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