©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Ca-Calmodulin Binds to the Carboxyl-terminal Domain of Dystrophin (*)

(Received for publication, July 17, 1995; and in revised form, January 11, 1996)

J. Todd Anderson R. Preston Rogers Harry W. Jarrett (§)

From the Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The unique COOH-terminal domain of dystrophin (mouse dystrophin protein sequences 3266-3678) was expressed as a chimeric fusion protein (with the maltose-binding protein), and its binding to calmodulin was assessed. This fusion protein, called DysS9, bound to calmodulin-Sepharose, bound biotinylated calmodulin, caused characteristic changes in the fluorescence emission spectrum of dansyl-calmodulin, and had an apparent affinity for dansyl-calmodulin of 54 nM. Binding in each case was Ca-dependent. The maltose-binding protein does not bind calmodulin, and thus binding resides in the dystrophin-derived sequences. Deletion mutation experiments further localize the high affinity calmodulin binding to mouse dystrophin protein sequences 3293-3349, and this domain contains regions with chemical characteristics found in the calmodulin-binding sequences in other proteins. The COOH-terminal domain provides sites of attachment of dystrophin to membrane proteins, and calmodulin binding may modulate these interactions.


INTRODUCTION

Dystrophin, the 427-kDa protein product of the Duchenne muscular dystrophy gene locus, is a cytoskeletal protein of the sarcolemma. Its structure is composed of four distinct domains(1) : an amino-terminal domain, which binds F-actin(2, 3, 4, 5, 6, 7) , a rod-like central domain, which makes up most of its length, a cysteine-rich domain homologous to alpha-actinin, and a unique COOH-terminal domain(1) . Dystrophin, in the presence of detergents, can be isolated in a complex with other sarcolemma proteins, the dystrophin-glycoprotein complex(8, 9) . This complex contains at least three proteins, syntrophin(10, 11, 12, 13) , beta-dystroglycan(14, 15) , and adhalin(13) , which interact with the COOH-terminal domain of dystrophin. The amino-terminal domain does not bind to any of the DGC (^1)components in gel blot overlay experiments (13) and presumably only interacts with F-actin. Thus, current models of dystrophin and the DGC(8, 13, 15, 16) propose that dystrophin has two types of attachment to the rest of the cytoskeleton: amino-terminal binding to F-actin and COOH-terminal binding to DGC proteins that anchor it to the membrane.

Chimeric fusion proteins containing dystrophin amino-terminal domain sequences bind calmodulin(2, 7) . Under some assay conditions (2) but not others(7) , calmodulin inhibits F-actin binding, suggesting Ca-calmodulin modulation of dystrophin's attachment to the cytoskeleton at this end of the molecule. Ca-calmodulin also has other effects within the DGC. Dystrophin is phosphorylated in vivo(17, 18) . A Ca-calmodulin activated protein kinase, probably a muscle isoform of CaM kinase II, copurifies with the DGC and phosphorylates dystrophin(19) . One site phosphorylated by the endogenous activity and by the brain isozyme of CaM kinase II has been localized to dystrophin's unique COOH-terminal domain(20) . The DGC also contains a protein phosphatase activity, which dephosphorylates dystrophin(19) , and phosphorylations of dystrophin's COOH-terminal domain catalyzed by CaM kinase II can be dephosphorylated by calcineurin, the Ca-calmodulin-dependent protein phosphatase type 2b(21) . Dystrophin is also phosphorylated in vitro by other protein kinases(22) , including Cdc2 kinase, which also phosphorylates the COOH-terminal domain of dystrophin(17) . Thus, Ca-calmodulin-dependent (19, 21) and independent (17, 22) phosphorylations occur in the COOH-terminal domain of dystrophin.

In Becker muscular dystrophy, dystrophin is shortened by genetic deletions, and the regions deleted have been correlated with the severity of disease(23) . Deletions that affect this unique COOH-terminal domain result in severe muscular dystrophy, suggesting an important role for this domain in normal muscle function. Since this domain interacts with DGC proteins to anchor dystrophin to the membrane and is phosphorylatable, these protein-protein interactions and their regulation may explain the severe consequences of deletions within the COOH-terminal domain.

Clearly, calmodulin by way of activated protein kinases and phosphatases can affect the COOH-terminal domain of dystrophin, but can it act more directly? Madhavan, et al.(24) have shown that dystrophin in DGC gel blot overlay experiments binds biotinylated calmodulin with an apparent affinity in the range of 6-60 nM. Based upon sequence comparison, they suggested calmodulin binding to sites located in both the amino- and COOH-terminal domains of dystrophin. The amino-terminal domain actually contains more than one calmodulin-binding site, but the apparent affinity here is about 2 µM(2) and cannot account for the higher affinity reported earlier(24) . Here, we investigate calmodulin binding to the COOH-terminal domain dystrophin sequences. While it appears that high affinity calmodulin-binding is indeed attributable to these sequences, the site of binding was not where predicted.


MATERIALS AND METHODS

Fusion Proteins

The cloning, expression, and purification of the maltose-binding protein-DysS9 chimeric fusion protein (13) and the MalF control containing only maltose-binding protein sequences (2) have been previously described. The pMALf plasmid used for some constructs has been previously described(2) . Common molecular biology methods were those described in (25) . The various deletion mutants of DysS9 (depicted in Fig. 1) were constructed as follows.


Figure 1: Fusion proteins containing dystrophin COOH-terminal domain sequences. Each of the chimeric fusion proteins is produced with the maltose-binding protein at its amino terminus (not shown). The mouse dystrophin sequences are shown to scale and are given in parentheses. The position of putative CBS 3 is also shown.



DysS9A

DysS9 in pMALc was restricted at the plasmid BamHI and the unique insert MscI sites. The resulting 446-bp fragment was gel-purified and ligated to a BamHI/StuI-restricted pMALf vector, which provides a necessary stop codon.

DysS9B

DysS9 in pMALc was restricted at the insert MscI site and at the plasmid (downstream) EcoRI site, and the resulting 815-bp fragment was gel-purified and ligated into a StuI/EcoRI pMALf vector.

DysS9E

The DysS9A plasmid, isolated by miniprep, was restricted at the insert NcoI site, and the overhang was filled with Klenow large fragment DNA polymerase I. After 65 °C heating to denature the polymerase, phenol:chloroform extraction, and ethanol precipitation, the DNA was further restricted at the plasmid (upstream) BamHI site, and the resulting 266-bp fragment was gel-purified and ligated into a BamHI/StuI-restricted pMALf vector.

DysS9F

DysS9 in pMALc was restricted at the insert DraIII site, and the overhang was blunted with the Klenow fragment 3`-exonuclease activity. After 65 °C heating to denature the exonuclease, phenol:chloroform extraction, and ethanol precipitation, the DNA was further restricted at the plasmid (upstream) BglII site, and the resulting 880-bp fragment was gel-purified and ligated into a BglII/StuI-restricted pMALf vector.

DysS9G

A portion of the blunted, DraIII-restricted DysS9 DNA (prepared for DysS9F) was restricted at the plasmid (downstream) XbaI site, and the resulting 1185-bp fragment was gel-purified and ligated to a StuI/XbaI pMALc vector.

DysS9H

DysS9 in pMALc was restricted with NcoI, and the overhang was filled with the Klenow fragment polymerase. After 65 °C heating to denature the polymerase, phenol:chloroform extraction, and ethanol precipitation, the DNA was further restricted at the plasmid (downstream) XbaI site, and the resulting 1018-bp piece was gel-purified and ligated to a filled EcoRI/XbaI pMALc vector.

Each protein was expressed and purified by maltose affinity chromatography using procedures previously described(2) . In all cases, authenticity was demonstrated by DNA sequencing as described previously (2) at both ends of the construct using pMALc sequence-based primers, which allow sequence at the plasmid-insert junctions to be determined. Furthermore, each fusion protein was of the expected size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the methods of Laemmli(26) .

Calmodulin

Calmodulin was purified from bovine brain by the procedure of Gopalakrishna and Anderson(27) . Calmodulin-Sepharose was prepared as described previously(28) . Briefly, 10 g. (wet weight) of Sepharose 4B was activated using 3 g of CNBr and coupled to 10 mg of bovine brain calmodulin in 100 mM sodium borate, pH 8.2, 1 mM MgCl(2), and 0.02 mM CaCl(2) at 4° for 24 h. The resulting calmodulin-Sepharose was then reacted with 0.5 M 2-aminoethanol in the same borate buffer and pH 8.2 for an additional 24 h prior to use. Calmodulin-Sepharose was packed in a 2-ml bed column and equilibrated in buffer AC (50 mM Tris, pH 8, 100 mM KCl, 3 mM MgCl(2), 1 mM CaCl(2)). For chromatography (at 4 °C), 1 ml of 0.2 mg/ml fusion proteins in buffer AC was applied to the equilibrated column and washed with 10 ml of buffer AC. The buffer was then changed to buffer AE (same as AC except for 1 mM EGTA replacing the calcium), and the column was eluted. One-ml fractions were collected. From each fraction, 0.5 ml was mixed with 0.5 ml of a 2-fold concentrated Bradford protein reagent (29) , and after 10 min the absorption at 594 nm was recorded as a measure of protein concentration. The column was stored at 4 °C in buffer AC containing 10 mM sodium azide and 1 mM 2-mercaptoethanol.

Dansyl-calmodulin was prepared by the procedure in (30) . It contained 1.1 mol of dansyl/mol of calmodulin. Spectra were collected using the SLM Aminco-Bowman series 2 spectrofluorometer. To measure binding, 3 ml of 9 nM dansyl-calmodulin in buffer MC (0.1 mg/ml ovalbumin, 10 mM MOPS, pH 7, 90 mM KCl, 1 mM CaCl(2)) was titrated by additions of 5 µM fusion protein in buffer MC. The fluorescence emission at 490 nm was measured with excitation at 345 nm (both slits at 16-nm band pass). An identical titration except in the absence of dansyl-calmodulin was performed, and these data were subtracted to correct for fluorescence resulting from the fusion protein alone. Emission spectra (excitation, 345 nm) were recorded in either buffer MC or buffer ME (the same as buffer MC except for 1 mM EGTA replacing calcium) in the absence and presence of either 200 or 400 nM DysS9 fusion protein and both slits at 8-nm band pass.

Biotinylated calmodulin was prepared as described(32) . Slot blots of the dystrophin fusion proteins were prepared using the Bio-Rad slot-blot apparatus and were blocked and probed with 250 nM biotinylated calmodulin using previously described procedures (31) .


RESULTS

Fig. 1depicts the sequences from the COOH terminus of mouse muscle dystrophin that were expressed as fusion proteins for this study. Within this 413-residue region, a smaller region is indicated. CBS 3 is a putative calmodulin-binding site that will be the subject of further discussion.

Fig. 2shows 1 µg of the purified fusion proteins applied to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The maltose binding protein sequences were expressed separately as the MalF protein, which has been previously described (2) . These sequences add about 43 kDa to the apparent molecular mass of each fusion protein. While the MalF protein and DysS9F appear to be the same size on this gel (Fig. 2), higher percentage acrylamide gels do resolve the two, and DysS9F is about 3-4 kDa larger as expected (data not shown). Each of the fusion proteins was of the expected size and obtained in a high state of purity. However, each of the purified fusion proteins has some smaller species that are shortened dystrophin fusion proteins since they were purified by maltose affinity chromatography, and all bind an antibody against the maltose-binding protein sequences (Fig. 2). Shorter protein species were also encountered previously with maltose fusion proteins containing amino-terminal dystrophin sequences(2) ; these presumably result from partial proteolysis. Since the maltose-binding protein must be relatively intact to allow purification and antibody staining, proteolysis must be primarily at the COOH terminus of the dystrophin sequences. The DysS9H protein is also of the correct size but shows somewhat more proteolysis (data not shown).


Figure 2: Each of the purified fusion proteins is of the expected size. One microgram of each fusion protein shown was applied to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue in the upper panel. In the lower panel, an identical gel was blotted to nitrocellulose and detected with a 1:100,000 dilution of serum antibodies against the maltose-binding protein. Goat antirabbit-alkaline phosphatase conjugates from Bio-Rad Laboratories at 1:1000 dilution provided the second antibody, and the procedures used were those in (31) . The position of molecular mass markers is given along with their mass in kilodaltons.



When 0.2 mg of DysS9, the intact COOH-terminal domain of dystrophin, is applied to calmodulin-Sepharose it binds in the presence of Ca and elutes in its absence (i.e. in the presence of the Ca chelator EGTA beginning at fraction 12) as shown in Fig. 3. In contrast, the MalF control protein passes through the column unretained, demonstrating that calmodulin-binding is a property of the dystrophin COOH-terminal sequences.


Figure 3: Fusion proteins containing dystrophin sequences 3293-3349 bind to calmodulin-Sepharose in a Ca-dependent manner. Chromatography of 0.2 mg of each fusion protein on a 2-ml calmodulin-Sepharose column is shown. Ca was present in all buffers initially and up to fraction number 11 when it was replaced with EGTA to elute the column. Protein was detected using the Bradford dye binding assay (29) and is shown as Absorption, 594 nm. The chromatograms from different experiments are displaced by one absorption unit so that they may be shown on the same figure for comparison.



It is also clear from Fig. 3that some of the DysS9 protein (<10%) did not bind to the column, and the cause of this was further investigated (data not shown). When higher amounts of DysS9 are applied to the column, the same proportion binds, showing that these results are not due to inadequate column capacity. When eluted protein is reapplied to the column, it all binds, while the fraction that is unretained by the column is unretained upon rechromatagraphy. The protein loaded, unretained, and retained by the column appears virtually identical when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, demonstrating that the unretained protein is not a contaminant or some proteolysed fragment of DysS9. These results suggest that while a large fraction (>90%) of the DysS9 protein binds calmodulin, a small fraction (<10%) is not active and is probably improperly folded.

The data in Fig. 3also demonstrate that calmodulin binding must be due to dystrophin protein sequences 3293-3349. The proteins that contain this sequence (DysS9, DysS9A, DysS9E, and DysS9G; see Fig. 1) all bind to calmodulin-Sepharose in a calcium-dependent manner, while those that lack this sequence (DysS9B, DysS9F, DysS9H, and MalF) do not (Fig. 3).

Dansyl-calmodulin has been used to characterize protein-calmodulin interactions by different laboratories(30, 33) . Upon Ca binding, the fluorescent emission (excitation, 345 nm) is enhanced, and the spectrum undergoes a dramatic blue-shift. In the absence of Ca, the emission maximum is at 530 nm, and this shifts to 494 nm upon Ca binding, and fluorescence is enhanced about 2.5-fold (Fig. 4). Upon binding a target protein, the fluorescent emission is further enhanced and blue-shifted, although by a lesser amount(33) . These changes were also observed with the dystrophin fusion proteins. In Fig. 4, when 200 or 400 nM DysS9 is added, the spectrum observed in the presence of Ca (but not in EGTA) undergoes further changes (Fig. 4), which agrees with the Ca dependence of the DysS9-calmodulin interaction demonstrated using calmodulin-Sepharose (Fig. 3). In the presence of Ca and 400 nM DysS9, the dansyl-calmodulin emission maximum is further blue-shifted to 490 nm and is enhanced 32% (Fig. 4). These observations with DysS9 closely parallel what has been observed with other well characterized calmodulin-binding proteins such as phosphodiesterase, calcineurin, and troponin I(33) .


Figure 4: The dystrophin DysS9 fusion protein causes characteristic changes in the emission spectrum of dansyl-calmodulin. The three lower tracings were all obtained in the 1 mM EGTA buffer; the upper three tracings are in 1 mM CaCl(2). The spectra shown are of 25 nM dansyl-calmodulin containing either no addition (thin line), 200 nM (thicker line), or 400 nM (thickest line) dystrophin DysS9 fusion protein. Excitation was at 345 nm, and both excitation and emission slits were set at 8-nm band pass.



While dansyl-calmodulin has previously been used at concentrations too high to accurately measure the affinity of protein binding(30, 33) , we have found that dansyl-calmodulin can also be used at much lower concentrations, which allow estimation of the binding affinity of DysS9 for calmodulin. In Fig. 5, 9 nM dansyl-calmodulin was titrated with DysS9 while measuring emission at 490 nm (excitation, 345 nm). The data points were fit by nonlinear least squares analysis to the Scatchard equation, and the line on the figure shows the resulting fit. This fit has a correlation coefficient (r) of 0.986, indicative of a good fit. Three separate experiments like Fig. 5yielded an average apparent dissociation constant of 54 ± 25 nM (mean ± S.D.) DysS9. In contrast, the MalF protein shows no binding (data not shown). However, since isolated DysS9 protein is not all full-length (Fig. 2) and not all capable of binding calmodulin (Fig. 3), this apparent dissociation constant is only an upper limit of the true dissociation constant. Furthermore, since the dansyl-calmodulin concentration (9 nM) is of appreciable magnitude relative to the dissociation constant, this apparent constant would be higher than the true dissociation constant. Thus, DysS9 binds dansyl-calmodulin with a dissociation constant less than 54 nM. This value agrees reasonably well with our previous estimates of the affinity of dystrophin for biotinylated calmodulin (i.e. between 6 and 60 nM), suggesting that the high affinity of intact dystrophin for calmodulin (24) may be attributed to the COOH-terminal domain. However, this conclusion is tentative since calmodulin binding to only amino- and COOH-terminal dystrophin sequences have been investigated and other, undiscovered binding sites may exist.


Figure 5: The dystrophin DysS9 fusion protein has an apparent affinity of 54 nM for dansyl-calmodulin. Nine nM dansyl-calmodulin in buffer MC was titrated with additions of the DysS9 fusion protein, and fluorescence was measured (closed circles, 345-nm excitation, 490-nm emission). The data from an identical titration in the absence of dansyl-calmodulin has been subtracted to correct for DysS9 fluorescence, and the fluorescence in the absence of DysS9 protein was subtracted to show only the fluorescent changes resulting from DysS9. Relative Fluorescence is the photomultiplier tube output in volts on a scale where the fluorescence of dansyl-calmodulin was set at 6 V; e.g. a relative fluorescence of 2 V represents a 33% increase in the fluorescence. The line shown is that obtained from fitting these data by nonlinear least squares analysis (using the PsiPlot software) to the Scatchard binding isotherm; for this experiment, the apparent binding affinity was 50.9 nM, which is close to the average value of 54 nM for three experiments.



The dystrophin DysS9 fusion proteins also bind biotinylated calmodulin as shown in Fig. 6. While the same mass of each protein was loaded for comparison, some of these are larger (e.g. DysS9) than others (e.g. DysS9E) and therefore represent fewer moles of protein for calmodulin to bind; this probably accounts for some of the differences in staining intensity observed. Binding resides in the amino-terminal end of DysS9 since DysS9A but not DysS9B binds biotinylated calmodulin. The further deletions resulting in DysS9E do not significantly affect binding, further limiting the location of the binding site. With DysS9F, little binding is observed over this concentration range, while DysS9G binds as expected (Fig. 6). Thus, in agreement with the experiment in Fig. 3, we conclude that high affinity calmodulin binding resides in 3293-3349 (i.e. CBS 3), sequences present in DysS9A, -E, and -G but absent in DysS9B and -F. The binding of biotinylated calmodulin is also calcium-dependent (data not shown).


Figure 6: Binding of biotinylated calmodulin to dystrophin DysS9 fusion proteins. Across the top is shown the nanograms of fusion protein that were applied to the nitrocellulose slot. After blocking, the blot was probed with 250 nM biotinylated calmodulin in BSA/Ca/TTBS (0.1 mg/ml bovine serum albumin, 50 mM Tris, pH 7.5, 0.5 M NaCl. 1 mM CaCl(2), 0.05% Tween-20) and detected with streptavidin-biotinylated alkaline phosphatase conjugates as described previously(31) . MalF contains only maltose-binding protein sequences and serves as a negative control.



When 10 µg of DysS9F was applied to blots such as in Fig. 6, more biotinylated calmodulin was bound than the comparable MalF control, indicating some presumably lower affinity binding occurring in DysS9F sequences (data not shown but submitted for review). Evidently, this lower affinity binding is insufficient to be retained by calmodulin-Sepharose (Fig. 3) or to be apparent at the lower amounts in Fig. 6. The significance of this weaker binding in DysS9F sequences is not clear.

The sequence responsible for high affinity calmodulin binding, dystrophin 3293-3349 (CBS 3), is shown in Table 1. This region of sequence shows characteristics typical of other calmodulin-binding sequences in that it is predominantly cationic and hydrophobic with aromatic residues interspersed. This entire protein sequence is identical in mouse and human dystrophin, suggesting strong conservation. CBS 3 and the calmodulin-binding sequences of skeletal muscle myosin light chain kinase, CaM kinase II, Ca-transport ATPase, and the two calmodulin-binding sequences previously found in the amino-terminal domain of dystrophin (CBS 1 and 2) all have these cationic-hydrophobic chemical characteristics. The region of the CBS 3 dystrophin sequence that is most similar to the other calmodulin-binding sequences is toward the COOH-terminal end, sequence positions 3313-3349, and it may be this region that actually binds the calmodulin. It is interesting to note that these sequences are those that make up most of exon 69 of dystrophin.




DISCUSSION

This report is the first to directly investigate calmodulin binding to the unique COOH-terminal domain of dystrophin. Here, calmodulin binding was found to be localized to dystrophin sequences 3293-3349 (CBS 3) and to have an affinity with an upper limit of 54 nM, consistent with the affinity previously estimated from DGC gel blots(24) . Since dystrophin's amino-terminal calmodulin-binding has an apparent affinity of about 2 µM(2) , highest affinity binding resides in the COOH-terminal domain. Previously, Madhavan et al.(24) , based upon sequence comparisons, had predicted calmodulin binding to sequences that correspond to 3366-3391 of mouse dystrophin. While this region also has chemical characteristics consistent with calmodulin binding, it does not appear to contribute to the high affinity binding observed here since its deletion in the DysS9E protein has little effect on the binding of calmodulin-Sepharose (Fig. 3) or biotinylated CaM (Fig. 6). Furthermore, these sequences are present in the DysS9H protein, which does not bind calmodulin-Sepharose (Fig. 3). Thus, it would appear that calmodulin binding resides in the sequences 3293-3349, not where previously predicted. This finding of a calmodulin-binding site not where previously predicted (24) and the unexpected discovery of CBS 2 in our previous report (2) suggest that while the chemical properties of calmodulin-binding sequences are necessary, they are not sufficient to ensure binding. Other attributes of these sites are responsible for high affinity binding; presumably these result from higher order structure.

The sequences contained in the DysS9 protein are those that compose the unique COOH-terminal domain of dystrophin(1) . Mutations in this region of dystrophin result in severe Becker muscular dystrophy phenotype (23) , suggesting that this region of dystrophin is critically important to normal muscle function. Sequences contained within the DysS9 protein have been shown to be important to dystrophin's interaction with sarcolemma membrane proteins. Specifically, these sequences bind to syntrophins(10, 11, 12, 13) , beta-dystroglycan(14, 15) , and adhalin(13) . Syntrophins also bind Ca-calmodulin(24) . The syntrophins are a triplet of homologous proteins present in the DGC (16) and bind to dystrophin sequences derived from exon 73 (10) or exons 73 and 74 (11, 12) (mouse dystrophin protein sequences 3438-3510). These syntrophin-binding sequences are adjacent to the dystrophin calmodulin-binding domain demonstrated here. beta-Dystroglycan apparently binds predominantly to sequences amino-terminal to those present in DysS9 but overlapping with part of DysS9(15) . Adhalin has also been found to bind to a similar region to that utilized by beta-dystroglycan, again overlapping with DysS9, and DysS9 binds adhalin (13) . Thus, the CBS 3 sequences located here are in a region of dystrophin that interacts with other proteins, and this topography suggests that calmodulin may modulate some of these interactions. Recently, we have shown that the DysS9 sequences are also phosphorylated at the RSDSS sequence (dystrophin 3613-3617) by CaM kinase II(20) . Ser, present in mouse dystrophin DysS9, is also apparently phosphorylatable by Cdc2 kinase(17) . The membrane interactions, nearby phosphorylation sites, and a calmodulin-binding site may help explain the severe disease resulting from mutations within this COOH-terminal domain.

While the function of calmodulin-binding to the COOH-terminal domain awaits discovery, some progress has been made toward defining a role for the amino-terminal binding. Bonet-Kerrache et al.(7) found that fusions containing dystrophin amino-terminal sequences bound to calmodulin-Sepharose and to calmodulin-coated microtiter plates, and binding was Ca-dependent. However, they did not find an effect of calmodulin on F-actin binding. In a subsequent investigation using different fusion proteins, calmodulin-binding to the amino terminus of dystrophin was again demonstrated and shown to be Ca-dependent. Additionally, calmodulin was found to competitively inhibit F-actin binding with half-inhibition occurring at 2 µM calmodulin in a microtiter plate-based assay(2) . Using the apparent affinities for F-actin and calmodulin measured in this latter report(2) , it can be calculated that under the cosedimentation assay conditions used by Bonet-Kerrache et al.(7) , calmodulin would have reduced F-actin binding by only a few percent (<10%) and may have escaped detection. Another bacterially expressed protein containing dystrophin's amino terminus (3) has at least 400-fold lower affinity for F-actin than was found in later studies (2, 6) or has been reported for full-length dystrophin (5) ; calmodulin's effect on F-actin binding would be expected to be even smaller, and calmodulin inhibited F-actin binding only to a small extent(35) . Dystrophin, found predominantly in muscle and nerve, has a homologue called utrophin (or dystrophin-related protein) found in other tissues. The amino-terminal domain of utrophin has recently been shown to bind calmodulin and F-actin, and furthermore, calmodulin inhibited F-actin binding(35) . Thus, calmodulin inhibits F-actin binding to utrophin (35) and to dystrophin(2) . It is interesting to note that calmodulin inhibits F-actin interactions with other cytoskeletal components in other cells, notably F-actin's interaction with spectrin (39, 40) and utrophin (35) (both members of the dystrophin gene family), the MARCKS protein(41) , and caldesmon (42) . Since dystrophin is an antiparallel dimer, these amino- and COOH-terminal events are adjacent and may share calmodulin or other components.

The CBS 3 sequence in human and mouse dystrophin are identical and are also quite similar in utrophin; 93% identity with utrophin is found at the protein level, and most replacements are conservative ones. This is also true of the other binding regions found in both dystrophin and utrophin. CBS 1 shows the lowest identity (64%), CBS 2 the next highest (86%), and CBS 3 the highest (93%). The putative actin-binding sites (2) are also conserved with ABS 1 (74%), ABS 2 (86%), and ABS 3 (86%), all showing high percentage identity between dystrophin and utrophin. The close similarity of these two proteins in calmodulin-binding, actin-binding, and cellular localization is thus reflected in similar protein sequence.

Despite a report to the contrary(34) , which has been discussed elsewhere(2) , the evidence that calmodulin binds to dystrophin sequences is now broadly based. Biotinylated calmodulin binds to dystrophin (24) and to fusion proteins containing its amino- (2) or COOH terminus (Fig. 6). Amino-terminal dystrophin fusion proteins also bind to calmodulin-Sepharose and to calmodulin-coated microtiter plates (7) , (^2)and calmodulin-binding inhibits F-actin binding by dystrophin (2) and utrophin(35) , although not under some assay conditions(7) . COOH-terminal dystrophin fusions bind to calmodulin-Sepharose (Fig. 3) and to biotinylated-calmodulin (Fig. 6); bind dansyl-calmodulin (Fig. 5) with an affinity (<54 nM) that is similar to that estimated for dystrophin in gel blot experiments (between 6 and 60 nM)(24) ; and cause characteristic changes in the dansyl-calmodulin fluorescence spectrum (Fig. 4) similar to that found for other well characterized calmodulin-binding proteins(30, 33) . Calmodulin binding to dystrophin and fusion proteins derived from it has also been uniformly calcium-dependent, as is true of most calmodulin-binding. For both the amino- (2) and COOH-terminal calmodulin-binding (Table 1), calmodulin-binding was localized to dystrophin sequences that have characteristics similar to calmodulin-binding sequences located in other calmodulin-binding proteins. While calmodulin-binding sequences are not strictly homologous, they do all share the chemical characteristics of being cationic, hydrophobic, and aromatic-rich sequences (see (31) for a further discussion of these characteristics). Furthermore, dystrophin is homologous to spectrin, fodrin, and utrophin, which are all also calmodulin-binding proteins (36, 37, 38) . Thus, dystrophin by numerous criteria is a Ca-dependent, calmodulin-binding protein with a relatively high affinity for calmodulin. While calmodulin-binding occurs at both the amino- and COOH-terminal ends of dystrophin, the highest affinity binding resides in the unique COOH-terminal domain of dystrophin. Closely associated with dystrophin is a calmodulin-dependent protein kinase that copurifies with dystrophin and phosphorylates it (19) at at least one site contained in the COOH-terminal domain(20) . This same COOH-terminal domain also binds calmodulin (this report), syntrophins(10, 11, 12, 13) , beta-dystroglycan (14, 15) , and adhalin(13) . Thus, calmodulin acting directly, or indirectly via protein phosphorylation, has the potential to regulate many of the interactions of dystrophin.


FOOTNOTES

*
This work was supported by the Muscular Dystrophy Association. 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.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Tennessee, 858 Madison Ave., Memphis, TN 38163. Tel.: 901-448-7078; Fax: 901-448-7360; hjarrett{at}utmem1.utmem.edu.

(^1)
The abbreviations used are: DGC, the dystrophin-glycoprotein complex; CaM kinase II, calmodulin-dependent multifunctional protein kinase type II; CBS, calmodulin-binding site; MOPS, 4-morpholinepropanesulfonic acid; bp, base pair(s).

(^2)
J. T. Anderson, R. P. Rogers, and H. W. Jarrett, unpublished data.


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