-309) inhibits a Ca/calmodulin-independent form of CaM kinase II, the inhibition being noncompetitive with respect to peptide substrate (syntide-2) but competitive with respect to ATP. The apparent K(i) values for CaMK-(281-309) were estimated from the results of Fig. 6to be 166 nM for the competitive inhibition with respect to autocamtide-2 (K(m) = 0.4 µM), 19 nM for the competitive inhibition with respect to ATP (K(m) = 30 µM), and 20 nM for the noncompetitive inhibition with respect to syntide-2 (K(m) = 8.5 µM), respectively. Thus, CaMK-(281-309), a synthetic peptide corresponding to the autoinhibitory domain, interacts with the substrate-binding site for the autophosphorylation site (Thr) as well as ATP-binding site.


Figure 5: Kinetic analyses of the inhibition of the 30-kDa fragment of CaM kinase II by AIP. The activity of the 30-kDa fragment of CaM kinase II was measured at varying concentrations of the indicated substrates at four or five different fixed concentrations of AIP, and the data were plotted as a double-reciprocal plot. A, assayed at varying concentrations of syntide-2 in the presence of 0 (bullet), 0.01 (circle), 0.02 (), 0,03 (), and 0.05 µM (box) AIP. B, assayed at varying concentrations of autocamtide-2 in the presence of 0 (bullet), 0.01 (circle), 0.02 (), 0.05 (), and 0.08 µM (box) of AIP. C, assayed at varying concentrations of CaMK-(281-289) in the presence of 0 (bullet), 0.003 (circle), 0.007 (), and 0.01 µM () of AIP. D, assayed at varying concentrations of ATP in the presence of 0 (bullet), 0.01 (circle), 0.015 (), 0.025 (), and 0.03 µM (box) of AIP, using syntide-2 (200 µM) as a peptide substrate.




Figure 6: Kinetic analyses of the inhibition of the 30-kDa fragment of CaM kinase II by CaMK-(281-309). The activity of the 30-kDa fragment of CaM kinase II was measured at varying concentrations of the indicated substrates at four or five different fixed concentrations of CaMK-(281-309), and the data were plotted as a double-reciprocal plot. A, assayed at varying concentrations of syntide-2 in the presence of 0 (bullet), 0.05 (circle), 0.1 (), and 0.2 µM () of CaMK-(281-309). B, assayed at varying concentrations of autocamtide-2 in the presence of 0 (bullet), 0.1 (circle), 0.2 (), 0.3 (), and 0.5 µM (box) of CaMK-(281-309). C, assayed at varying concentrations of ATP in the presence of 0 (bullet), 0.05 (circle), 0.1 (), 0.2 (), and 0.3 µM (box) of CaMK-(281-309), using syntide-2 (200 µM) as a peptide substrate.



Further experimental support for our contention was provided by fluorescence analysis of the binding of NBD-AIP to the 30-kDa fragment. Fig. 7shows the emission spectra of NBD-AIP at an excitation wavelength of 472 nm before and after the addition of the 30-kDa fragment. Both spectra showed maxima at 521.6 nm, but the addition of the 30-kDa fragment caused a significant decrease in the fluorescence, while bovine serum albumin caused no fluorescence change (data not shown), indicating the specific interaction of the fragment with NBD-AIP. Effects of various substrates on the interaction between NBD-AIP and the fragment were examined by monitoring the changes in the fluorescence intensity at 521.6 nm at an excitation wavelength of 472 nm, as summarized in Table 2. The addition of micromolar concentrations of autocamtide-2 and CaMK-(281-309) completely blocked the quenching of the NBD-AIP fluorescence induced by the binding of the 30-kDa fragment, whereas syntide-2, synapsin I site 3 peptide, and ATP/Mg did not significantly affect the quenching even at millimolar concentrations, suggesting that autocamtide-2 and CaMK-(281-309) competed with NBD-AIP for binding to the 30-kDa fragment but the other substrates did not, in good agreement with the results of the kinetic analyses as shown in Fig. 5and 6.


Figure 7: A change in the fluorescence emission spectrum of NBD-AIP upon binding to the 30-kDa fragment of CaM kinase II. Fluorescence emission spectra of NBD-AIP were recorded at an excitation wavelength of 472 nm before (curve a) and after (curve b) the addition of the 30-kDa fragment of CaM kinase II, as described under ``Experimental Procedures.'' The spectra were corrected for dilution.





Effects of Syntide-2 and Autocamtide-2 on Autothiophosphorylation of CaM Kinase II

Since our experimental results so far described indicate that the binding site of CaM kinase II for the autophosphorylation site (Thr) in the autoinhibitory domain is distinct from that for the ordinary exogenous substrate, it would be expected that autocamtide-2 is a more potent inhibitor for the autothiophosphorylation than is syntide-2. As shown in Fig. 8, the autothiophosphorylation, which should occur only at Thr under the experimental conditions used(24) , was markedly inhibited by as little as 9 µM autocamtide-2, but such a marked inhibition was not observed with as much as 0.3 mM syntide-2. The K(m) values of the native CaM kinase II for autocamtide-2 and syntide-2 were estimated to be 2.3 and 7.7 µM, respectively (data not shown). These results also support for the contention that the binding site of CaM kinase II for the autophosphorylation site (Thr) is distinct from the ordinary substrate-binding site.


Figure 8: Effect of syntide-2 and autocamtide-2 on autothiophosphorylation of CaM kinase II. CaM kinase II (16.9 nM as holoenzyme) was autothiophosphorylated at 5 °C for 30 s (lane 1) or 1 min (lane 2) in the reaction mixture containing 40 mM Hepes-NaOH (pH 8.0), 0.4 mM CaCl(2), 0.1 mM EGTA, 0.5 µM calmodulin, 5 mM magnesium acetate, 0.01% Tween 20, and 50 µM [-S]ATPS (0.12 mCi/ml), with the following additions: A, none; B, 0.3 mM syntide-2; C, 9 µM autocamtide-2. The autothiophosphorylation reaction was terminated by adding 16.3 mM EDTA, and the mixture was mixed with an equal volume of a solution consisting of 125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.002% bromphenol blue, and 130 mM beta-mercaptoethanol, followed by boiling for 2 min, and then aliquots were subjected to SDS-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel. The gel was visualized by fluorography.




DISCUSSION

Previous reports from our own and other laboratories(26, 27, 28) demonstrated that autophosphorylation of CaM kinase II, presumably occurring at multiple sites of the enzyme, causes a progressive decrease in the enzyme activity, and thereafter Lai et al.(29) suggested that the inactivation is due to a decrease in the thermal stability of the autophosphorylated enzyme. More recently, Colbran (30) reported that autophosphorylation at Thr, gradually occurring in the absence of Ca/calmodulin, results in inactivation of the enzyme. In the present paper, we show that autothiophosphorylation at Thr, which is involved in the activation of the enzyme(18, 19, 20, 21, 22, 23, 24) , caused a marked decrease in the thermal stability. The active 30-kDa chymotryptic fragment of CaM kinase II(31, 41) , devoid of the autoinhibitory domain containing the calmodulin-binding and inhibitory domains and also the autophosphorylation site of Thr, was also very labile (Fig. 1). The thermal inactivation of the two labile CaM kinase II preparations, the autothiophosphorylated enzyme and the 30-kDa fragment, was prevented by autocamtide-2 and CaMK-(281-289), synthetic peptides modeled after the sequence around the autophosphorylation site of Thr, and CaMK-(281-309), a synthetic peptide corresponding to the sequence of the autoinhibitory domain, but it was not prevented by ordinary protein and peptide substrates such as MAP2, myosin light chain, syntide-2, and synapsin I site 3 peptide, while it was also prevented by ATP/Mg ( Fig. 3and 4, and Table 1). These results, taken together with the general contention that the autoinhibitory domain of CaM kinase II suppresses the enzyme activity by interaction with the catalytic site(50) , suggested that the autophosphorylation site located in the autoinhibitory domain not only inhibits the enzyme activity but also stabilizes the enzyme by interacting with the second substrate-binding site specific for the autophosphorylation site, which is distinct from the substrate-binding site for ordinary exogenous substrates such as MAP2, myosin light chain, syntide-2, and so on. The possibility of the existence of the two distinct substrate-binding sites in the catalytic site of CaM kinase II, one for ordinary exogenous protein and peptide substrates and the other for the endogenous substrate, the autophosphorylation site (Thr), has been suggested from our previous observation (31) that the inactivation of CaM kinase II by Ca/calmodulin is prevented by autocamtide-2 but not by syntide-2 or MAP2. In the present study, this was confirmed by the kinetic data that the inhibitions of the enzyme by AIP and CaMK-(281-309), respectively, were competitive with respect to autocamtide-2 but noncompetitive with respect to syntide-2 ( Fig. 5and 6), and was further confirmed by the fluorescence analysis that the quenching of NBD-AIP induced by the 30-kDa fragment of CaM kinase II was completely blocked by autocamtide-2 or CaMK-(281-309) but was not affected by syntide-2 or synapsin I site 3 peptide at all (Table 2). This was also suggested by the fact that autocamtide-2 inhibited the autothiophosphorylation of Thr much more strongly than did syntide-2 (Fig. 8). The K(m) value for autocamtide-2 was reduced from 2.3 µM to 0.4 µM by truncation of the autoinhibitory domain, whereas the K(m) for syntide-2 was not changed significantly. This observation might be explained by two different substrate-binding sites; the binding of autocamtide-2 to its binding site is inhibited by the autoinhibitory domain in the native enzyme, but the binding of syntide-2 to its binding site is not inhibited.

Of particular interest is a novel synthetic peptide inhibitor, AIP (KKALRRQEAVDAL), which was made by the substitution of Ala for the phosphorylation site of autocamtide-2 (KKALRRQETVDAL), Thr^9. This peptide was a potent inhibitor with a K(i) value as low as 2-8 nM for the 30-kDa fragment of the enzyme. The kinetic data of the inhibition by AIP (Fig. 5) and the results of fluorescence analysis with NBD-AIP (Table 2), together with the results that autocamtide-2 inhibited the autothiophosphorylation of Thr more strongly than did syntide-2 (Fig. 8), indicated that AIP may bind to the substrate-binding site for the autophosphorylation site (Thr) but not bind to the substrate-binding site for the ordinary exogenous substrates, thereby inhibiting the enzyme activity. Thus, AIP will be a useful tool for studying the regulatory mechanism of CaM kinase II through the autophosphorylation of Thr in the autoinhibitory domain of the enzyme. It will be also interesting to examine the question of whether AIP is a specific inhibitor for CaM kinase II, because no real specific inhibitor for CaM kinase II has so far been reported(51) . The specificity and the detailed mechanism for the inhibition by AIP are under investigation in this laboratory.

In contrast to AIP, CaMK-(281-309), a synthetic peptide corresponding to the sequence of the autoinhibitory domain containing the autophosphorylation site and calmodulin-binding site, inhibited the enzyme activity in a competitive manner with respect not only to autocamtide-2 but also to ATP, while it inhibited the enzyme in a noncompetitive manner with respect to syntide-2 (Fig. 6), suggesting that the autoinhibitory domain of the enzyme may interact with both the substrate-binding site for the autophosphorylation site (Thr) and the ATP-binding site.

Pears et al.(52) found that deletion of the pseudosubstrate sequence of protein kinase C made it so unstable that it was not possible to purify the recombinant enzyme. More recently, Faux et al.(32) reported that the pseudosubstrate sequence of smooth muscle myosin light chain kinase plays an important role in stabilizing the enzyme, and they predicted that other enzymes regulated by pseudosubstrate mechanism may also utilize the pseudosubstrate sequence to stabilize the structure. Here we show that CaM kinase II was also stabilized by a similar mechanism, utilizing the autophosphorylation site, the endogenous substrate, instead of the endogenous pseudosubstrate sequence. Knighton et al.(53) have proposed a model for the regulatory mechanism of myosin light chain kinase by the pseudosubstrate sequence that the pseudosubstrate sequence occupies the substrate binding site. However, the results that substrate peptides, such as MLC-(1-23) and MLC-(11-23)A, can protect the enzyme from thermal inactivation but their potency is not as great as that of the pseudosubstrate sequence (32) may reflect the existence of the endogenous pseudosubstrate-binding site different from the ordinary substrate-binding site.


FOOTNOTES

*
This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan and by grants from the Smoking Research Foundation and the Byotai Taisha Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: CaM kinase II, calmodulin-dependent protein kinase II; AIP, autocamtide-2-related inhibitory peptide; ATPS, adenosine 5`-O-(3-thiotriphosphate); HPLC, high performance liquid chromatography; MAP2, microtubule-associated protein 2; NBD, 7-nitrobenz-2-oxa-1,3-diazole-4-yl.


ACKNOWLEDGEMENTS

We thank Dr. Takashi Daiho (Dept. of Biochemistry II, Asahikawa Medical College) for valuable advice in performing fluorescence spectroscopy.


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