An Enzymatically Active Cross-linked Complex of Calmodulin and

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 264, N o. 26, Issue of September 15, PP. 15552-15555,1989 Printed in U.S.A.

0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

An Enzymatically Active Cross-linked Complex of Calmodulin and Rabbit Skeletal Muscle Myosin Light Chain Kinase* (Received for publication, January 17, 1989)

Henry G . Zot$ and David Puetts From the Reproductive Sciencesand Endocrinology Laboratories, Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami,Florida 33101

The interaction between bovine testes calmodulin 1987; Kennelly et al., 1987; Pearson et al., 1988). Occupancy and rabbit fast skeletal muscle myosin light chain ki- of the catalytic site sterically prevents phosphorylationof the nase was investigated with the zero-length cross-link- substrate, and binding to calmodulin removes the calmodulin ing reagent N,N’-dicyclohexylcarbodiimide. A cross- binding domain from the catalytic site. This mechanism is linked product of 110 kDa was produced only in the fully consistent with the preparation of a calmodulin-indepresence of Ca2+.The reaction mixture was separated pendent form of the kinase by proteolytic excision of a linear on diethylaminoethyl cellulose, and a fraction contain- sequence from the C terminus (Tanaka et al., 1980; Edelman ing the cross-linked complex of calmodulin and myosin light chain kinase wasfound to havean elevated kinase et al., 1985; Pearson et al., 1988). To further investigate this catalytic site blockade mechaactivity in the absence of Ca2+, which constituted approximately 50%of the maximally stimulated kinase nism we have initiated studies to cross-link calmodulin at the activity of control, and additional kinase activity in calmodulin binding domain of MLCK. The same approach the presence of Ca2+,which constituted the remaining has been used successfully by others to produce calmodulin 50%of control activity. Calmodulin addedexogenously complexes with enzymes (Kincaid, 1984; Yamamoto et al., to the cross-linked complex had no effect on the meas- 1988). In thispaper, the Ca2+-dependentcovalent association ured Ca2+dependence or the maximal extent of kinase of calmodulin with MLCK using the “zero-length” crossactivity, which is consistent with the cross-linking of linking reagent DCC (Carraway and Koshland, 1972), sepacalmodulin in close proximity to a regulatory region of ration of the reaction products on DEAE-cellulose, and the myosin light chain kinase. Moreover, the results are measurement of kinase activity of a fraction containing a consistent with a mechanism whereby the association cross-linked MLCK are reported. The results generally supof calmodulin is sufficient to stimulate kinase activity port theproposed mechanism of catalytic site blockade (Kemp and the binding of Ca2+to bound calmodulin increases et al., 1987; Kennelly et al., 1987; Pearson et al., 1988) and catalytic efficiency. suggest that Ca2+regulates calmodulin bound to MLCK. EXPERIMENTALPROCEDURES

Protein Preparations-Calmodulin was prepared from frozen bovine testes as previously described (Toda et al., 1985) except that phenyl-Sepharose chromatography (Charbonneau et al., 1983) was substituted for the final purification instead of ACA 44. Myosin light chain kinase was purified from fresh rabbit back and leg muscle essentially by methods previously described (Yazawa and Yagi, 1978; Crouch et al., 1981). Crude myosin light chains were prepared from rabbit skeletal muscle myosin (Kielley and Harrington, 1960) by the method of Pires and Perry (1977). Contaminating calmodulin was removed from crude light chains by chromatography on DEAEcellulose in 6 M urea. Methods-Protein concentration was determined either by colori* This work was supported by National Institutes of Health Grant metric assay (Smith et al., 1985) or by using published extinction GM 35415 and by Grant 8729 G1A from the American Heart Assocoefficients for MLCK (Crouch et al., 1981) and calmodulin (Klevit, ciation, West Florida Regional Affiliate. A preliminary account of 1983). SDS-PAGE was performed by the method of Laemmli (1970); portions of this work was presented at the20th Miami Bio/Technolsamples for electrophoresis were diluted into a 125 mM Tris-HC1 ogy Winter Symposium ((1988) ICSU Short Rep. 8, 161). The costs of publication of this article were defrayed in part by the payment of buffer, pH 6.8, containing 0.1% sodium dodecyl sulfate, 5% glycerol, page charges. This article must therefore be hereby marked “aduer- 0.03% bromphenol blue, and 1 mM EDTA, and protein bands were stained with Coomassie Blue R-250. Free Ca2+was determined from tisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate the binding constants of EGTA and ATP (Smith and Martell, 1974) this fact. $ Present address: Dept. of Cell Biology and Anatomy, The Johns by an iterative computer method (Robertson and Potter, 1984). Kinase activity was determined from the rate of 32Ptransferred Hopkins University School of Medicine, 725 North Wolfe St., Baltifrom [T-~*P]ATP (Amersham Corp.) to myosin light chains by the more, MD 21205. method of Corbin and Reimann (1975) except thatthe filters § T o whom correspondence should be addressedREPSCEND Labs (D-5), University of Miami School of Medicine, P. 0. Box (Schleicher and Schuell) were not exposed to ethanol. The conditions for the assay were based on the optimal conditions for MLCK activity 016960, Miami, FL 33101. ‘The abbreviations used are: MLCK, myosin light chain kinase determined previously (Blumenthal and Stull, 1980). The reaction from rabbit fast skeletal muscle unless otherwise specified DCC, was started by the addition of ATP, incubated for 5 min at 30 “C, N,N’-dicyclohexylcarbodiimide; MOPS, 3-(N-morpholino)propane- and stopped by spotting 20 r l on filter discs and immediately imsulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic mersing the discs into an ice-cold solution of 5% trichloroacetic acid acid SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- and 1%potassium pyrophosphate. Net counts/min were calculated by subtracting the counts/min of control filterswhich were prepared phoresis.

Recently two groups have demonstrated that synthetic peptides corresponding to the high affinity calmodulin binding domains of MLCK’ and smooth muscle MLCK compete with the substrate for the catalytic site of the enzyme (Kemp et al., 1987; Kennelly et al., 1987). Based on these and other results a common mechanism for calmodulin activation has been proposed whereby the calmodulin binding domain of the kinase can bind either internally to the catalytic site of the enzyme or to calmodulin when Ca2+is present (Kemp et al.,

15552

Cross-linking MLCK of Calmodulin and

15553

from identical reactions without kinase and converted to picomoles fromthespecif'icactivit~ofthe['y-iP]ATPadded.Inallcasescontrol experiments were performed under the same conditions to establish of the assay: that the enzyme activitywas linear over the time course these data are not shown.

and calmodulin. A shorter incubation time of 3 h was used in ordertolimittheproduction of highermolecular weight species. Earlier studies had shown that calmodulin was retained strongly onDEAE-cellulose while MLCK bound rather weakly (Yazawa and Yagi, 1978). Having properties of both calmodulin and MLCK, the 110-kDa product was expected to RESULTS ANDDISCUSSION have an intermediate affinity for DEAE. Fig. 2 shows the Cross-linking of Calmodulin and MLCK-Calmodulin and chromatogram from the best preparative cross-linking reacMLCK were incubated with DCC, and a major cross-linked tion that was separated onDEAE-cellulose. Aliquots from species of 110 kDa was obtained only in the presence of Ca" fractions were taken across the chromatogram and separated (Fig. 1). The yield of the 110-kDa band was increased by on SDS-PAGE (data not shown). Fraction62 was chosen for increasing the ratioof calmodulin to MLCK during thereac- furtheranalysis of kinaseactivityusingthecriteriathat tion; however,even high ratios did not produce detect.able uncross-linkedcalmodulin, whichwas visible infractions cross-linking in t,he absence of Ca'+ even after an incubation beyond82,did not significantly contaminate this fraction. time of 18 h (Fig.1).Higher molecular weight speciesappeared The cross-linked complex from two other preparations was in low yield as the calmodulin to MLCK ratio was increased. completelyseparatedfromuncross-linkedMLCKbutnot In view of the high affinity between calmodulin and MLCK completelyseparatedfromuncross-linked calmodulin on (Crouch et al., 1981), it may seem surprising that maximal DEAE-cellulose. cross-linking occurred a t higher ratios of calmodulin to enComparison of Kinase Activity of Fractions 11 and 62zyme. This, however, can he explained if DCC, which is quite Timecourseexperimentsdemonstratedthattheassayas insoluble in aqueous solution, binds to one or both of the described in Fig. 3 was linear for a t least 10 min with 2 ng of hydrophobic p0cket.s on calmodulin that become exposed fol- unreacted MLCK, 2 ng of uncross-linked MLCK from fraclowing Ca')+hinding (Babu etal., 1988; Strynadka and James, tion 11, and 2.6 ng of 110-kDaprotein from fraction 62. would Compared with unreacted MLCK, the protein from fraction 1988). If this is true, then DCC-mediated cross-linking represent a fine balancebetween possible inhibition of bind- 11 had a basal activity which was not significantly different ing, assuming that the hydrophobic pockets are important in over the time course of the assay and a maximal activity enzyme recognition, and covalent attachmentfollowing initial whichwas 60% lower. I t was assumed that differences in helix-helix interaction, with reaction of DCC and a carboxyl kinetic parametersbetween unreacted MLCK and the protein group to form an 0-acyl urea,which could then react with an in fraction 11 resulted from the sameprocesses which affected amino group, serving as a nucleophile. The 110-kDa compo- the activity of the MLCK component in fraction 62. These nent, which corresponds to the M , expected for a 1:l molar controlexperiments provided the basis for comparingthe complex of calmodulin and MLCK, and the Ca2+ dependence kinase activity of the protein from fractions11 and 62. of cross-linking suggested that calmodulin was bound a t t h e The kinase activity of fraction 62 was found to be significalmodulin binding domain of MLCK. cantly elevated in the absence of Ca'+ compared to fraction Preparation of the 110-kDa Product-Separation of the 11 (Fig. 3 A ) . More protein from fraction 62 than fraction 11, products of the cross-linking reactionwas attempted in order 2.6 versus 2.0 ng, was used to compensate for the difference to isolate the 110-kDa product from uncross-linked MLCK in apparent Mr. Since the concentration of the MLCK component was the same, this striking difference in basal activity between fractions 62 and 11 cannot be easily explained as a difference in enzyme levels. The kinase activity of both fractions was stimulated by Ca'+, althoughto very different extents. While the kinase activity of fraction 62 was stimulated only approximately 2-fold (Fig. 3 A ) by Ca2+, the kinase activity of fraction 11 did not differ significantly from zero

-

E 0.6 C

0 N N

-0

4

w

0 2

202 U 0 v)

m 00

a

Fraction Number

FIG. 2. Separation of cross-linkedreaction products on ) calmodulin (20 p ~ in) 20 DEAE-cellulose. To MLCK (2p ~ and Reaction tubes (25-pI final volume) were prepared with 3 pM MLCK, mM MOPS, pH 7.0, 0.2 mM EGTA, 1.2 mM CaCI,,, and 0.001% (w/v) 20 mM MOPS, pH 7.0, 140 mM KCI, 1.0 mM EGTA, 0.005% (w/v) NaN,,, DCC was addedto a final concentration of 0.2 mM (0.02% (w/ NaN:,, 2% isopropyl alcohol, and 0.2 mM DCC, incubated for 18 h at v) isopropyl alcohol final) and the reaction (20 ml) incubated for 3 h 'L., r C, and suhsequently diluted into sample huffer for electrophoresis.a t 25 "C. The reaction was terminated by the addition of200 mM Aliquots (10 p l ) were takenfromeachreactionandrunonSDSglycine and 2 mM EDTA, followed by dialysis with three changes PAGE (5-20!6 linear acrylamide).The calmodulin concentration was (1000 ml) of a 20 mM MOPS buffer, pH 7.0, containing 75 mM KC1, ?I p M ( l n n ~ 1s and ij), 6 pM (lanes 2 and 6 ) , 15 pM (lanes 3 and 7 ) , or 2.0 mM EGTA, and0.01% (w/v) NaN:, at 4 "C. The sample was loaded 30 pM (lanes 4 and 8 ) . Reactions which included Ca2+ contained 1.2 on a column of DEAE-cellulose(0.5-cmdiameterand 2-ml bed mM CaCI,, (lanes 5 - 8 ) . As a control DCC was not included in the volume) a t 4 "C. The column was washed with 20 ml of the same reaction (lane 9);lane IO contains molecular weight markers ( A , 116 buffer and the protein eluted with a200-ml linear gradient of KC1 t o kDa; H , 97.4 kDa; C, 66 kDa; D,45 kDa; E, 29 kDa). a final concentration of 300 mM. Fractions of 2 ml were collected. FIG. 1. Cross-linking of calmodulin and MLCK with DCC.

0

Cross-linking MLCK of Calmodulin and

15554

about 1 nM and has the following amino acid sequence (Blumenthal et al., 1985), Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Vai-Ser-Ala-Ala-AsnArg-Phe-Lys-Lys-Ile-Ser-Ser-Ser-Gly-Ala-Leu-Met

-8.5

-5 5

-6.5

-7.5

-4.5

Log Calcium (Ml

-0.5

0.5

1.5

25

Log Calmodulin (nM added)

FIG. 3. CaZ+and exogenous calmodulin dependence of a cross-linked complex of MLCK and calmodulin. Aliquots containing 2.0 ng of fraction 11 or 2.6 ng of fraction 62 containing 0.3 pmol of protein (Smith et ul., 1985) were diluted into a 25-p1 total volume that included 50 mM MOPS, pH 7.0, 10 mM MgC12, 1.0 mM EGTA, 1.0 mM ATP, 0.05 mM myosin light chains, and 1.2 pCi of [y32P]ATP.A , kinase activity was measured as a function of free Ca2' for fraction 11 (A), fraction 62 (+), and fraction 62 plus calmodulin (0).For fraction 11 and fraction 62 plus calmodulin, 100 nM calmodulin was included in the assay. B, kinase activity was measured as a function of added calmodulin for fraction11 (A) and fraction 62 (e); the reactions contained 10p~ free Ca2+.In both A and B, the symbols and burs are the means and standard error of the means for three determinations.

for Caz+below 0.4 p~ and was stimulated at least 20-fold over the activity measured at 0.6 p~ Ca2+. Another preparation of cross-linkedcomplex that was assayed exhibited elevated kinase activity in the absenceof Ca2+and additional activity in the presence of Ca2+; contamination with uncross-linked calmodulin prohibited further quantification. Analysis of Ca2+-dependent kinase activityrevealed significant differences in the kinetics of activation by the protein from fractions 11 and 62. First, the midpoint for Ca2+ stimulation of fraction 62, which was measured at 0.35 p~ in Fig. 3A, was approximately &fold less than the midpoint forCa2+ stimulation of fraction 11, which was measured at 1.7 p~ in Fig. 3A. Second, the slope of the Ca2+ response was significantly less for fraction 62, which had a Hill coefficient of 0.9 (Fig. 3A), thanfor fraction 11,which had a Hill coefficient of 2.0 (Fig. 3A). The distinct kineticsof Ca2+activation together with the Ca2+ dependenceof cross-link formation, the apparent 1:1 molar stoichiometry, and the insensitivity to exogenous calmodulin(Fig. 3B)areconsistentwith calmodulin bound at thecalmodulin binding domain of MLCK. The sequence of the calmodulin-binding region of rabbit skeletal muscle MLCK hasbeen identified (Blumenthal et al., 1985) and is known to be located at thecarboxyl terminus of the enzyme (Takio et al., 1986; Roush et al., 1988). The 27residue peptide (M13) binds to calmodulin with an affinityof

and it hasbeen shown that a synthetic peptide (M5), containing the first 17 amino acid residues and terminating in glycylamide, also binds to calmodulin (Kennelly et al., 1987). Although there is no crystallographic structure of a calmodulin complex with a peptide or enzyme, several models have been proposed for calmodulin-peptide complexes (O'Neil and DeGrado, 1985; Persechini and Kretsinger, 1988; Chin and Brew, 1989).The O'Neil-DeGrado and Chin-Brewmodels are based ona linear centralhelix of calmodulin, which occurs in the crystal structure (Babu et al., 1985, 1988; Kretsinger et al., 1986); there are some similarities in these models, but substantive differences also exist. The Persechini-Kretsinger model places a bend in the central helix a t Ser-81 and, like the O'Neil-DeGrado model, emphasizes electrostatic interactions between the bound peptide and calmodulin. In contrast, the Chin-Brew model attaches more importance to nonpolar interactions with thetwo hydrophobic pockets of calmodulin (Babu et al., 1985, 1988; Kretsinger et al., 1986), which are believed to be important in the binding of phenothiazines and other hydrophobic compounds (Jackson et at., 1987; Faust et al., 1987; Strynadka and James,1988). Our results cannot distinguish these quite different models, but it isof interest to speculate on the possible sites of crosslinking between calmodulin and skeletal muscle MLCK. Assuming that some or all of the DCC-mediated cross-linking involves the above sequence, the following sites could potentially form cross-links in the Persechini-Kretsingermodel (M = M13; C = calmodulin): (M)K5-(C)D78, (M)K6-(C)E84or (C)E85,(M)K18-(C)E114,and(M)KlS-(C)Ell or (C)E14. This model predicts no significant interaction between the carboxyl-terminal 6 residues of M13 and calmodulin. In contrast, the Chin-Brewmodel would allow a cross-link between the a-carboxyl group of M13 and Lys-21 of calmodulin. The O'Neil-DeGrado model predicts that helix G of calmodulin is important in the binding of amphipathic peptides, while the other two models would predict only limited, if any, direct contact between the bound peptide and this region of calmodulin. The 'H NMR data of Klevit et al. (1985) on a calmodulinM13 complex offersstrong support to the involvement of both globular lobes of the protein in peptidebinding. The present experimental results have demonstrated that a functional calmodulin-MLCK cross-linkedcomplex can be prepared. From theabove discussion of possible contact sites between calmodulin and M13, it is clear that identificationof the sitesof cross-linking between calmodulin and MLCKmay provide important information on the molecular nature of the complex, and such studies are under way. Identification of the sites of cross-linking may also be important in determiningif DCC first binds to the hydrophobic pockets of calmodulin before cross-linking. If so, the strong basic character of the calmodulin-binding region of skeletal muscle MLCK (cf. M13 sequence) would suggest that a carboxyl on calmodulin is activated, following by nucleophilic attack from peptide amino groups. The exceptions would be cross-linking of the MLCK a-carboxyl group, as favored in the Chin and Brew (1989) model, or cross-linking of calmodulin to other residues in MLCK that are not partof the M13 sequence. Summary-The results indicate that DCC is capable of producing a Ca2+-dependent cross-link between calmodulin and MLCK, possibly because DCC, a cyclohexyl compound, is localized at the high affinity binding site of calmodulin (Mann and Vanaman, 1988). A cross-linked product of cal-

Cross-linking MLCK of Calmodulin and modulin and MLCK was separated on DEAE-cellulose and found to have two modes of kinase activity. Approximately 50% of maximal kinaseactivityappearedto be produced through the simple association of calmodulin and MLCK. This result is consistent with the proposed mechanism in which thebinding of calmodulinreleases the calmodulin binding domain of the kinase from the catalytic site (Kemp et al., 1987; Kennelly et al., 1987; Pearson et al., 1988). The remaining Ca2+-dependent kinase activity of the cross-linked complex suggests that Ca2+ can regulate the activity of calmodulin associated with MLCK. Acknowledgments-It is a pleasure to thank David Chin and Dr. Keith Brew for helpful discussion and for providing results prior to publication. REFERENCES Babu, Y. S., Sack, J. S., Greenhough, T. J., Bugg, C. E., Means, A. R., and Cook, W. J. (1985) Nature 3 1 5 , 37-40 Babu, Y. S., Bugg, C. E., and Cook, W. J. (1988) J. Mol. Biol. 2 0 4 , 191-204

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