PHYSIOLOGICAL REVIEWS
Vol. 79, No. 3, July 1999
Printed in U.S.A.
Apocalmodulin
LUIS A. JURADO, PRIYA SETHU CHOCKALINGAM, AND HARRY W. JARRETT
Department of Biochemistry, University of Tennessee, Memphis, Tennessee
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I. Introduction
A. Role of calmodulin
B. Apocalmodulin binding within the cell
II. Structure of Apocalmodulin
A. Complex formation
III. Known Apocalmodulin Binding Motifs
A. IQ motifs
B. Synergistic binding of noncontiguous sequences
C. Probability of other types
IV. Apocalmodulin-Binding Proteins
A. Actin-binding proteins
B. Cytoskeletal and membrane proteins
C. Enzymes
D. Receptors and ion channels
V. Conclusions
Jurado, Luis A., Priya Sethu Chockalingam, and Harry W. Jarrett. Apocalmodulin. Physiol. Rev. 79: 661– 682,
1999.—Intracellular Ca21 is normally maintained at submicromolar levels but increases during many forms of
cellular stimulation. This increased Ca21 binds to receptor proteins such as calmodulin (CaM) and alters the cell’s
metabolism and physiology. Calcium-CaM binds to target proteins and alters their function in such a way as to
transduce the Ca21 signal. Calcium-free or apocalmodulin (ApoCaM) binds to other proteins and has other specific
effects. Apocalmodulin has roles in the cell that apparently do not require the ability to bind Ca21 at all, and these
roles appear to be essential for life. Apocalmodulin differs from Ca21-CaM in its tertiary structure. It binds target
proteins differently, utilizing different binding motifs such as the IQ motif and noncontiguous binding sites. Other
kinds of binding potentially await discovery. The ApoCaM-binding proteins are a diverse group of at least 15 proteins
including enzymes, actin-binding proteins, as well as cytoskeletal and other membrane proteins, including receptors
and ion channels. Much of the cellular CaM is bound in a Ca21-independent manner to membrane structures within
the cell, and the proportion bound changes with cell growth and density, suggesting it may be a storage form.
Apocalmodulin remains tightly bound to other proteins as subunits and probably hastens the response of these
proteins to Ca21. The overall picture that emerges is that CaM cycles between its Ca21-bound and Ca21-free states
and in each state binds to different proteins and performs essential functions. Although much of the research focus
has been on the roles of Ca21-CaM, the roles of ApoCaM are equally vital but less well understood.
I. INTRODUCTION
External signals often lead to transient increases in
intracellular Ca21 (13). Cells have elaborated exquisite ways
of controlling and utilizing the gradient of ion concentration
across the plasma membrane and have developed an intracellular messenger system by adopting Ca21 signaling. Increases in the cytoplasmic Ca21 concentration lead to binding of Ca21 by intracellular regulatory proteins (33), an event
that initiates a wide variety of cellular processes. One of
many Ca21-binding regulatory proteins, calmodulin (CaM),
0031-9333/99 $15.00 Copyright © 1999 the American Physiological Society
is a ubiquitous, multifunctional protein that can bind to at
least 30 different target enzymes and proteins (see Tables 1
and 2) including Ca21-transport ATPase, phosphodiesterase,
myosin light chain kinase (MLCK) and other CaM-dependent
protein kinases, calcineurin (phosphoprotein phosphatase
2b), and nitric oxide synthase (61, 93, 141). Although much
of the research focus has been on the Ca21-bound form,
cellular Ca21-binding proteins such as CaM can exist primarily in two states, the other being effectively Ca21 free. Either
state can bind a different (or overlapping) set of target
proteins and in that way signal cellular responses.
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TABLE
Volume 79
1. Some well-characterized Ca21-calmodulin binding proteins
Group
Protein kinases
Phosphoprotein phosphatase
Second messenger metabolism
Metabolism
Other
Putative Function
Phosphorylase b kinase
Myosin light chain kinase
CaM kinase I
CaM kinase II
CaM kinase III
CaM kinase IV
Calcineurin (2b)
Type I adenylate cyclase
cAMP phosphodiesterase
NO synthase
Inositol 3-kinase
Ca21-transport ATPase
G protein-coupled receptors
Dystrophin
Spectrin/band 4.1
Fodrin
Caldesmon
MARCKS protein
Syntrophin
NAD kinase
Microtubules
Regulate glycogen metabolism
Regulate smooth muscle contraction
Multifunctional
Multifunctional, regulate over 30 enzymes
Translation?
Multifunctional
Metabolic regulation/cell cycle
Produce cAMP
Degrade cAMP
Produce NO
Produce IP4
Decrease cytosolic Ca21
Various signaling
Inhibit F-actin binding
Inhibit F-actin binding
Inhibit F-actin binding
Inhibit F-actin binding
Inhibit F-actin binding
Inhibit dystrophin binding?
Convert NAD to NADP
Inhibit tubulin polymerization
CaM, calmodulin; NO, nitric oxide; IP4, inositol 1,3,4,5-tetrakisphosphate.
Calmodulin was discovered as an activator of cyclic
nucleotide phosphodiesterase in brain and heart (24, 65).
It was subsequently rediscovered several times, since
many Ca21-dependent cellular processes were eventually
shown to involve the same Ca21-binding protein. As a
result, there was confusion in the nomenclature in the
early literature, since the protein was referred to by several different names (e.g., Ca21-dependent regulator,
modulator protein, Ca21-dependent modulator, activator
protein, troponin C-like protein). However, the name
CaM, first suggested by W. Y. Cheung, has found general
acceptance. Calcium-free CaM is referred to as apocalmodulin (ApoCaM).
Calmodulin is widely distributed in nature and is
ubiquitous in eukaryotic cells. Calmodulins isolated from
diverse organisms are remarkably similar in biological,
chemical, and physical properties (78, 92). There is 100%
identity in its amino acid sequence among vertebrates,
with multiple genes encoding identical CaM (43). This
high degree of conservation may be essential for the
maintenance of interaction with a diverse family of CaMbinding proteins (30). It is a small (16.7 kDa), very acidic
(isoelectric point ;4), relatively stable, and heat-resistant
protein. It contains four Ca21-binding sites per molecule
(53, 69). One of the properties of CaM, which was discovered earlier and is by now extremely well documented
(155), is the conformational change that takes place upon
binding Ca21. It transmits the Ca21 signal by binding to
and activating numerous enzymes central to cellular regulation (27). Although CaM could potentially exist in 16
different Ca21-bound states (i.e., ApoCaM and CaM with
1, 2, 3, or 4 Ca21 bound at various combinations of the 4
sites), most enzymes that are activated by Ca21-CaM re-
quire that three or four Ca21 be bound (29). Thus much of
our discussion can be simplified by considering primarily
two states of CaM: ApoCaM and “Ca21-CaM,” the form
containing three or four Ca21 that activate certain enzymes. What makes CaM more interesting is not merely
how many, but also how varied are its binding proteins.
Binding is not to a conserved motif: Ca21-CaM binding
domains in different target proteins show very little similarity in primary sequence. Instead, these binding sites
are amphipathic a-helices, typically ;20 residues in
length, with basic and hydrophobic residues intermingled.
Typically, one or more aromatic residues are also found
near the amino-terminal end of this region, which are
important to binding. This is an unusual sort of specific
molecular recognition (157). Interestingly, the binding
sites for ApoCaM show greater homology.
Table 1 lists Ca21-CaM binding proteins that have
been reasonably well characterized. With only two exceptions (phosphorylase b kinase and syntrophin), we do not
discuss the proteins in Table 1 further. The interested
reader should consult the numerous current reviews of
Ca21-CaM (19, 30, 91, 93, 100, 106, 141, 142). Among these
reviews are an issue of a journal (19) and a volume of a
series (91) that give details of individual CaM-protein
interactions. Calmodulin can also interact with proteins in
a Ca21-independent manner (118). This review discusses
the structure of ApoCaM and how it differs from Ca21CaM, the roles of ApoCaM in the eukaryotic cell, and
those proteins for which there is substantial evidence of
ApoCaM binding. These ApoCaM-binding proteins are
listed in Table 2.
The binding of CaM to proteins in the presence of
Ca21 chelators (e.g., EGTA or EDTA) is frequently re-
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Cytoskeletal/muscle
Enzyme or Protein
TABLE
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2. Apocalmodulin-binding proteins
Class
Actin-binding proteins
Cytoskeletal and membrane
proteins
Name
Brush-border myosin I
Myr4
P190
Neuromodulin
Enzymes
Receptors and ion channels
IQGAP
Phosphorylase b kinase
Adenylyl cyclase
Inducible nitric oxide
synthase
Glutamate
decarboxylase
cGMP-dependent protein
kinase
Inositol 1,4,5trisphosphate receptor
SR Ca21 release channel
Actin-activated ATPase, shedding
membranes, twisting or moving the
microvillus itself, moving microvillar
membrane proteins and transporting
vesicles
ATP-dependent binding to F-actin
Actin-activated ATPase
Reversible CaM storage, regulation of
GTP binding to Go, and regulation of
phosphatidylinositol metabolism
Unidentified
Unidentified
Unidentified
Currently unknown; clustering Na1
channels
Unidentified
Regulation of glycogen metabolism
cAMP production
Nitric oxide synthesis
Decarboxylation of glutamate to CO2
and g-aminobutyrate
cGMP- and cAMP-dependent
phosphorylation
Inositol 1,4,5-trisphosphate binding
21
Release of Ca
from the SR
Reference No.
23
9
40
1, 127, 139
46
28
121
140
11
152
107
154
CaM, calmodulin; SR, sarcoplasmic reticulum.
ferred to as ApoCaM binding, although the metal-bound
state of CaM is often not known. This may be a significant
problem. In Figure 1 is illustrated a simple model for the
interaction of a binding protein (E) and CaM in the presence and absence of Ca21.1 It is a rather simple matter to
show that in such a scheme, K1K2 5 K3K4, where K is a
dissociation constant. This equivalence means that all of
the various binding equilibria are linked to one another.
This concept of linkage is an old one (149). Although the
overall average Ca21 affinity of CaM (K1) is probably ;11
mM under conditions approaching physiological (29), this
is not necessarily the Ca21 affinity of CaM bound in a
complex with another protein. If a protein (E) preferentially binds Ca21-CaM (i.e., K2 , K4), this binding will
increase CaM’s Ca21-binding affinity in the complex. Because of this effect, CaM binding to enzymes may occur at
extremely low Ca21 concentrations, concentrations possible even in the presence of a chelator. Thus whether
binding is to Ca21-CaM or to ApoCaM must be directly
1
A more complete model is given by Cox (29) that takes into
account Ca21 binding to each of the four Ca21-binding sites on CaM and
the effect on enzyme binding. A simpler model is discussed here by
considering only a weighted average Ca21 binding obtained from the
Ca21 concentration at which Ca21 binding was half-maximal in Figs. 1
and 6 in Cox (29). Although the more complex model is more accurate,
the simplified model is given here, since it makes the concept of linkage
more obvious.
demonstrated and cannot always be inferred from the
presence or absence of a chelator.
A few specific examples may be helpful. Taking simply the concentration of Ca21 required for half of CaM’s
binding sites to be occupied, MLCK shifts CaM’s apparent
affinity for Ca21 from the ;11 mM mentioned above to
;0.6 mM. When the effect on individual Ca21-binding sites
was calculated using a current model, the binding of one
site was changed by nearly 3,000-fold (29). Inducible nitric
oxide synthase (iNOS) is isolated from macrophages in
buffers containing EGTA and is found tightly bound to
CaM. A CaM binding site has been demonstrated between
amino acids 504 and 532 in the primary structure (2, 120,
140). A synthetic peptide of this sequence binds CaM, and
the binding is highly Ca21 dependent as shown by the
concentration at which half-maximal binding to dansylCaM occurs (2). The affinity of the peptide for CaM in 0.1
mM Ca21 (K2) is ;1,000-fold greater than it is in the
virtual absence of Ca21 (K4). These peptide binding studies and the scheme in Figure 1 suggest that the binding of
CaM to these sequences could increase CaM’s affinity for
Ca21 by 1,000-fold: from ;11 mM (see above) to about 11
nM Ca21. When a chimera was constructed in which these
iNOS sequences were used to replace the normal CaMbinding sequences of neuronal NOS (nNOS), half-maximal
activation occurred at ;4 nM Ca21 instead of the 200 –300
nM found for the wild-type nNOS (120). Inducible NOS
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Neurogranin
PEP-19
Igloo
Syntrophin
Proposed Role(s)
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1. CaM, calmodulin; E, binding protein; K, dissociation con-
has additional sequences that also contribute to CaM
binding (120), and the shift in CaM’s Ca21 affinity may
result from a complex linkage of multiple binding events.
However, this shift nevertheless does occur in the direction predicted by the scheme in Figure 1, and of the
predicted magnitude. Inducible NOS is maximally active
at concentrations of Ca21 calculated to be as low as 0.1
nM (120), yet Ca21 and EGTA do affect activity in other
studies (140). Thus whether CaM or ApoCaM is bound by
iNOS cannot really be known with any certainty until the
Ca21 composition of pure, active enzyme is finally measured.
Another valuable point to be made from this linkage
is that a protein’s affinity for Ca21-CaM (relative to its
affinity for ApoCaM) can cause changes in the Ca21 affinity of the CaM-target protein complex. Thus, as target
proteins have evolved to bind preferentially one form of
CaM over another, the same process also alters the Ca21
concentrations to which the protein will respond. Calmodulin-dependent processes can thus respond to different
Ca21 concentrations (dissociation constant values of 1028
to 1025 M), that are determined by a combination of
CaM’s intrinsic affinity for Ca21 and its affinity for a
particular binding protein.
In this regard, it is also important to realize the limits
of chelators. Although the dissociation constant of
EGTA42 (the fully deprotonated form) for Ca21 is an
impressive 10 pM, the actual binding is highly pH sensitive, and this value would only be approached at quite
alkaline pH (20). At pH 7, the calculated dissociation
constant is actually only ;0.2 mM (calculation not
shown), and CaM complexes may actually have a greater
Ca21 affinity in many experiments.
Thus the affinity of CaM for Ca21 can be much
greater in the presence of its target proteins. Because it is
difficult to ensure adequate sequestration of Ca21 under
some experimental conditions, it can be expected that
some of the binding attributed to “ApoCaM” may actually
turn out to be Ca21 dependent. Nevertheless, it is clear
that true ApoCaM binding does occur. For example, neuromodulin binds CaM in EGTA, and this binding is disrupted by Ca21 (4). Because neuromodulin binds preferentially in low Ca21 or its absence (i.e., K4 , K2), it would
be expected to decrease CaM’s Ca21 affinity, and the
EGTA used (4) should be adequate to ensure Ca21-free
CaM. Thus, in this case, it is clear that ApoCaM binding is
a neuromodulin property.
The reader should be aware though that the Ca21bound state of ApoCaM is not determined in the literature
reviewed here. There is not a single case we are aware of
in which a protein-ApoCaM complex was shown to be
Ca21 free. Lacking these crucial data, we have been
forced here to utilize less certain criteria for ApoCaM
binding. The criteria used are as follows.
1) The first criterion is when a protein is purified
through a number of steps utilizing adequate amounts of
chelator and CaM remains associated. This criterion applies to phosphorylase b kinase, iNOS, and several other
proteins discussed in sections IIIB and IVC.
2) The second criterion is when the protein-binding
affinity in the presence of chelator is greater than or equal
to that measure in the presence of Ca21. This is to be
contrasted to the case with most Ca21-CaM binding proteins where chelator usually diminishes affinity by several
orders of magnitude. This criterion applies to neuromodulin, neurogranin, and many others.
3) The third criterion is when the affinity for ApoCaM
is such that binding may be physiologically important. For
example, the Bordetella pertussis adenylate cyclase binds
CaM under conditions that are even less favorable than
the low intracellular Ca21 concentrations present in the
host eukaryotic cell (52).
These criteria are adequate for the purposes of this
review, but the reader should approach this topic critically. To aid in this, our discussion of the ApoCaM binding
proteins (see sect. IV) provides details of the evidence that
binding is Ca21 independent.
A. Role of Calmodulin
Calmodulin is involved in the regulation of cellular
processes as diverse as platelet aggregation, cell-cell interactions, cell proliferation, smooth muscle contraction,
neurosecretion, and glandular secretion.
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FIG.
stants.
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APOCALMODULIN
complementation groups. Each group showed different
characteristic functional defects in actin organization,
CaM localization, nuclear division, or bud emergence.
Calmodulin performs two essential functions in S.
cerevisiae (16, 34, 103, 104, 129). Calmodulin plays an
important role in polarized yeast cell growth via an interaction with an unconventional type V myosin, Myo2p (17).
An essential mitotic function requires an interaction between CaM and a 110-kDa protein component of the
spindle pole body, the yeast equivalent of the microtubule
organizing center (47, 126). Very recently, Moser et al.
(95) reported that CaM is localized to the spindle pole
body of Schizosaccharomyces pombe and performs an
essential function in chromosome segregation.
The terminal phenotype of a CaM-deficient S. cerevisiae indicated difficulty in nuclear division (102). In S.
pombe, a CaM R116F substitution results in a proteolytic
attack of this mutant CaM, and resultant CaM shortage
causes difficulties in sporulation (132).
The above studies show that in yeast CaM is essential
and serves important roles in the cell cycle. The same is
also true in other organisms and cell types. Increased
CaM concentration transiently accelerates proliferation,
and antisense RNA-induced decrease in CaM transiently
arrests cell cycle progression in mouse C127 cells (115).
Calmodulin concentration doubles at the G1/S boundary
in cultured mammalian cells (21). Braam and Davis (14)
found that the transcription of CaM and similar genes
increased dramatically and rapidly upon mechanical stimulation of Arabidopsis and related this induction to thigmomorphogenesis. Depletion of Aspergillus nidulans
CaM causes a failure to progress from a nimT23 cell cycle
block at the G2 to M phase boundary (83).
Diversity of functional consequences of CaM mutations has been observed previously among CaM mutants
of Paramecium (70). Kink et al. (70) examined CaM and
its gene from the wild-type and viable mutants of Paramecium tetraurella. The mutants selected for their behavioral aberrations had little or no defects in growth
rate, secretion, excretion, or motility. They could be
grouped according to whether they underreact or overreact behaviorally to certain stimuli, reflecting their respective loss of either a Ca21-dependent Na1 current or a
Ca21-dependent K1 current. Their results indicated that
the sites defined by these mutations are important in
membrane excitation but not in other biological functions.
Thus CaM is essential in a disparate group of organisms, but is this due to a role of Ca21-CaM or ApoCaM?
Mutant S. cerevisiae CaM that do not bind detectable
levels of Ca21 support normal rates of cell growth and
division (48). They also localize in a manner identical to
wild-type CaM throughout the cell cycle (16). Vertebrate
CaM with analogous mutations also support normal
growth of S. cerevisiae, indicating a Ca21-independent
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The immediate physiological consequences of CaM
action have been demonstrated in several cell types. In
smooth muscle cells, Ca21/CaM-dependent activation of
MLCK initiates muscle contraction (128). In mammalian
skeletal muscle and liver, Ca21-CaM activation of phosphorylase kinase induces glycogenolysis (27). The discovery that immunosuppressive drugs such as cyclosporin
cause inhibition of the Ca21/CaM-dependent protein
phosphatase calcineurin suggests a role for CaM in T-cell
activation (80). Hemenway et al. (56) reported that calcineurin plays a role in the regulation of cation transport
and homeostasis with their study on vph6 mutants of
Saccharomyces cerevisiae. In plants, CaM activates NAD
kinase and may play an essential role in regulating photosynthesis (62). In Paramecium, CaM regulates motile
behavior (70).
Although many of the above activities require Ca21,
the Ca21 dependence of many other cellular roles of CaM
is less clear. To discuss this, some background of the
genetic analysis of CaM must be given. These studies
show CaM to be essential for cell viability and that
ApoCaM serves an essential role(s).
Mutational analysis of S. cerevisiae was carried out
to explore the function CaM plays during cell growth and
division. Yeast CaM has all the attributes expected of a
Ca21 receptor. Calmodulins from yeast and vertebrates
have structural, biochemical, and biophysical similarity
(125) and are functionally conserved (35, 101). In addition
to having a high affinity for Ca21 (84) and changing conformation to reveal hydrophobic surfaces upon binding
Ca21 (36), yeast CaM activates both mammalian and yeast
enzymes in a Ca21-dependent manner (84, 105). Also,
vertebrate CaM can substitute for yeast CaM in vivo (35,
101, 112). A Ca21/CaM-dependent protein kinase (82, 94,
108) and a Ca21/CaM-dependent protein phosphatase (31)
have been identified and purified from yeast. Harris et al.
(55) identified residues critical for CaM action in yeast
cell growth by gain-of-function mutations in a human
CaM-like protein.
Disruption of the unique gene encoding CaM in budding yeast (36), fission yeast (133), or Aspergillus nidulans (116) is lethal. Yeast cells with mutations that cause
a complete loss of CaM function suffer many defects
simultaneously. As a result, it is difficult to tell which are
direct and which are secondary consequences of the loss.
Furthermore, the more dramatic effects of the loss of
CaM function may mask defects that are more subtle or
slow to develop. Ohya and Botstein (103) used the techniques of yeast genetics to generate temperature-sensitive
mutations that abolish single functions of CaM, while
leaving the others intact. Examination of 14 temperaturesensitive yeast mutants bearing one or more phenylalanine to alanine substitutions in the single essential CaM
gene of yeast (CMD1) revealed diverse essential functions. Their mutations were classified into four intragenic
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B. Apocalmodulin Binding Within the Cell
Calmodulin is a major constituent protein of the cytoplasm and membranes and is present in or on organelles
(54, 62, 66, 77, 114). In the microscopic localization, CaM
is found associated with membrane structures including
the plasma membrane, nuclear membrane, and on the
outer (cytoplasmic) surface of mitochondria and the endoplasmic reticulum. In the rat cerebellum, these structures were found to contain much of the CaM-antibody
staining and, at the synapse, the postsynaptic membrane
was found to contain abundant CaM (77).
These localization studies have also been complemented with cell fractionation experiments (62, 66, 134).
One advantage of cell fractionation is that it allows testing
the Ca21 dependence of binding to these cell constituents.
The particulate fraction of tissues often contains much of
the total CaM present in the cell, an observation that
supports the microscopic localization to membranes. This
has been particularly well characterized in the brain
(134). Homogenization of brain in Ca21-containing buffers
followed by centrifugation at 105,000 g results in recovery
of ;42% of the cell’s CaM in the particulate fraction.
Further fractionation reveals that the majority of this CaM
is in the microsome fraction, with a sizable portion also
found along with mitochondria. Extraction of this microsome fraction repeatedly with EGTA shows that ;70% is
reversibly bound in a Ca21-dependent manner but that the
remaining 30 –35% is not extractable with EGTA and is
only solubilized after treatment with nonionic detergents
such as Lubrol (66, 134) or Triton (124). Overall, in brain,
;15% of the cell’s CaM is bound to membranes in this
Ca21-independent bound fraction (134). Calmodulin is
;0.5% of the total protein present in brain; thus bound
ApoCaM is a major brain protein (134). Because this
fraction is not released from membranes after repeated
extraction with EGTA or EDTA, we refer to it here as
particulate ApoCaM.
When a variety of tissues were assayed, the percentage of total CaM found as particulate ApoCaM was shown
to vary from a low of ;11% in testis to 63% in spleen.
Several observations argue that this is an important pool
of cellular CaM. It probably does not represent entrapment, since cytoplasmic marker enzymes have shown to
not copurify with it (124). By several criteria, the purified,
particulate ApoCaM is identical to soluble CaM in its
properties, suggesting that it is the same protein tightly
bound to the membrane in a Ca21-independent fashion
(124). This particulate ApoCaM may represent storage of
CaM that is released as part of the cell cycle. The fraction
of the cellular CaM in this particulate CaM pool varies
with cell density in tissue culture and is different in
cancerous and noncancerous cells. Chinese hamster
ovary cells were grown to a density of 2–9 3 106 cells/dish
and showed that as cell number increased there was an
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function fundamental to the CaM molecule (48). Moser et
al. (96) did an analysis of CaM in the fission yeast S.
pombe to explore the universality of a Ca21-independent
function for CaM during cell proliferation. They assessed
the abilities of both Ca21-binding site mutants and heterologous CaM to support proliferation. Saccharomyces
pombe requires at least one intact Ca21-binding site in
contrast to S. cerevisiae. Substitution of a valine for the
conserved glutamic acid in position 12 of all four of the
EF-hand Ca21-binding sites of CaM yielded a protein that
failed to support proliferation. The same mutant S. pombe
protein allowed the growth of S. cerevisiae. The importance of Ca21 binding may reflect essential Ca21-dependent CaM functions in S. pombe not present in S. cerevisiae. Alternatively, essential functions that are not Ca21
independent in S. cerevisiae may be Ca21 dependent in S.
pombe.
The fact that S. pombe does require Ca21 binding by
CaM was exploited to assess the importance of each Ca21
binding site in vivo. Moser et al. (96) also analyzed the
Ca21-binding properties of S. pombe CaM by NMR and
showed that the relative affinity of each site for Ca21 does
not parallel the functional importance of that site. Furthermore, despite the observed differences in the Ca21binding properties of CaM from S. pombe and vertebrates,
CaM from vertebrate sources can substitute for that of S.
pombe. Thus, although CaM serves an essential role, it
seems likely that ApoCaM may sometimes be what is
essential and Ca21 binding, while required for many CaM
functions, may not always be prerequisite to life itself.
There are certainly many candidates for essential
cellular functions of CaM that may not require Ca21 binding to the protein. Numerous studies suggest that CaM is
required for microtubule function, and it has been demonstrated that the ability of CaM to bind to the mitotic
apparatus is not dependent on the presence of Ca21 (131).
Endocytosis in yeast requires CaM but is apparently Ca21
independent (75). Calmodulin may regulate a protein
phosphorylation cascade that results in chromosome segregation (37, 68, 117). However, the Ca21 transients proposed to trigger this cascade were later found not to be
required for chromosome segregation (67). Calmodulin
may also regulate a nuclear contractile system that includes actin in proliferating liver cells (8). In contrast to
Ca21-CaM, ApoCaM binds to and can regulate a variety of
proteins whose target sequences are far more restricted.
Neuromodulin, neurogranin, and unconventional myosins
(e.g., intestinal brush-border myosin I) and several enzymes interact with CaM in the absence of Ca21 (1, 10, 40,
45) binding CaM tightly at low Ca21 concentration or in its
absence (22, 148).
Thus Ca21 binding is not necessary to some of the
vital roles CaM serves in the cell, and understanding the
roles of cellular ApoCaM is necessary to our understanding of the cell cycle and viability itself.
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II. STRUCTURE OF APOCALMODULIN
Calmodulin is an internally homologous protein. The
complete sequence can be divided into roughly four segments, and each one-fourth of the structure is homologous to the others. The first (amino terminal) quarter is
most similar to the third, and the second is most similar to
the fourth (carboxy terminal). This structure is thought to
21
FIG. 2. Comparison of Ca /calmodulin (CaM) and apocalmodulin
(ApoCaM). Left: rat testis Ca21/CaM [Brookhaven National Laboratory
protein database (PDB) file 3CLN]. Right: Xenopus ApoCaM (PDB file
1CFC). (Species differences are unimportant, since all vertebrate CaM
are identical.) Structures were analyzed using Swiss PDB Viewer software. Structures are shown with amino-terminal (N) domains in similar
orientation. C, carboxy terminal.
have arisen by gene duplication of an ancestral single
Ca21-binding domain protein to give a two domain ancestor first, which again duplicated to give the current four
domain protein (144). Each of these four domains consists of an a-helix, a loop where Ca21-binding occurs, and
then a second a-helix. The helices orient the loops for
Ca21 binding. This structure is the “EF hand” Ca21-binding motif named for the Ca21-binding site between the E
and F helices of parvalbumin (73). Thus CaM having four
EF-hand Ca21 binding sites would be expected to have a
total of eight a-helical segments. This is essentially correct except that the fourth and fifth helices combine to
form a long “central helix.”
The Brookhaven database currently has 30 structure
files relevant to CaM. A complete discussion of all this
structural data is beyond the scope of this review, but
some generalizations may help the reader appreciate the
overall structure and the effects of Ca21 binding and
protein binding on structure. Figure 2 shows the structure
of Ca21-CaM and ApoCaM in similar orientation. The
overall shape of CaM is often referred to as a “dumbbell.”
Each half (consisting of 4 a-helices and 2 Ca21-binding
sites) folds into a compact globular structure (where the
weights of a dumbbell would be) and the fused central
helix makes a connecting “bar” (7). This coarse description is sufficient for many purposes, but a more useful
description is of a dumbbell with two ends shaped more
like soup ladles, the bowl of the ladle ends forming a
hydrophobic “cup” in Ca21-CaM (42, 74) and connected
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increase in soluble CaM. Soluble CaM increased twofold,
whereas the particulate fraction decreased by one-third
(41). More soluble CaM is extracted from cancer cells
than from noncancerous cells (122, 138, 145), whereas the
particulate ApoCaM fraction is more abundant in noncancerous cells than cancerous ones (138). In the case of
certain hepatomas, the ratio of soluble CaM to particulate
ApoCaM is changed from 0.8 in normal liver to 9.8 in
hepatoma (138). These results are consistent with the
hypothesis that soluble CaM, necessary for rapid cell
growth, is derived to some extent from the particulate
CaM pool.
The binding site for this ApoCaM has not been rigorously identified; however, in brain it seems likely this
binding may be primarily the activity of two proteins,
neuromodulin and, to a lesser extent, neurogranin. Neuromodulin is a 57-kDa protein found only in neural tissue
that binds CaM in both the presence and absence of Ca21.
Cimler et al. (25) found that the particulate fraction of
bovine cerebral cortex contained 61 pmol neuromodulin,
and Kakiuchi et al. (66) found 129 pmol particulate
ApoCaM/mg membrane protein. Thus this area of brain
contains enough neuromodulin to bind about one-half of
the CaM that remains after extensive EGTA washing.
Neurogranin is present in brain at only 2–5% of the mass
of neuromodulin (10) but is about sevenfold lower in
molecular mass so it could bind about one-third as much
CaM as neuromodulin. Neurogranin and neuromodulin
are both confined to nerve tissues, both have a quite
similar IQ motif CaM binding site, both are phosphorylated by protein kinase C, and in both cases, the phosphoprotein no longer binds CaM.
It is clear that regulation by CaM may be exerted both
independently and dependently of Ca21 (118). The transformation from Ca21-independent to Ca21-dependent
binding is apparently due to subtle substitutions in the
recognition motif. Calmodulin can therefore perform under highly localized, fluctuating Ca21 concentrations that
require Ca21-dependent and -independent interactions for
membrane cycling, movement, and regulation. Detailed
studies on ApoCaM promise to yield insights into the
mechanism of activation for the entire family of EF-hand
Ca21-binding proteins. This review deals with the structural evaluation, target proteins, and recognition motifs of
ApoCaM.
667
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JURADO, CHOCKALINGAM, AND JARRETT
A. Complex Formation
Calcium-CaM binds to and activates smooth muscle
and skeletal muscle MLCK and CaM kinase II. In all three
cases, the region of the enzyme sequence that binds CaM
has been identified (59, 89, 90). The structure of the
complexes of Ca21-CaM with peptides representing each
of these sequences has been solved and shows the significance of these differences between Ca21-CaM and
ApoCaM. When each peptide binds, the hydrophobic
faces and surrounding sequences at each end of Ca21CaM contact the peptide and have many favorable inter-
actions with it. To do this, the central helix bends. Indeed,
a useful concept is that the central helix serves as a
flexible tether between these two globular ends (110),
positioning them for favorable interactions with enzyme
binding sites. Thus the inaccessibility of these hydrophobic residues in ApoCaM and the blockage by the aminoand carboxy-terminal helices mentioned above probably
account for why Ca21-CaM binds to and activates these
enzymes while ApoCaM is ineffective.
What happens when ApoCaM is bound by proteins
adapted for its binding is unknown. The structure of a
fragment representing the regulatory domain of scallop
myosin with two light chains (one essential and the other
regulatory) bound has been reported (151). Because these
light chains are homologous to CaM and remain associated when myosin is isolated from EGTA solution, this
binding may be analogous to ApoCaM binding. Indeed,
there are similarities between the regulatory light chain
and both the Ca21-CaM and ApoCaM structures. For example, the regulatory light chain is in a conformation that
resembles a state intermediate between Ca21-CaM and
ApoCaM in that many of the hydrophobic residues still
form a core structure at each end, but some are also
directed toward and in contact with the myosin sequences. The bound structure is more relaxed as in Ca21CaM, and the terminal “blocking” sequences are oriented
out away from the central helix in a nonblocking position.
The bound region of myosin is a-helical, which is also true
of peptides when bound by Ca21-CaM (59, 88, 89); however, in the case of myosin, the region of peptide a-helix
in contact with myosin light chains is longer and the
contacts more extensive. Recently, Houdusse and Cohen
(57) presented a model for ApoCaM binding based on
these scallop myosin results. The scallop sequence contains an IQ motif, and IQ motifs do indeed bind ApoCaM
(23, 118). The model shows a more open CaM structure
with the blocking termini in a nonblocking position away
from the central helix. Apocalmodulin is shown as interacting over a longer length of protein sequence than do
the Ca21-CaM binding models and with sequences within
the IQ motif itself and other sequences both amino and
carboxyl terminal to it. The authors also suggest that Ca21
dependence or independence of binding may result from
interactions with the carboxy terminal half of the IQ motif
(57). Because the model (57, 58) is theoretical and based
on homologous myosin light chain binding data, its accuracy is not known, although it seems plausible. However,
other studies have shown that CaM can interact with
proteins in recognizably different ways. More than a single sequence can bind to CaM at the same time (32, 76),
and CaM does not always assume the same conformation
while bound to different recognition sequences (136).
Thus bound (Apo)CaM may assume conformers about
which we currently know little. Although the Houdusse
and Cohen model is clearly a first step toward understand-
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by a “flexible tether” instead of a bar (109 –111). This
description is accurate only for Ca21-CaM, and the differences in ApoCaM are significant.
The differences between Ca21-CaM and ApoCaM are
numerous, but a few useful generalizations can be made.
1) Apocalmodulin is overall a more compact structure. Calcium-CaM has an open, “relaxed” structure. Figure 2 shows a depiction of both forms with the aminoterminal globular ends in similar orientation.
2) The position of the hydrophobic residues in the
two structures is perhaps most striking. In ApoCaM, these
hydrophobic residues predominantly pack together between a-helical segments in each globular end and form a
hydrophobic core in the interior of the structure. In contrast, in Ca21-CaM, these residues swing out from the
space between helical segments and form a hydrophobic
cup structure at each ladle end (facing out, adjacent, and
perpendicular to the central helix) accessible from the
outside.
3) In ApoCaM, nearly half a dozen residues in the
amino and carboxy terminus (e.g., from 8 to 15 and 140 to
146), which form the first and last a-helices of CaM, are
roughly parallel to the central helix of CaM, providing a
“second bar” to its dumbbell. Furthermore, the central
helix is interrupted in the center (at Ser-81) by a nonhelical region that is bent. In contrast, these terminal peptides are swung out away from the central helix in Ca21CaM, and the central helix is not bent in the crystal
structure (see Fig. 2). This allows access to the ladle
“hydrophobic cups” at each end of Ca21-CaM that were
blocked off by these terminal segments in ApoCaM. The
two globular ends of ApoCaM are also twisted by about
180° relative to each other when compared with their
position in Ca21-CaM (e.g., Gly-40 and Leu-112, residues
in different globular ends, are on the same side of the
molecule in ApoCaM and roughly 180° apart in Ca21CaM). This twisting of the two ends and the position of
the terminal helices is illustrated in Figure 2.
4) In summary, ApoCaM has a hydrophobic core,
which becomes exposed as two hydrophobic faces in
Ca21-CaM.
Volume 79
July 1999
APOCALMODULIN
ing ApoCaM binding, there are almost certainly bound
states of CaM about which we currently know little.
III. KNOWN APOCALMODULIN BINDING
MOTIFS
A. IQ Motifs
The sequence IQXXXRGXXXR (1-letter amino acid
abbreviations where X denotes any amino acid) defines
this motif, but the motif is rather loosely adhered to in
many cases. For example, the isoleucine in the first position is frequently a different branched chain amino acid
(Leu or Val) or, rarely, a methionine. The arginines in both
the sixth and the terminal position are sometimes lysine
or histidine, and the seventh position glycine is frequently
not conserved. Despite this lack of strict conservation,
there is little doubt that this is a recognizable protein
motif that binds CaM or homologous proteins including
troponin C and myosin light chains. An extensive compilation of observed IQ motifs, many known only by homology, is given by Rhoads and Friedberg (118). IQ motifs
were first identified as ApoCaM binding sites; however, it
is now clear that IQ motifs can show different levels of
Ca21 dependency. Several selected IQ motifs of different
types are shown in Table 3.
The IQ motif is repeated one to six times in muscle
and nonmuscle myosins (23). Normal muscle myosins
(i.e., myosin II) contain a globular ATPase domain connected to a helical tail region by a short “neck” domain.
Nonmuscle myosins (i.e., myosin I) have a similar head
and neck structure, although the tail domain may be
nonhelical and possess binding properties not normally
associated with myosin. IQ domains are found in this
neck region of myosins. In normal muscle myosin, the IQ
motifs provide binding sites for essential and regulatory
myosin light chains, and thus this neck region is thought
to be critical for regulation of myosin’s biological function. Because myosin light chains, troponin C, and CaM all
belong to the same gene family and have similar structures, binding by alternate members within this family has
been observed.
The IQ motif is also sometimes adjacent to or overlapping phosphorylation sites phosphorylated by protein
kinase C and dephosphorylated by calcineurin and phosphorylation inhibits CaM binding in vitro (5).
The Ca21 sensitivity of CaM binding by IQ domains
appears to be highly variable. For example, neuromodulin
binds to CaM-Sepharose in the absence of Ca21 and is
eluted with Ca21-containing buffers (4). Thus neuromodulin binds preferentially to ApoCaM. In other cases such as
the IQ-GAP2 proteins and the Myr-4 myosin, binding may
be Ca21 dependent or Ca21 independent, sometimes at
adjacent sites within the same protein.
B. Synergistic Binding of
Noncontiguous Sequences
Another type of ApoCaM binding is that exemplified
by PbK and probably the prokaryotic adenylyl cyclase
toxins. Phosphorylase b kinase in skeletal muscle is activated by cAMP or Ca21. The active enzyme phosphorylates glycogen phosphorylase b and converts it to an
active form that catalyzes the phosphorolysis of glycogen
to glucose-1-phosphate. Activation by cAMP was characterized early (72), but the activation by Ca21 was not well
understood until CaM was discovered to be a subunit of
this enzyme (28). Glycogen phosphorylase b kinase has
the subunit structure (abgd)4, where d is CaM. The d-subunit CaM remains tightly associated even in the presence
of chelators and requires strongly denaturing conditions
to remove CaM. The rate of exchange with 14C-labeled
CaM is only ;15% per week at 4°C and 2 mM EDTA (113)
and is also indicative of tight, Ca21-independent association. This CaM subunit is tightly bound by the catalytic
g-subunit in the holoenzyme. The g-subunit has an aminoterminal presumptive catalytic domain (residues 20 –276)
homologous to other protein kinases and a carboxy-terminal domain (residues 276 –386) that is presumed to be a
regulatory region of the enzyme. This putative regulatory
region was synthesized in its entirety as a series of 18
overlapping 25-mer peptides, and each was assayed for its
ability to bind CaM in the presence of Ca21 and inhibit a
MLCK assay or to inhibit the CaM-facilitated renaturation
of denatured g-subunit (32). On the basis of these criteria,
CaM binding was found to reside in two noncontiguous
regions of sequence centered at two peptides named
PhK5 (residues 342–366) and PhK13 (302–326). Both peptide sequences are shown in Table 3. The binding of either
peptide is Ca21 dependent, and both peptides can bind
CaM simultaneously (32). Interestingly, antibodies against
PhK5 and PhK13 have the ability to activate and inhibit,
respectively, PbK holoenzyme and the isolated gd-subunit
complex (143). Low-angle scattering data also suggest
different modes of binding of each to Ca21-CaM. It should
be remembered from a previous section where CaM’s
structure is discussed that when CaM binds peptides representing binding domains of myosin light chain kinase
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Currently known ApoCaM binding falls into two
classes: 1) the IQ motif found in myosins, neuromodulin,
neurogranin, and other proteins; and 2) a concerted binding exemplified by CaM binding to the catalytic (g) subunit of glycogen phosphorylase b kinase (PbK) and probably also the prokaryotic adenylyl cyclase toxins.
Although these represent the currently known themes,
there are likely others, since proteins are known that bind
ApoCaM with no obvious adherence to either (99).
669
670
TABLE
JURADO, CHOCKALINGAM, AND JARRETT
Volume 79
3. Types of CaM-binding sequences
Ca21-dependent CaM binding
Mouse a1-Syn CBS-C
(480-503)
Mouse Dys CBS-1
(18-42)
Mouse Dys CBS-2
(104-125)
Mouse Dys CBS-3
(3293-3349)
Skeletal muscle MLCK
(577-603)
CaM kinase II
(292-318)
PKTMVFIIHSFLSAKVTRLGLLA
1 **A**1 A*
1* 1* **
KKTFTKWINAQFSKFGKQHIDNLFS
11 A 1A*
A 1A 1 1*2 *A
HKLTLGLIWNIILHWQVKNVMK
11* * **A
***1A *1 **1
RYRSLKHFNYDICQSCFFSGRVAKGHKMHYPM
1A1 *11A A2*
AA
1* 1 11*1A *
KRRWKKNFIAVSAANRFKKISSSGALM
111A11 A* *
1A11*
**
KKFNARRKLKGAILTTMLATRNFSVGR
11A
111*1
**
**
1 A * 1
Neuromodulin
(31-58)
Neurogranin
(24-51)
Myr4-1
(700-721)
AHKAATKIQASFRGHITRKKLKGEKKGD
11
1*
A1 1* 111*1 211 2
ANAAAAKIQASFRGHMARKKIKSGERGR
1*
A1 1* 111*1
21 1
RVVLFLQKVWRGTLARMRYKRT
1***A* 1*A1
* 1*1A11
Ca21-dependent IQ motif
Myr4-2
(722-743)
IRS-1 (IQ1)
(106-126)
IRS-1 (IQ2)
(534-554)
KAALTIIRYYRRYKVKSYIHEV
1
* **1AA11A1*1 A*12*
WYQALLQLHNRAKAHHDGAGG
AA
** *1 1 1 112
KVDTAAQTNSRLAPTRLSLG
1*2
1*
1* *
Undetermined Ca21 dependency IQ motifs
IQGAP2-1
(660-689)
IQGAP2-2
(690-719)
IQGAP2-3
(720-749)
IQGAP2-4
(750-779)
SEELLLRFQATSSGPILREEFEARKSFLHE
22***1A
**122A2 11 A*12
QEENVVKIQAFWKGYKQRKEYMHRRQTFID
22 **1*
AA1 A1 112A*111
A*2
NTDSVVKIQSWFRMATARKSYLSRLQYFRD
2 **1*
AA1*
11 A* 1* AA12
HNNEIVKIQSLLRANKARDDYKTLVGSENP
1
2**1*
**1
1 122A1 **
2
Ca21-independent concerted
PhK5
(342-366)
PhK13
(302-326)
iNOS
(503-534)
LRRLIDAYAFRIYGHWVKKGQQQNR
*11**2 A A1*A 1A*11
1
GKFKVICLTVLASVRIYYQYRRVKP
1A1** * **
*1*AA A11*1
RRREIRFRVLVKVVFFASMLMRKVMASRVR
1112*1A1***1**AA
***11**
1*1
Unknown type
Rabbit a1-Syn-1
(106-126)
Rabbit a1-Syn-2
(1-38)
RENKMPILISKIFKGLAADQT
12 1* *** 1*A1 *
2
MASGRRAPRTGLLELRAGTGAGAGGERWQRVLVSLAED
*
11
1
**2*1
21A 1*** * 22
Chemical properties of each sequence are depicted using *, 1, 2, and A to denote aliphatic hydrophobic, basic, acidic, and aromatic
hydrophobic amino acids, respectively. Numbers in parentheses are amino acid sequence positions shown for each protein. Mouse a1-syntrophin
(Syn) CBS-C is from Newbell et al. (99). Mouse dystrophin (Dys) CBS-1 and -2 are from Jarrett and Foster (63), and Dys CBS-3 sequences are from
Anderson et al. (3). Myosin light-chain kinase (MLCK) sequence is M13 peptide from rabbit enzyme taken from Edelman et al. (38). CaM kinase II
sequence is from rat brain enzyme deduced from cDNA (12). Neuromodulin and neurogranin sequences are from Apel et al. (5) and Baudier et al.
(10), respectively. Myr4-1 and -2 were taken from Bahler et al. (9). Insulin receptor substrate-1 (IRS-1) IQ1 and IQ3 were taken from Munshi et al.
(97). IQGAP2-1, -2, -3, and -4 were taken from Brill et al. (15). Glycogen phosphorylase kinase PhK5 and PhK13 sequences were taken from Dasgupta
et al. (32). Inducible nitric oxide synthetase (iNOS) sequence are deduced from cDNA sequence reported in Xie et al. (150). Rabbit a1-Syn-1 and -2
were taken from results of Iwata et al. (60) and Genebank database accession number U01243.
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Ca21-independent IQ motif
July 1999
APOCALMODULIN
B. pertussis can (119). Apparently, two different forms of
the enzyme are produced by B. pertussis, which are both
;200 kDa. Both are catalytically active, CaM activated,
and immunologically similar but can be separated by gel
filtration chromatography. One of these forms enters lymphocytes, whereas the other cannot (119).
Why the adenylyl cyclase can bind ApoCaM (as opposed to Ca21-CaM) may be adaptive to the conditions
inside the eukaryotic cell. The CaM concentration inside
eukaryotic cells is often in the range of 1–10 mM, and
unstimulated cells typically have free concentrations of
Ca21 of 0.1 mM or less; thus the unstimulated cell would
have abundant ApoCaM, and it is under these conditions
that the toxin adenylyl cyclase must be active. Because
prokaryotes like B. pertussis do not produce CaM, the
CaM requirement ensures that the enzyme obtains high
activity only when it leaves the bacteria and enters the
host. This is an interesting system in which eukaryotic
CaM serves as a “fail safe” mechanism reminiscent of
zymogens that ensures that only the exported protein
becomes fully catalytically active.
The domain organization of the enzyme shows two
CaM binding sequences reminiscent of PbK. Although the
intact adenylyl cyclase is ;200 kDa, shorter secreted
forms (43– 61 kDa) have been frequently isolated which
are CaM activated and catalytically active, and derived
from the amino terminus of the holoenzyme. The 43-kDa
form has been particularly well studied. It binds Ca21CaM with an apparent dissociation constant of 0.14 nM
and binds CaM in the absence of Ca21 (i.e., in 2 mM
EGTA, pH 8) with an apparent dissociation constant of 15
nM. Both of these apparent binding constants are so much
lower than the CaM concentrations usually measured in
eukaryotic tissues, and the enzyme would likely bind CaM
independent of Ca21 in vivo. When this 43-kDa form is
trypsin-digested in the presence of CaM, cleavage gives
rise to a 25- and 18-kDa fragments. Both fragments remain
associated with CaM, and this trimer is catalytically active
(76). The 25-kDa fragment is from the amino terminus of
the 43-kDa form and thus from the amino terminus of the
holoenzyme. It has a low catalytic activity in the absence
of the 18-kDa carboxy-terminal fragment and represents
much of the catalytic domain of the enzyme (50). Trypsin
digestion of the enzyme in the absence of CaM rapidly
inactivates the enzyme and gives rise to a 24-kDa fragment which, unlike the 25-kDa fragment, does not apparently bind CaM (76). The 18-kDa form (residues 236 or
238 –399) has no catalytic activity and represents a regulatory region of the enzyme. It contains Trp-242, which
has been shown by site-directed mutagenesis to be essential for high-affinity CaM binding. The fragments associate
only in the presence of CaM to restore much of the
original catalytic activity of the undigested enzyme (50).
Thus, like PbK, the B. pertussis adenylyl cyclase
apparently has at least two regions, present in the 25- and
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(MLCK) or CaM kinase II, CaM changes from an extended
dumbbell to a more compact structure as the central helix
bends and the two globular ends of CaM associate with
the peptide. This contraction upon binding can be observed by low-angle scattering. PhK5 causes a contraction
of CaM similar to that found with the MLCK peptide, but
PhK13 binds to an extended conformer of CaM. When
both peptides are bound, CaM is in an extended conformer (136). Thus the binding of CaM to PbK appears to
be fundamentally different than its binding to MLCK or
CaM kinase II.
Thus, although high-affinity binding to either PhK5 or
PhK13 (i.e., 20 nM and 6.5 nM, respectively) (32) is Ca21
dependent, binding of the two is synergistic, and although
the individual affinities in the absence of Ca21 may be
low, the synergism of binding makes binding to the g-subunit resistant to changes in Ca21. For example, if two
independent binding events occur with different regions
of sequence and binding is noncooperative, the individual
affinities would multiply. Thus, even if each region binds
in the absence of Ca21 with quite low affinities (e.g., 1024
M), the product of two such independent binding events
(e.g., 1028 M) could be substantial and yield a quite stable
complex making strong denaturation necessary for dissociation. Thus Ca21 binding can still affect each individual
binding event and regulate activity while maintaining a
stable complex with the g-subunit independent of Ca21.
A similar mechanism may pertain to the Ca21-independent binding of CaM to the prokaryote adenylyl cyclase toxins produced by Bordetella pertussis and Bacillus anthracis, the causative agents of whooping cough
and anthrax, respectively. Both toxins bind CaM in the
presence and absence of Ca21. Because prokaryotes do
not produce CaM, presumably the requirement for CaM
here serves the purpose of ensuring that catalytic activity
is maximal only within the eukaryotic host cell where
these toxins function. These two adenylyl cyclases are
homologous to one another and markedly different from
mammalian adenylyl cyclases. More is known about the
B. pertussis enzyme. The protein secreted by the bacteria
has a molecular mass usually reported to be ;200,000 Da.
This form of the enzyme has been highly purified from an
overexpressing B. pertussis strain, the molecular mass is
176 kDa, and it is activated half-maximally by 0.3 nM CaM
in the presence of Ca21 and by 20 nM in the absence of
Ca21 (49). This form of the enzyme is capable of entering
the eukaryotic cell in a process that requires extracellular
Ca21 (49). Because Ca21 binds to the enzyme and affects
its physical and chemical properties (88), extracellular
Ca21 may be required to produce the conformer that
crosses into the cell. Posttranslational modification is
apparently also required, since expression of the recombinant gene in Escherichia coli produces a protein which,
while catalytically active and CaM dependent, cannot enter lymphocytes while the recombinant gene expressed in
671
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JURADO, CHOCKALINGAM, AND JARRETT
18-kDa peptides, that bind CaM, and this probably accounts for the high affinity for CaM in the presence or
absence of Ca21. A significant difference, however, is that
CaM should properly be considered a subunit of PbK,
whereas the adenylyl cyclase must acquire its CaM from
its host.
Inducible nitric oxide synthase also has at least two
sequence regions that bind CaM and possibly represents a
further case of this concerted binding mechanism but is
discussed separately in section IVC with the other NOS
isozymes.
C. Probability of Other Types
two other sites at a1-syntrophin-(1O38) and -(106O126)
(see Table 3). All binding was found to be Ca21 dependent
(60). Several observations may be relevant to this discrepancy, some published and others not. We have found that
our His-Tag syntrophin fusion protein is rapidly degraded
by E. coli proteases and that the purified proteins are
proteolysed primarily at the carboxy terminus. This proteolysis has been described (99), and a rapid batch purification procedure was developed (63, 85) to lessen proteolysis. Unless great care is taken, the SU domain and the
carboxy-terminal CaM-binding site it contains may become truncated, and CaM binding would not be detected.
The shortest SU domain construct used by Iwata et al.
(60) was about 49 kDa, and truncation of the complete
24-residue CaM-binding sequence would alter the molecular mass by only ;5%, which would have been difficult to
detect. Both groups agree that CaM binds within syntrophin sequences 4 –174. Newbell et al. (99) did not determine the precise location or number of these sites,
whereas Iwata et al. (60) localized two sites to a1-syntrophin-(1O38) and -(106O126). The two groups disagree on
whether the site(s) binds CaM only in the presence of
Ca21 (60) or in both the presence and the absence of Ca21
(99). However, Newbell et al. (99) had presented data that
syntrophins aggregate, that aggregation is affected by
Ca21, and that aggregation affects CaM binding. More
recently, we have found that syntrophins self-associate to
form large aggregates that pellet in the ultracentrifuge.
Aggregation is affected by micromolar Ca21 concentrations, and these aggregates no longer bind CaM in
amounts stoichiometric with syntrophin monomer (Oak
and Jarrett, unpublished data). Thus CaM binding can be
affected by aggregation, and the Ca21 dependence of
aggregation can interfere with CaM binding as had been
proposed by Newbell et al. (99). There may be two CaM
sites in this sequence region as determined by Iwata et al.
(60). However, the effect of Ca21 on aggregation (which
inhibits CaM binding) can make ApoCaM binding appear
to be Ca21 dependent. Indeed, Newbell et al. (99) showed
that fusion proteins containing only these ApoCaM binding sequences bind to CaM-Sepharose in Ca21 and elute in
EGTA because of this aggregation phenomenon. However, in either case, the sequence(s) within syntrophin(4O174) do not contain either an IQ motif or other readily
discernible Ca21-CaM binding sequences. Such Ca21-CaM
binding sequences are basic, hydrophobic, and frequently
aromatic-rich stretches of ;20 amino acids in length.
Acidic residues and prolines within this amphipathic,
a-helical region are rare (see Table 3). The sequences
identified by Iwata et al. (60) are compared with several
Ca21-dependent and Ca21-independent binding sites that
have been identified in Table 3. Except for short stretches,
these sequences differ from this Ca21-dependent paradigm and from the IQ motif. Interesting is the appearance
of a proline in the midst of the most basic sequence
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Although IQ motifs and noncontiguous sites represent
two known types of ApoCaM binding, there must certainly
be others. Proteins have been discovered that bind ApoCaM
for which there is no recognizable motif. One example recently encountered is the syntrophins (99).
Syntrophins were first discovered in the postsynaptic
membrane of the Torpedo electric organ (44) and are
constituent proteins of a membrane complex, the dystrophin glycoprotein complex (DGC), which is defective in
various forms of muscular dystrophy. There are a1-, b1-,
and b2-syntrophins that are ;50% identical with one another in sequence. They differ somewhat in their charge
properties with a1-syntrophin being more acidic than the
more basic b-syntrophins (135). The b-syntrophins are
found in a variety of nonmuscle tissue including nerve,
and syntrophins also bind to Na1 channels isolated from
either brain or muscle (46).
Syntrophins have a complex domain structure consisting of a syntrophin unique (SU) domain at the carboxy
terminus, two pleckstrin homology (PH) domains, and a
single PDZ domain. The PH domains were discovered in
the major protein kinase C substrate protein of platelets,
pleckstrin. This domain is found in several membraneassociated proteins. The PDZ domains are found in membrane-associated proteins such as the postsynaptic density 95-kDa protein and nNOS [see Gee et al. (46) for
discussion]. Syntrophins bind to CaM (87), to dystrophin
(the gene product of the Duchenne muscular dystrophy
gene) (39, 71), and to F-actin (60), and they also selfassociate (86, 153). Calcium-CaM inhibits the syntrophindystrophin interaction (99). In one study (99), CaM was
found to bind to syntrophin in the presence and absence
of Ca21. One binding site is the carboxy-terminal 24 residues of a1-syntrophin (CBS-C, Table 3), a sequence
highly conserved in all syntrophins. Calmodulin binding
to this site was observed only in the presence of Ca21. A
second kind of binding was localized to a1-syntrophin(4O174), which bound either Ca21-CaM or ApoCaM (99).
Somewhat different results were found by another group.
They found no binding within the SU domain and found
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APOCALMODULIN
regions in each sequence and the paucity of aromatic
residues. Thus either syntrophin contains a previously
unknown type of ApoCaM binding site as we have suggested (99) or they contain a new type of Ca21-CaM
binding that is currently unknown. In either case, much
will be learned about CaM binding by further study of
syntrophin.
IV. APOCALMODULIN-BINDING PROTEINS
A. Actin-Binding Proteins
Unconventional myosins represent a good example
of Ca21-independent CaM binding. These proteins contain
a “generic” motor domain that converts chemical energy
into directed mechanical force by hydrolyzing ATP, binding actin, and translocating along actin filaments (9, 23).
In contrast to conventional muscle myosins, where two
different light chains stabilize the regulatory domain (RD)
in the neck region of the molecule, unconventional myosins are a diverse group of molecular motors in which one
to six regulatory light chain subunits or CaM are bound
tandemly to the RD (58). Intestinal brush-border myosin I
(BBMI), an unconventional myosin from vertebrates that
binds three or four CaM, has been found to retain its
ability to bind these molecules even in the absence of
Ca21 (130). Evidence that Ca21 lowers CaM’s affinity for
BBMI came from studies showing that this ion seems to
alter the proteolytic digestion of the myosin heavy chain.
Calmodulin activates the Mg21-ATPase activity of BBMI
in the absence of actin, and it inhibits in vitro motility.
Brush-border myosin I has several IQ motifs in its neck
region RD that is thought to be responsible for CaM
binding. There is a family of similar CaM-binding unconventional myosins in other vertebrate tissues including rat
kidney brush borders, two from brain, adrenal cortex, and
smooth muscle (23).
Recently, a novel subclass of myosin I (myr4) was
identified from rat brain, demonstrated to bind CaM, and
showed by sequence analysis to be not closely related to
BBMI (9). Calmodulin’s association with myr4 was demonstrated by detection of CaM in preparations of affinity
purified myr4 by immunoblotting. Immunoprecipitation
with myr4 antibodies also yields a band comigrating with
authentic CaM. With the use of fusion proteins for myr4,
encompassing various portions of the two IQ motifs and
CaM overlay assays, it was found that both myr4 IQ motifs
bind CaM. The amino-terminal IQ motif (amino acids
700 –721) binds CaM only weakly in the presence of free
Ca21 but strongly in the absence of free Ca21. In contrast,
the carboxy-terminal IQ motif (amino acids 722–743)
binds CaM strongly in the presence of free Ca21, but only
weakly in the absence of free Ca21. Thus two adjacent IQ
motifs differ in the Ca21 requirements for optimal CaM
binding. Studies with the myr4 fusion protein that contained both IQ motifs (amino acids 500 –743) showed the
binding of CaM in the absence and presence of free Ca21
in a manner equivalent to the sum of the two separate IQ
motifs. These results demonstrate that IQ motifs can exhibit drastically different dependencies on Ca21 for CaM
binding; IQ motifs can bind CaM in a Ca21-independent
manner, and with a few differences in sequence, become
Ca21 dependent (9).
Other CaM-binding unconventional myosins are Dilute, p190, and MYO2. These proteins share certain structural features. The biochemistry of the p190-CaM complex
from vertebrate brain has been the best characterized.
P190, which is also present in several nonbrain tissues,
was originally identified as a CaM-binding protein enriched in brain actomyosin preparations, and partially
purified from EGTA extracts as a complex with regulatory
light chains. To map the CaM-binding domain of p190,
sequences comprising the head, tail, and neck domains
were expressed as bacterial fusion proteins and probed in
an 125I-labeled CaM gel overlay assay. The results showed
that the fusion protein containing the neck domain of
p190 exhibits substantial CaM binding activity in the absence of Ca21, whereas no detectable binding of CaM was
observed for the head or the tail domain fusion proteins.
These results suggest that CaM binding sites of p190 map
to the IQ motifs contained within this neck region (40).
It is clear from all these studies that the IQ motifs of
unconventional myosins are the regions responsible for
binding of ApoCaM or Ca21-CaM and that the neck region
of myosins is the most variable region of the molecule. It
is also clear that whether an IQ domain binds CaM in a
Ca21-dependent or -independent fashion depends on
modifications of this basic sequence motif that are only
beginning to be characterized.
B. Cytoskeletal and Membrane Proteins
Cytoskeletal and membrane proteins also bind CaM
in the absence of Ca21. Syntrophin, a peripheral protein of
sarcolemma, is discussed in section IIIC. Neuromodulin
(also referred to as P-57 or GAP-43) is a neurospecific,
membrane-associated protein. Palmitoylation of two cysteine residues accounts for its membrane association
(156). Neuromodulin binds to CaM with at least 10-fold
higher affinity in the absence of Ca21 than in its presence
(25). This protein was originally detected by cross-linking
experiments between azido 125I-CaM with detergent-solubilized cerebral cortex membranes in the presence of
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Usually the binding of CaM to proteins is Ca21 dependent, but ApoCaM binding occurs with various actinbinding proteins (e.g., myosins), cytoskeletal and membrane proteins, enzymes and channels, and receptors.
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JURADO, CHOCKALINGAM, AND JARRETT
ter substitution of Ser-41 by Asp or Asn. The result is Asn
mutant retains its affinity for CaM-Sepharose, while the
Asp mutant does not bind CaM-Sepharose (22).
Neurogranin (or RC3) also binds CaM in the absence
of Ca21. This protein, purified from bovine forebrain, has
a molecular mass of 7.8 kDa and forms dimers and higher
oligomers in the absence of reducing agents (10). Database searches between the amino acid sequence of neurogranin and other protein sequences revealed a striking
homology between neurogranin and the known phosphorylation site/CaM-binding domain of neuromodulin. On the
basis of this homology, the possible interaction of neurogranin with immobilized CaM was tested (10). Neurogranin also binds to CaM-Sepharose in the absence of Ca21
and is eluted with Ca21. As was found with neuromodulin,
CaM binding to neurogranin inhibits its phosphorylation
by protein kinase C. Phosphorylation of neurogranin by
protein kinase C and trypsin digestion results in a peptide,
IQASFR, phosphorylated on the serine residue. This sequence is part of the conserved IQ motif present in both
neuromodulin and neurogranin. Because of the strict conservation of the CaM binding and phosphorylation site in
neuromodulin and neurogranin, the authors suggest that
neurogranin might have a similar function of sequestering
CaM at specific locations in the brain and releasing it in
response to increase in Ca21 and/or to phosphorylation by
protein kinase C (10).
PEP-19, a small 7.6-kDa neuron-specific protein,
which contains a consensus motif similar to the CaM
binding domains of neuromodulin and neurogranin have
also been demonstrated to bind CaM in a Ca21-independent manner. Slemmon et al. (123) demonstrated that
PEP-19 inhibits the CaM-dependent nNOS activity with an
EC50 of 8 mM, showing that PEP-19 binds CaM with
moderate affinity. PEP-19 was shown to bind immobilized
CaM in the absence and presence of Ca21 with an apparent dissociation constant of 1.15 and 1.25 mM, respectively. Calmodulin binding was localized to an IQ motif at
PEP-19 amino acids 36 – 60. Specific substitution of conserved amino acids within the consensus IQ motif of
PEP-19, neurogranin, and neuromodulin abolish CaM
binding. Interestingly, sequences upstream and downstream of the motif itself also affect binding. The PEP-19
IQ motif contains a Ser in the same relative position as the
corresponding Ser in neuromodulin and neurogranin;
however, in this case, the residue is not phosphorylated in
vitro by protein kinase C, suggesting that PEP-19 constitutes a PKC-independent pathway for CaM regulation
(123).
Igloo, a neuromodulin-related protein from Drosophila, binds CaM (98), but whether this interaction is preferentially Ca21 dependent or Ca21 independent was not
directly demonstrated. Igloo and neuromodulin share
some sequence similarities. Most striking are its three IQ
motifs that are similar to the ones in neuromodulin. The
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excess EGTA (4). Radioimmunoassay experiments also
demonstrated the presence of this protein in retina and
spinal cord (25). Because of its unusual higher affinity for
ApoCaM compared with Ca21-CaM, an easy purification
protocol using CaM-Sepharose affinity chromatography
was developed. This involves binding of neuromodulin to
CaM-Sepharose in the presence of excess EGTA, a Ca21
chelator, and elution from the affinity column by buffers
containing excess of Ca21 (4). Purification to apparent
homogeneity was accomplished by solubilization from
membranes, DEAE-Sephacel chromatography to remove
endogenous CaM, and two CaM-Sepharose columns
(6Ca21). Neuromodulin is the only protein in the membrane preparation with higher affinity for ApoCaM than
Ca21-CaM. Neuromodulin binds a single CaM (4). Regulation of CaM binding to neuromodulin is exerted by
phosphorylation at a serine residue (Ser-41, see Table 3)
in a Ca21-dependent manner and phosphorylation prevents neuromodulin binding to CaM-Sepharose. Also,
CaM decreases the rate of phosphorylation of neuromodulin, suggesting that a residue nearby or involved in
the binding of CaM to neuromodulin is the one modified
(1). Identification of the phosphorylation site by using
recombinant neuromodulin gave as a result a peptide of
the sequence IQASFR. Serine-41 is the residue phosphorylated, and this sequence is the amino terminus of neuromodulin’s IQ motif (5), explaining why phosphorylation of
neuromodulin dramatically lowers its affinity for CaM. It
has been proposed that neuromodulin binds and concentrates CaM at specific sites within the cell and releases it
locally in response to stimulation by phosphorylation
and/or to increase in free Ca21 (1, 22). Phosphorylation
may be more important, since at physiological ionic
strengths, the difference neuromodulin’s affinity for CaM
is only lessened about fivefold by Ca21 (81). In addition,
phosphoneuromodulin is a substrate for calcineurin
(phosphatase 2B) and phosphatase 2A, indicating that the
concentrations of CaM in neurons may be controlled by a
phosphorylation/dephosphorylation cycle (5, 22). Characterization of the CaM binding domain to neuromodulin
was also studied by constructing a deletion mutant neuromodulin, designated NM(DCM), which lacks the IQ motif amino acids 39 –55 and examined for binding to CaMSepharose (22). The wild-type neuromodulin adsorbed to
CaM-Sepharose in the presence of excess of EDTA and
eluted with Ca21, while NM(DCM) did not adsorb to
CaM-Sepharose in the presence or absence of Ca21. This
resulted in the identification of the sequence
Q39ASFRGHITRKKLKGEKK56 required for CaM binding;
this domain contains the only phosphorylation site (Ser41) that affects CaM binding to neuromodulin. The adjacent Phe-42 apparently interacts hydrophobically with
CaM, an interaction that is disrupted by the introduction
of negative charge to Ser-41. This proposal was also supported by studying the neuromodulin/CaM interaction af-
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APOCALMODULIN
C. Enzymes
Some properties of the CaM binding enzymes PbK,
adenylyl cyclase, and iNOS have been discussed previously. Glycogen phosphorylase b kinase, an enzyme that
catalyzes the phosphorylation of glycogen phosphorylase
and serves a key regulatory role in glycogen metabolism,
contains CaM as an integral subunit (the d-subunit). The
quaternary structure is (abgd)4. The d-subunit CaM remains associated even after prolonged exposure to Ca21
chelators such as EGTA and EDTA (28). Skeletal muscle
PbK can also bind four additional extrinsic CaM, referred
to as d9, in a Ca21-dependent fashion. Troponin C, a
member of the CaM homology family, also binds and can
substitute for d9, albeit with lower affinity. The greater
abundance of troponin C in skeletal muscle makes it
likely this interaction is physiologically relevant (26). The
linked function between Ca21 and PbK binding to CaM
(d9) has been particularly well characterized (18).
Bordetella pertussis adenylyl cyclase binds, and is
activated by, CaM in a Ca21-independent manner. This
enzyme is the first reported example of Ca21-independent
stimulation of any enzyme (52). When the enzyme was
rendered Ca21 free by desalting the enzyme in buffers
containing EGTA and including the chelator in the assay
mixture, cyclase activity increased 23-fold upon addition
of CaM. Half-maximal activation occurred at 24 nM of
CaM in EGTA and 0.1 nM in the presence of Ca21. Calmodulin activated the enzyme in EGTA to about twice the
activity found in Ca21. Bordetella pertussis adenylyl cyclase also binds CaM-Sepharose in the presence of 5 mM
EGTA (52).
Inducible (macrophage) NOS has also been reported
to be a Ca21-independent CaM binding protein. The enzyme is isolated with a tightly bound CaM even in EGTA,
and extrinsic CaM is not required for maximal activity.
Inducible NOS is maximally active at Ca21 concentrations
as low as 0.1 nM in vitro (120) and is thus probably
maximally active in vivo even at the low intracellular Ca21
concentrations encountered in unstimulated cells. However, because Ca21 activates the enzyme relative to the
activity in EGTA in some studies (140), the Ca21-bound
state of the intrinsic CaM is uncertain. Amino acids 503–
532 of the mouse iNOS consist almost exclusively of the
characteristic hydrophobic/basic regions of Ca21-CaM
binding sites. Comparison of this sequence with the corresponding region of constitutively expressed NOS
(cNOS), which is a Ca21- and CaM-dependent protein,
shows only 43% identity of the 21-amino acid residues that
are designated as the presumptive CaM-binding site of
cNOS, suggesting that these differences allow iNOS to
bind CaM at low concentrations (150). Two synthetic
peptides corresponding to the CaM-binding domain of
iNOS [P29, NOS-(504O532), and P34, NOS-(499O532)]
were subsequently used to study Ca21-dependent and
Ca21-independent binding of CaM. Both peptides bound
CaM both in the presence as well as in the absence of
Ca21 (i.e., in the presence of chelator EGTA). The binding
affinity of CaM in the presence of Ca21 was reported with
a dissociation constant of ;1 nM; this binding affinity is
lower, but still remarkably high (45– 85 nM) in the presence of EGTA (2). A chimeric NOS, constructed by replacement of the CaM-binding sequence of endothelial
NOS (residues 493–512) with aligned sequence from iNOS
(residues 501–532), results in a functional protein that is
activated by CaM in either Ca21 or EGTA, but these
changes do not produce irreversible CaM binding, sug-
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three IQ modules of Igloo (IGL) were denoted V, T, and S
for the amino acid abbreviation of the residue in the
putative protein kinase C phosphorylation position found
for neuromodulin. Database searches revealed additional
homology of these three modules of IGL to PEP-19, neurogranin, and the cephalochordate polypeptide Ca21 vector target protein. On the basis of this sequence homology, and the fact that protein kinase C phosphorylates the
IQ motif regions in other proteins, recombinant fusion
proteins containing glutathione S-transferase and regions
of IGL were produced and tested as a substrate for PKC.
Phosphorylation by PKC occurred only within the S module, and mutation of this Ser to an Ala prevented phosphorylation. Surprisingly, the threonine residue of IGL
fusion protein used was not phosphorylated in vitro by
protein kinase C. The GST-IGL fusion proteins were found
not to bind vertebrate CaM-Sepharose. However, with the
use of Drosophila CaM and the yeast two-hybrid system,
interaction with IGL and with each individual IQ motif
module was demonstrated.
The IQGAP proteins are homologous to the GTPaseactivating proteins (GAP) of the small G proteins (such as
Ras and Rho), which also contain four apparent IQ motifs.
Human IQGAP1 and IQGAP2 (a liver-specific protein) are
the currently known IQGAP proteins (146). Despite the
homology, there is currently no evidence that they have
GAP activity, although IQGAP2 binds Cdc42 and RacI.
Recently, the possible interaction of the IQGAP2 four IQ
motifs with calmodulin was studied (15). Immunoprecipitation studies and the yeast two-hybrid system showed
the interaction of CaM with full-length IQGAP2. This interaction was disrupted when a truncated IQGAP2 lacking the IQ motif sequences was tested. This CaM interaction with IQGAP2 occurs even in 1% Triton and high salt
concentrations. IQGAP2 binds CaM very strongly in the
presence of Ca21 and to a lesser extent in the absence of
Ca21. These results demonstrate an interaction of IQ
motifs with both Ca21-CaM and ApoCaM and suggest that
these interactions may provide a link between CaM-mediated processes and the small G protein signaling pathways.
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JURADO, CHOCKALINGAM, AND JARRETT
says of cGMP-dependent protein kinase showed that CaM
increased maximum binding of this enzyme in the absence of Ca21 without altering the Michaelis constant for
ATP. It was found that in the presence of CaM concentrations producing maximum stimulation, this enzyme
was specifically activated by a low concentration of
cGMP. Adenosine 39,59-cyclic monophosphate also partially activated the kinase. Thus CaM may play an important role in the regulation of cyclic nucleotide levels in the
cell in the absence of Ca21 (152).
D. Receptors and Ion Channels
Inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R)
and ryanodine receptor Ca21-release channels bind and
are apparently regulated by ApoCaM. Patel et al. (107)
found a specific and reversible binding of 125I-CaM to
purified cerebellar InsP3R in the absence of Ca21. Equilibrium competition binding experiments showed that
ApoCaM binding to InsP3R occurs at multiple sites; one
class binds only Ca21-CaM and another binds equally well
to Ca21-CaM or ApoCaM. Further experiments showed
that the Ca21-CaM antagonists calmidazolium and N-(6aminohexyl)-5-chloro-1-naphthalenesulfonamide
(W-7)
are much less effective in preventing binding of 125I-CaM
to InsP3R in the absence of Ca21 than in its presence. In
the presence or absence of Ca21, CaM inhibits InsP3
binding and InsP3-induced Ca21 release under all conditions tested. These studies suggest an ability of InsP3R to
sense levels of CaM and regulate Ca21 action in cerebellum.
Calmodulin also binds the skeletal muscle sarcoplasmic reticulum (SR) Ca21-release channel in the presence
of EGTA (154). Incubation of purified heavy SR vesicles
from skeletal muscle in the presence of EGTA with the
affinity-labeling derivative 125I-benzophenone-CaM results
in the identification of a single major band of Mr .450,000
corresponding to a complex between CaM and the Ca21
channel protein. With the use of a rhodamine-wheat germ
CaM (Rh-CaM) and fluorescence anisotropy, the binding
of the Rh-CaM in the presence of Ca21 or EGTA to the
Ca21 channel protein was further studied. At low ionic
strengths, binding of ApoCaM is minimal; however, at
physiologically relevant concentrations of KCl, the stoichiometry of Rh-CaM binding in the presence of EGTA is
about four to six CaM-binding sites per subunit for this
homotetrameric channel. The apparent dissociation constant in EGTA is 8.6 6 0.8 nM CaM. Interestingly, when 1
mM MgCl2 is present, the addition of Ca21 (0.1 mM)
decreases CaM binding by nearly threefold (154). In these
early reports, CaM was found to inhibit Ca21 release by
the channel protein.
A recent report of the binding of 125I-CaM to the
skeletal muscle SR Ca21 release channel found some
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gesting that other regions of iNOS (outside of residues
501–532) are required (140). A further study to test directly whether the predicted CaM-binding region of iNOS
(amino acids 501–532) accounts for its Ca21 independence used a chimera protein composed of reciprocally
exchanged amino acids 503–532 of iNOS and the corresponding residues 725–754 of nNOS. The results of this
study showed that both chimeras required 7–10 nM free
Ca21 to bind CaM and that Ca21 is essential for enzymatic
activity for both chimeras. Additional results by using the
indicator fura 2 showed that the concentration of free
Ca21 required for half-maximal activity (EC50) are ;0 for
iNOS, 200 –300 nM for nNOS, and 7–10 nM for the chimeras. Thus sequences outside the 503–532 region of iNOS
are essential for its apparently irreversible binding of CaM
(120).
Molecular procedures for screening for CaM-binding
proteins in plants using 35S-labeled recombinant CaM as a
probe resulted in the identification of a petunia protein of
58 kDa with a high amino acid sequence similarity to the
53-kDa glutamate decarboxylase (GAD) from E. coli (11).
Recombinant GAD from petunia catalyzes the conversion
of glutamic acid to g-aminobutyric acid and also binds
CaM, whereas the E. coli GAD does not have CaM-binding
activity. The CaM-binding domain on petunia GAD resides
at the carboxy end of this protein, a region that is not
present in the E. coli GAD. The authors concluded that
intracellular levels of Ca21 via CaM may regulate g-aminobutyric acid synthesis in plants (11). A related study
reported the identification of a 62-kDa CaM-binding protein from fava bean seedlings with GAD activity. These
authors reported Ca21-dependent CaM activation of the
fava bean GAD activity (79). Activation of the petunia
enzyme by CaM is also Ca21 dependent; however, CaM
binds to petunia GAD even in the absence of Ca21, and
CaM-GAD cross-linking is also Ca21 independent. A synthetic peptide corresponding to the petunia GAD carboxyterminal sequence (amino acids 470 – 495) binds CaM as a
stable complex in the presence of Ca21, but not in its
absence. Using carboxy terminus-deleted mutants of GAD
(by removal of 2 Lys residues, Lys-494 and Lys-495) results in a reduction of cross-linked complexes in the
absence of Ca21, suggesting that these lysine residues
might facilitate electrostatic interactions between GAD
and acidic CaM residues in a role similar to that of Lys in
other CaM-target sites (6). Thus CaM binding to petunia
GAD occurs in the presence or absence of Ca21 as is the
case for other enzymes described in this review (e.g., PbK,
iNOS, Ca21-release channels), but Ca21, presumably binding to CaM, is required for enzyme activation (as with PbK
and perhaps iNOS). Apocalmodulin binding occurs in
plants as well as in animals and fungi.
Guanosine 39,59-cyclic monophosphate-dependent
protein kinase has also been reported to be activated by
CaM in a Ca21-independent fashion (152). Enzymatic as-
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APOCALMODULIN
677
V. CONCLUSIONS
The literature of ApoCaM, like that of Ca21-CaM, is
extensive; in an effort to be concise, we have not mentioned all of the studies of which we are aware. For
example, we have not discussed “CaM-binding” sites
known only by homology for which there is currently no
experimental confirmation. Rather, we have focused on
those studies that pointed out some unique aspects to
ApoCaM biochemistry, physiology, or cell biology. What
emerges from this focused approach is, we hope, a more
easily grasped appreciation of the complexity of these
interactions. Some aspects of that complexity are dealt
with in Figure 3. Relative to Ca21-CaM, ApoCaM can bind
more tightly (e.g., neuromodulin, neurogranin), equally
tightly (e.g., one site on the SR Ca21-release channel,
probably one or more of syntrophin’s binding sites), or
less tightly (e.g., MLCK and most of the other Ca21dependent CaM-binding proteins).
Binding can be of such high affinity as to be essentially irreversible (PbK, iNOS, myosin I, petunia GAD) or
low affinity and freely reversible (neuromodulin, most
Ca21-dependent CaM-binding proteins). Reversible
ApoCaM binding that is inhibited by Ca21 may serve the
role of storing CaM at specific sites within the cell ready
for release when needed (1). This hypothesis finds support in those studies that show the particulate ApoCaM
fraction changing with cell density while the cytoplasmic
pool increases (41) or the inverse relationship between
these two pools found in normal and neoplastic cells (122,
138, 145). This hypothesis is the origin of the top portion
of Figure 3, where the binding of ApoCaM to neuromodulin (presumably other proteins serve such a role in non-
FIG. 3. CaM, calmodulin; ApoCaM, apocalmodulin; PKC, protein
kinase C; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum.
nerve tissue) is shown as reversibly modulated by either
increased intracellular Ca21 and protein phosphorylation,
here by protein kinase C. Apocalmodulin binding to iNOS
or Ca21-release channels likely alters the activity of these
proteins under the low intracellular Ca21 concentrations
found in unstimulated or resting cells, enhancing activity
in low-Ca21 environments. Thus ApoCaM itself probably
alters the activity of a subset of enzymes directly. Calcium-CaM also alters the activity of other enzymes. These
separate effects of CaM are also shown schematically in
Figure 3. Finally, at the bottom of Figure 3 is shown
schematically ApoCaM binding to the Ca21-release channel. (Apo)calmodulin also regulates Ca21 itself. This continues another theme in CaM research known since it was
discovered that Ca21-CaM regulates the plasma membrane Ca21-transport ATPase (51, 64). Cytoplasmic Ca21
is regulated by CaM. This Ca21 also binds CaM and affects
its activity. Calmodulin and Ca21 regulate one another’s
cellular activity.
An interesting feature of ApoCaM binding is that
when binding sequences are localized and peptides synthesized, these peptides are bound preferentially by Ca21CaM. This observation suggests that binding to ApoCaM
utilizes many of the same kind of protein-protein interactions that account for Ca21-CaM binding. This is not
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important new results (137). In this study, the tetrameric
Ca21 release channel protein bound with nanomolar affinity: 4 CaM in the presence of Ca21 and 16 CaM in the
absence of Ca21. More importantly, the authors found
that the binding of CaM activates Ca21 release at low
Ca21 concentrations, which approximate those found in
resting muscle cells (100 –150 nM), whereas CaM inhibits
Ca21 release when Ca21 levels are increased to the micromolar to millimolar levels anticipated during the contractile process. The rate at which CaM dissociates from
the channel is quite slow relative to the rate of contraction
in the presence or absence of Ca21, suggesting that during
contraction, the CaM-bound state of the channel is probably constant, at least during the initial stages of contraction. What emerges is a model in which each subunit of
the channel binds about four ApoCaM in the resting muscle, and this sensitizes the channels to Ca21 release. When
the muscle contracts, three of these CaM begin dissociating, and the one remaining binds Ca21 and further Ca21
release is inhibited (137).
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JURADO, CHOCKALINGAM, AND JARRETT
Some target proteins have such a poor affinity for
ApoCaM that binding probably does not occur to any
great extent at cellular CaM concentrations until Ca21
enters the cell to produce Ca21-CaM. Proteins that use
this paradigm exist in either a CaM-bound or CaM-unbound state, and they function differently in the two
states. For other proteins, however, there is a third state
in which they bind ApoCaM. This appears to be the case
with the SR Ca21-release channel (137) and, from a certain point of view, is certainly the case for PbK. The only
forms of PbK known that lack (d) CaM are inactive; those
that contain ApoCaM are active and modulatable in a
variety of ways (e.g., phosphorylation by other protein
kinases, pH changes in the range of 6.8 – 8, etc.), and those
which contain Ca21-CaM are in a third, different state that
is most active under some conditions. Thus CaM not only
signals the presence of Ca21, it can also signal its relative
absence by directly binding a target protein as ApoCaM.
When ApoCaM binding is added to the linkage between
Ca21 and target protein binding to CaM, what emerges is
a signaling system that can respond to virtually any Ca21
concentration. The target protein itself can change the
Ca21-binding properties of CaM so that the complex is
responsive to either lower or higher Ca21 concentrations
than CaM alone. Calmodulin is thus capable of responding
to virtually any physiologically relevant Ca21 concentration, and the response can be differently adapted for each
target protein by adapting the target protein itself. Added
to this sensitivity for Ca21 is the modulation of the concentration of free CaM itself that results from the binding
and release of CaM in response to cellular Ca21 or phosphorylation. The amount of neuromodulin in nerves, its
phosphorylation state, and the intracellular Ca21 concentration may all affect the concentration of free CaM available for binding to other target proteins. There seems to
be no aspect of CaM function that cannot readily be
altered by the cell. This degree of flexibility is astounding.
Calmodulin is essential for life in eukaryotes (36, 116,
133). However, Ca21 binding may not always be essential
(48). Thus ApoCaM must serve at least one essential role
in the cell. That Ca21 binding is essential in other organisms (96) does not refute this essential role for ApoCaM
but rather suggests that there must be essential roles for
both forms of CaM. What this essential ApoCaM role(s)
may be is currently unknown.
Understanding ApoCaM is as important as understanding Ca21-CaM, and we are much further behind in
the former. Both forms of CaM serve essential roles in life,
and to understand Ca21 signaling, we can neglect neither.
We thank Drs. Tayebeh Pourmotabbed and Bruce Martin
for much helpful discussion.
This work was funded by the Muscular Dystrophy Association.
Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on July 4, 2017
surprising, since these ApoCaM binding sequences usually have the same characteristics of being hydrophobic
and cationic, as are the Ca21-CaM binding sequences. The
IQ motif (IQXXXRGXXXR) has a framework structure
that is hydrophobic (Ile) and cationic (Arg) and added to
this basic framework; many of the intervening (X) or
flanking residues are frequently hydrophobic, sometimes
aromatic, or cationic (see Table 3). Indeed, the IQ motif
can be either a preferentially Apo- or Ca21-CaM binding
site, even within the same protein (e.g., Myr-4). Consider,
for example, these three sites: IQ motif 1 (Apo) and 2
(Ca21 dependent) of Myr-4 and the CaM-binding sequence
from MLCK (Ca21 dependent). One of these binds preferentially ApoCaM, whereas the other two bind Ca21-CaM;
however, if they are unlabeled, which is which would not
be currently predicted. We are only now beginning to
appreciate how ApoCaM binding occurs, and our knowledge is very imperfect.
However, the two kinds of binding while sharing
some similarities are also likely to be quite different. The
model that has emerged from the structure of Ca21-CaM
bound to peptides from MLCK and CaM kinase II (59, 89,
90) show a compact “clam shell” CaM bent about the
middle of its central helix and enveloping the amphipathic, a-helical peptide derived from these target enzymes. This compact, clam-shell model does not readily
accommodate the binding of two, noncontiguous peptides
such as those from PbK or the more extended conformation suggested for this complex by the low-angle X-ray
scattering data (136). Because this binding of multiple,
noncontiguous sites must also extend to iNOS, the B.
pertussis adenylyl cyclase, and probably many other examples of ApoCaM binding, a new model that accommodates this kind of binding must be constructed. This kind
of binding can be referred to as a kind of “trimeric binding,” since two examples of it, CaM-PhK5-PhK13 of PbK
and CaM-T25-T18 of B. pertussis adenylyl cyclase, would
consist of trimers of CaM and two peptides. The only
structural model for ApoCaM binding is based on a regulatory light chain binding the regulatory domain IQ motifs
of scallop myosin (58). This model also shows a clamshell CaM enveloping an amphipathic, a-helical IQ motif
over a somewhat more extended length of a-helical sequence. These observations suggest that there may be
another kind of ApoCaM binding, referred to above as
trimeric, about which we have little or no understanding
at a structural level.
Eukaryotic cells utilize Ca21 signaling. The cell maintains low Ca21 when quiescent (about 0.1 mM), and this
increases upon stimulation. In these two states, CaM is for
the most part ApoCaM and Ca21-CaM, respectively. It was
clearly possible that CaM-mediated signaling could have
occurred by binding only one of these two CaM states.
This kind of two-state (on-off, flip-flop) CaM signaling
clearly does occur with a multitude of target proteins.
Volume 79
July 1999
APOCALMODULIN
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