Download Calmodulin and Cell Function

497
Clinical Science (1984) 66,497-508
EDITORL4L REVIEW
Calmodulin and cell function
S . TOMLINSON’, S . MACNEIL’, S . W. WALKER’, C. A. OLLIS’, J. E. MERRITT3
AND
B. L. BROWN’
’
‘Department of Medicine, University Clinical Sciences Centre, Northern General Hospital, Sheffield, Department of
Chemical Pathology, Northern General Hospital, Sheffield and ’Department of Human Metabolism and
Clinical Biochemistry, The Medical School, Sheffield, U.K.
Introduction
The importance of calcium in the regulation of cell
function has become increasingly recognized in the
past 20 years.. It is now known that changes in
intracellular calcium concentration, like changes in
adenosine 3’:5’-monophosphate (cyclic AMP), are
of crucial importance in stimulus-response coup
ling. Thus, the second messenger theory originally
proposed by Sutherland et al. [l], in which a
hormone or nerve impulse is fust messenger and
cyclic AMP the second intracellular messenger, has
been expanded to include calcium ions as well as
cyclic nucleotides. Moreover, there is now evidence
Correspondence: Dr S. Tomlinson, Department
of Medicine, University Clinical Sciences Centre,
Northern General Hospital, Sheffield S5 7AU, U.K.
in many cellular systems that calcium ions and
cyclic nucleotides act as dual interrelated messengers [2], and, therefore, some cellular processes
are regulated by calcium as well as by cyclic
nucleotides, as shown in Table 1. Evidence has
accumulated that it is changes in free intracellular
calcium concentration that are, in some way,
responsible for activation of the enzymes involved
in the processes shown in t h i s Table.
The resting concentration of free intracellular
calcium is between lo-’ and lO-’mol/l (0.01-0.1
pnolll) and an increase in this concentration by
ten- to a hundred-fold effects the appropriate
response [3]. Changes in the intracellular free
calcium can be derived from either extracellular or
intracellular sources. Calcium channels allow
calcium to enter the cell from the extracellular
TABLE1. Cellular processes regulated by calcium, calmodulin and cyclic nucleotides
Cellular process
Insulin secretion
Thyroid secretion
pituitary secretion
Adrenal secretion
Neurohormone secretion
Intestinal secretion
Cell proliferation
Cell architecture
Lysosome release
Smooth muscle contraction
Lymphocyte mediated cytotoxicity
Prostaglandin synthesis
Disassembly of microtubules
Histamine release
Ciliary motility
Fast axonal transport
Neurohormone synthesis
Neurohormone super sensitivity
Phagocytosis
Initiation of DNA synthesis
Cast
dependent
Role for
calmodulin
cyclic
nucleotides
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S. Tomlinson et al.
fluid [4]; these calcium channels can be voltage
dependent (for example in glucose stimulated
insulin release and in secretion of some pituitary
hormones, such as prolactin) or agonist dependent
(for example in adrenergic and cholinergic effects
in the salivary glands). There is increasing evidence
that hydrolysis of the membrane phospholipid,
phosphoinositol, is connected with mechanisms
for opening agonist dependent calcium channels
PI.
Although some cells can respond to stimuli
even in the absence of extracellular calcium, it
has been established that increases in intracellular
calcium do still occur. The sources of increased
cytosolic free calcium are probably the mitochondria, endoplasmic reticulum, plasma membrane and possibly the nucleus of the cell. If an
increase in intracellular calcium activates a specific
calcium dependent process, the activation must be
terminated by a corresponding reduction in the
intracellular free calcium concentration. This is
achieved by a number of mechanisms: first, there
are so-called energy requiring calcium pumps
located in the cell membranes which transport
calcium from the intracellular to the extracellular
compartment; second, translocation of the calcium
from cytosol to cellular organelles can occur and,
third, calcium binding molecules not involved in
the activation process may also modulate intracellular free calcium concentration.
The question arises: how does calcium act as a
second messenger? It has become clear over the
last 10 years that many calcium dependent events
are mediated by its binding to and activation of a
ubiquitous intracellular calcium binding protein
called calmodulin. The calcium-calmodulin complex is then able to activate a wide variety of
enzymes, including those which affect cyclic
nucleotide metabolism.
TABLE2 . Chronological order of findings about
calmodulin
Year
Discovery
1970
Protein activator of phosphodiesterase
discovered
Protein found to actjvate Ca2+,Mg2'dependent phosphodiesterase
1973-1974 The protein's mechanism of action elucidated
The protein activates brain adenylate cyclase
1975
Trifluoperazine found to inactivate the
1976
protein
1977
The protein activates Ca2+-ATPase
The protein is the 8 subunit of phosphorylase
1978
kinase
The protein activates myosin lightchain
kinase
The protein activates NAD kinase
Galmodulin is given as a proper name
Calmodulin activates Caz+dependentprotein
kinase
Calmodulin enhances microtubule disassemblv
1979-1980 Calmoduh regulates synthesis and release
of neurotransmitters
Amino acid sequence of calmodulin
elucidated
Calmodulin activates glycogen synthase
kinase
Calmodulii important in DNA synthesis and
cell division
Naphthalene sulphonamides (W compounds)
increasingly recognized as more specific
inhibitors of calmodulin
Calmodulin involved in prostaglandin
synthesis
Immunoassays for calmodulin developed
? Calmodulin activation of adenylate cyclase
possibly a general phenomenon
1981-1983 Calmodulin activates guanylate cyclase
Calmodulin increasingly recognized as
important in stimulus-secretion coupling
in endocrine cells
DNA sequence of calmoduli elucidated
Alterations in calmodulin in disease:
increased activity in cystic fibrosis;
increased concentration in psoriasis and in
experimental diabetes
Calmodulin
A brief summary of the chronological order in
which calmodulin's role has been elucidated is
shown in Table 2. The protein was first discovered
as a heat-stable, dissociable activator of brain
cyclic nucleotide phosphodiesterase independently
by Cheung and Kachiuchi et al. in 1970 [6,7]. In
1973 Teo & Wang [8] elucidated the protein's
mechanism of action and showed that the two
proteins described by the original investigators
were, in fact, identical. Subsequently, Brostrom
et al. [9] found that the protein activated brain
adenylate cyclase and Levin & Weiss in 1976 [lo]
described the inactivation of the protein by trifluoperazine, which provided a useful tool for the
study of the role of calmodulin in cellular systems.
In 1978, the now accepted name calmodulin was
coined by Cheung et al. [l 11.
It is remarkable that calmodulin is very widely
distributed, being found in both the plant and
animal kingdoms and probably in all eukaryotic
cells. Brain and testicular tissue are particularly
rich sources of the protein but possibly the richest
source is the electroplax of the electric eel, where
calmodulin accounts for 5-10% of the total
protein [ 121.
Properties of calmodulin
Calmodulin is a heat-stable, acidic polypeptide of
148 amino acids. It contains four calcium binding
Calmodulin and cell function
TABLE 3. Properties of calmodulin
Straight chain polypeptide; 148 amino acids
Mol. wt. 16 500
Isoelectric point. pH approximately 4 .O
Calcium binding, four sites; affinity approximately
mol
Internal amino acid homology at calcium binding sites
Binds to phenothiazines: Caz* dependent
Resistant to denaturation
sites with affinities for calcium in the range which
corresponds closely to the free intracellular
calcium concentration in the stimulated state. The
primary structure of all calmodulins studied so far
is very similar and biological studies show that it
is neither tissue nor species specific. Some of the
important properties of calmodulin are summarized in Table 3. It is of interest that the four
calcium binding domains in calmodulin show nearly
identical amino acid sequences and it seems likely
that the protein has arisen by gene duplication.
Each domain has the basic ‘EF hand’ structure
as determined by X-ray crystallography [ 131. The
protein has a similar amino acid sequence, particularly in the calcium binding regions, to a number
of other calcium binding proteins known as the
troponin C super-family of calcium binding proteins. Not only is the basic structure of calmodulin
similar throughout the plant and animal kingdoms,
but recent evidence suggests that base triplets
coding for amino acids are also conserved since
DNA coding for electric eel calmodulin hybridizes
to DNA from several other species [14,15].
Mechanism of action of calmodulin
Current evidence suggests that under normal conditions calmodulin activity is not altered by
changes in its concentration within the cell but
Inactive
mlrnodulin
Active
oalrnodulin
499
mainly by changes in the concentration of free
intracellular calcium [161. Under resting conditions, the concentration of intracellular free
calcium is too low to allow for any significant
binding of calcium to calmodulin. When a cell
is stimulated, there is an increase in intracellular
free calcium concentration due to the movement
of calcium through channels in the plasma membrane or its release from intracellular membranes
or organelles. Calcium then binds to calmodulin,
which undergoes a conformational change, allowing interaction with inactive enzyme to form an
active complex. A reduction in intracellular free
calcium concentration by extrusion from the cell
(brought about by calmodulin-dependent Ca2’ATPase) or by translocation within the cell results
in dissociation of the active calmodulin-enzyme
complex, thus decreasing enzyme activity. This
simplified scheme is illustrated in Fig. 1. However,
it must be emphasized that there may be other
factors which regulate calmodulin’s activity, in
addition to alterations in intracellular free calcium
concentration. As well as enzymes that bind to
calmodulin and are thus activated, there are other
proteins to which calmodulin will bind and these
proteins themselves may regulate the activity of
calmodulin [171. One example of such an inhibitory protein is calcineurin, which has been found
in brain. Calcineurin is composed of two subunits:
a large molecular weight subunit which binds to
calmodulin, and a small molecular weight subunit
which itself is a calcium binding protein. This
complex can bind to and inactivate calmodulin,
thus preventing, for example, activation of cyclic
nucleotide phosphodiesterase or adenylate cyclase.
However, recent work has shown that calcineurin
itself has an enzymatic function since it appears
indistinguishable from protein phosphatase 2b
[18]. A third mechanism for the regulation of
lnactiw
receptor protein
Active
receptor protein
FIG. 1. Mechanism by which calmodulin mediates the biological action of calcium ions
is depicted in this highly schematic diagram. Neither calcium alone nor calmodulin
alone is active. The binding of 4 Ca2+ to calmodulin changes the shape of the protein.
As a result, calmodulin is able to interact with an enzyme (‘receptor protein’), which
is thereby activated.
500
S. Tomlinson et al.
calmodulin activity may be by the localization of
the protein within the cell. This has been described
both during cell division [19] and in the capping
of cell surface receptors for concanavalin A in a
human lymphoblastoid cell line [20]. It is possible
that this relocation of calmodulin is in some way
influenced by cyclic AMP. Conversely, calmodulin
may promote relocation of its own binding proteins
within the cell, as has been described in exocytosis
[21]. Finally, chemical modification of the calmodulin molecule itself may influence its activity; for
example, carboxylmethylation of calmodulin
occurs in intact cells and there is evidence that
carboxylmethylated calmodulin shows reduced
stimulation of cyclic nucleotide phosphodiesterase
[22]. The regulation of calmodulin’s activity is
clearly an area of great importance and one might
speculate (see below) that it could be abnormalities
in the regulation of this activity which might be
important in some disease processes.
Inhibitors of calmodulin activity
One of the advantages of using these drugs is that
they cross cell membranes and, therefore, can be
used in studies of whole cell systems.
Specific inhibitors of calmodulin, such as
monoclonal antibodies against the protein, can
only be used provided that calmodulin in the test
system is available to the antibody. A further disadvantage of antibodies is that they tend to have
lower affinity for calmodulin than calmodulin has
for its receptor proteins or enzymes and, therefore,
a large amount of antiserum may be required to
exert its inhibitory effect. The calmodulin binding
protein, calcineurin, may be more useful in such
studies since it is specific and binds with high
affinity to the protein. However, even using this
specific inhibitor, one has to bear in mind that it
also has intrinsic activity of its own which may
affect the system under investigation.
Calmodulin and cyclic nucleotide synthesis and
degradation [29]
Calmodulin was first described as a protein activaA major observation for subsequent studies of the tor of cyclic nucleotide phosphodiesterase [6, 71.
function of calmodulin was that phenothiazines Subsequently, it was found that as well as influencbind to and inactivate calmodulin in the presence ing cyclic nucleotide degradation, calmodulin
of calcium [lo]. After this initial observation, it could also influence cyclic nucleotide synthesis by
was found that many other drugs, including the activation of adenylate cyclase in brain [9, 111;
butyrophenones, tricyclic antidepressants, some more recently, it has been reported that calmodulin
dopamine antagonists, some opiate derivatives, is involved in the activation of adenylate cyclase
0-receptor blockers and local anaesthetics, could in guinea-pig sperm [30], pancreatic islets [3 1, 321,
inhibit, with varying potencies, the activity of the bovine adrenal medulla [33], a vasopressin sensitive
protein [23-251. The mechanism of this inhibition adenylate cyclase in a pig kidney cell line [34] and
has been partially elucidated; when calcium binds in rat testicular germ cells [35]. In addition, our
to calmodulin it exposes a hydrophobic domain own recent studies suggest the involvement of
which appears to be essential for interaction with, calmodulin in mouse B16 melanoma adenylate
and subsequent activation of, its receptor proteins cyclase activity [36] and human thyroid adenylate
or enzymes [26]. Phenothiazines appear to bind to cyclase activity [37]. Interestingly, although prothis exposed domain and interfere with the bind- karyotes do not appear to have calmodulin, activaing of calmodulin to its receptor proteins. tion of prokaryote adenylate cyclase by calmodulin
Unfortunately, none of these drugs is specific for has recently been reported in Bordetella pertussis
calmodulin; this has two consequences. The first [381.
is that the therapeutic effect of the drugs is unExperimentally, we have found it easier to
likely to be due to their ability to inhibit calmodu- demonstrate calmodulin stimulated phospholin; indeed, using a variety of chlorpromazine diesterase activity in a cell, using calmodulin
analogues, no correlation between tranquillizer depleted cytosol, than to demonstrate calmodulin
activity and calmodulin inhibition was found [27]. activation of membrane adenylate cyclase, because
Secondly, although the drugs can be used to indi- of the difficulty of removing calmodulin from cell
cate a possible role for calmodulin in cellular membranes. Reports of attempts to remove calmoprocesses, investigators must be aware that the dulin from particulate preparations vary considerdrugs have a variety of other effects related to ably, with up to 76% being removable from brain
their hydrophobicity rather than their specific membranes [39], but none was removable from
effect on calmodulin. There is evidence that a guinea-pig sperm membranes [30]. In our own
new agent N-(6-aminohexyl)-5 -chloro-1-naphtha- experience, addition of exogenous calmodulin
lene sulphonamide (W7) which we have used in to membranes containing calmodulin produces
some of our own studies is more specific for no further activation of adenylate cyclase. This
calmodulin than previously described drugs [28]. makes it easier to understand why the earliest
Calmodulin and cell firnction
demonstrations of calmodulin activation of
adenylate cyclase used solubilized enzyme from
which calmodulin was removed by chromatography
[9],and why the number of tissues in which
calmodulin has been demonstrated to activate
adenylate cyclase is still relatively few. With respect
to guanosine 3': 5'-monophosphate (cyclic GgP)
synthesis and degradation, guanylate cyclase
activity in Tetrahymena pynformis and Paramecium has now been shown to be activated by
calmodulin [40,40a] and cyclic GMP is known to
be hydrolysed by calmodulin dependent phosphodiesterase activity in brain.
It is therefore clear that calmodulin is involved
in cyclic AMP metabolism and, indeed, cyclic AMP
itself may modulate the calcium signalling system
[4]. Cyclic AMP may not only be important in
influencing the opening of calcium channels but
may also play a role in releasing calcium from
-
h
.5
intracellular stores and regulating intracellular
translocation of the calcium-calmodulin complex.
In attempting to understand the relationship
between the calcium and cyclic AMP signalling
systems, one is presented with the apparent
paradox that the enzymes for synthesis and degradation of cyclic AMP can both be activated by
calmodulin in the same cell. For example, we have
shown that there is not only calmodulin dependent adenylate cyclase in the mouse B16 melanoma,
but also a calmodulin dependent phosphodiesterase
(see Fig. 2). This is also true of other cell systems.
A partial and simplified explanation for this may
lie in the sequential activation concept proposed
by Cheung [41]. This is dependent upon the fact
that calmodulin dependent adenylate cyclase will
be located in the cell membrane, whereas the
calmodulin activated phosphodiesterase will be in
cytosol. Stimulus promoted entry of calcium ions
h
.5
( a ) Adenylate cyclase
501
(6)Phosphodiesterase
B
*
b)
ea
2a
T
Q
c
NaF
-
+C+W?
No treatment
-
- l+C+WJ
CDR depletion
~
FIG. 2. ( a ) Inhibition of basal activity and m S H (
molt and NaF (10 mmol/l)stimulated adenylate cyclase activity in mouse B16 melanoma cultured cell lysates
by the calmodulin antagonist prochlorperazine (PCP) (100 pmol/l). Results shown are
means f SEM of triplicate determinations of a typical experiment in which the inhibition produced by PCP is most marked for hormone stimulated activity ( 8 5 % ) compared
with that produced for basal (73%)or NaF-stimulated enzyme activity (43%).Hatched
histograms, controls; open histograms, in the presence of PCP. (b) Effects of calmodulin
and the calmodulin antagonist W7 on phosphodiesterase activity. The left-hand set of
histograms show inhibition of untreated soluble cyclic AMP phosphodiesterase activity
of mouse B16 melanoma by W7 (5 x 10-5mol/l) (4- W7).Addition of 1pg of calmodulin/
ml (1.5 x lO-'mol/l) (+ C) had no significant effect on enzyme activity. The righthand set of histograms shows the same soluble phosphodiesterase activity after passage
over fluophenazine Sepharose 6B gel, which removes most of the endogenous calmodulin. It can be seen that when endogenous calmodulin was removed, addition of
exogenous calmodulin (1.5 x lO-'mol/l) (+ C) now activated phosphodiesterase
activity and addition of W7 (5 x 10-5mol/l) (4- W7) had no effect on basal enzyme
activity. CDR, Calcium dependent regulator, i.e. calmodulin.
502
S. Tomlinson et al.
into the cell membrane would be the primary
signal resulting in activation of calmodulin and
then stimulation of adenylate cyclase. Subsequent
increase in intracellular calcium concentration
would activate calmodulin in the cytosol, leading
to stimulation of calmodulin dependent phosphodiesterase which would then hydrolyse cyclic
AMP, terminating the cyclic AMP signal. In addition, there is some evidence that the two enzymes
have different requirements for calcium; calmodulin dependent adenylate cyclase activity appears
to require low concentrations of calcium and higher
concentrations (10-5mol/l and higher) are, in fact,
inhibitory to the enzyme [42].
Thus it is now clear that the two signalling
systems are interrelated and play a fundamental
role in cellular metabolism. Some of the relationships between the two systems are considered in
subsequent sections on the role of calmodulin in
stimulus-response coupling, neurotransmitter
release and neuronal function.
Calmodulin and secretion
It is now well established that calcium ions play a
major role in hormone secretion. There is good
evidence that calmodulin mediates calcium regulated secretion of insulin. Release of insulin from
the /3 cell requires the presence of extracellular
calcium ions [43,44]. Calmodulin is present in
the /3 cell of the islets of Langerhans and phenothiazines can inhibit insulin secretion [45]. Both
calmodulin activated adenylate cyclase and cyclic
AMP phosphodiesterase have been found in islet
cells [45]. This is rather indirect evidence that calmodulin has a role in insulin secretion; however,
more direct evidence comes from the fact that
calmodulin can stimulate specific protein phosphorylations in islet cells, independent of those
stimulated by cyclic AMP [46].
The secretion of anterior pituitary hormones
appears to be dependent on calcium ions [47-501.
A calcium dependent activator of phosphodiesterase has been found in anterior pituitary tissue and
calmodulin has been purified from anterior
pituitaries of freshly killed pigs [51]. A cyclic
nucleotide phosphodiesterase activated by calmodulin has been identified in pituitary tissue [52].
In addition, inhibitors of calmodulin, such as the
phenothiazines and the naphthalene sulphonamide
W7, have been shown to inhibit thyrotropin (TSH)
secretion, prolactin secretion [53-551 and luteinizing hormone secretion from pituitary cells [56]. In
some of these studies the drugs had the same order
of potency on inhibition of hormone secretion as
reported for their effect on inhibition of calmodulin activated phosphodiesterase [25].
As in the islets of Langerhans, there also appear
to be calmodulin specific protein phosphorylations
in anterior pituitary cells. We have shown that one
of the major proteins phosphorylated in the
presence of calmodulin has an approximate molecular weight of 53 000 daltons. The phosphorylation of this protein was also enhanced by cyclic
AMP in the absence of calcium, suggesting that it
might be a substrate for two independently
regulated protein kinases. Interestingly, similar
results have been obtained with pancreatic islets
[57] and so there is the possibility that the same
(or similar) proteins exist in both tissues, and are
subject to similar regulation.
The thyroid gland is of particular interest
because, unlike the /3 cells of the islets or anterior
pituitary cells, TSH-stimulated thyroid hormone
release is not dependent on the presence of extracellular calcium. Nevertheless, extracellular calcium
is required for a number of other TSH-stimulated
intracellular processes in the thyroid (for example,
glucose oxidation and iodide binding to protein)
and these processes are also affected by manipulations which alter intracellular calcium levels. It
appears, therefore, that calcium is involved in
thyroid cell metabolism [58-601.
However, there are few reports of the presence
and possible role of calmodulin in thyroid cell
metabolism. Calcium dependent activators of
phosphodiesterase have been found in the thyroid
but these were not, at that time, identified as
calmodulin [61,62]. Our own studies have shown
that calmodulin is present in the human thyroid
and we have also shown that calmodulin is possibly
involved in thyroid cell cyclic nucleotide metabolism [63] (Fig. 3). Recently, calmodulin dependent phosphodiesterase has been found in dog
thyroid slices [64]. In addition, calmodulin binding proteins have been found in thyroid cell
membranes [65].
There are very few data on the function of
calmodulin in other endocrine tissues. Calmodulin
has been detected in the adrenal cortex by an
immunofluorescent technique [66] and calmodulin
may be involved in the synthesis of corticosteroids
[67]. The protein may also be important in the
stimulatory action of potassium and angiotensin I1
on aldosterone secretion since these processes are
completely blocked by trifluoperazine [68].In the
adrenal medulla calmodulin is probably important
in exocytosis of chromaffin granules [21]. In other
secretory systems there is evidence that phenothiazines can inhibit intestinal ion secretion [69],
histamine release from mast cells [70], serotonin
release from platelets [71] and protein secretion
from polymorphonuclear leucocytes [72]. The
role (if any) of calmodulin in parathyroid hormone
W7 (pmolll)
FIG. 3. Effects of W7 on TSH (50 m-units/ml)-stimulated cyclic AMP production by
cultured human thyroid cells. Cells were incubated for 60 min at 37'C. Each point is
the mean f SEM of triplicate cultures. 0, Unstimulated cyclic AMP accumulation;
0, TSH-stimulated cyclic AMP accumulation. At low concentrations W7 appears to
enhance cyclic AMP production, possibly as a result of inhibition of calmodulin
dependent phosphodiesterase in the cells. At higher concentrations there is a dose
dependent inhibition of cyclic AMP production, presumably related to inhibition of
calmodulin dependent TSH-stimulated adenylate cyclase in the thyroid cell membranes.
secretion and in calcitonin secretion has yet to be
established [73,74].
Calmodulin, neutrotransmitter release and neuronal
function
Many studies have indicated that calmodulin may
mediate the neuronal functions of calcium ions.
Calcium in micromolar concentrations can stimulate the release of several neurotransmitter substances, including acetylcholine, noradrenaline and
dopamine from preparations of synaptic vesicles
[75]. These neurotransmitter effects have been
shown to be mediated by calmodulin and inhibited
by calmodulin inhibitors such as trifluoperazine
and phenytoin. The rate limiting steps in the biosynthesis of serotonin and noradrenaline are
catalysed by tryptophan-5-mono-oxygenaseand
tyrosine-3-mono-oxygenaserespectively;both these
enzymes are activated by calmodulin dependent
protein kinase [76]. It has also been shown that
calmodulin can regulate calcium stimulated phosphorylation of several proteins in synapse structures [75]. Calmodulin appears to play a part in
dopaminergic activity in the brain and could be
interacting with guanyl nucleotides at various sites
on the dopamine sensitive adenylate cyclase [77].
The available evidence, therefore, clearly indicates
that calmodulin is involved in neurosecretion and
in neuronal function.
Calmodulin and cellular proliferation
An increase in cytosolic calcium is a control signal
for the initiation of DNA synthesis [78] and it
may be that calmodulin is involved in this event
[79]. During cell division it has been shown by
immunofiuorescence that calmodulin localizes on
the cellular spindle, which itself is composed of
microtubules, and there is evidence that calmodulin is important in the calcium dependent assembly
and disassembly of microtubules [80]. Calmodulin
is increase.d in transformed cells [81,82] and levels
of calmodulin have been shown to be elevated in
hepatomp and in regenerating liver; in addition,
calmodulin can stimulate DNA synthesis in isolated
liver cells [83-861. Furthermore, calmodulin
antagonists, particularly when present at the GI-S
transition phase, will inhibit cell cycling [82,87].
Our own studies, using a number of calmodulin
antagonists, have indicated that calmodulin may
be important in agonist-stimulated DNA synthesis
in lymphocytes (see Fig. 4) [88].
One of the possible ways in which calciumcalmodulin could regulate DNA synthesis is by
activation of a protein kinase. Iwasa et al. [89]
have shown effects of calmodulin on histone phosphorylation and, hence, possibly on gene expression. Calcium-calmodulin itself also controls the
activity of ornithine decarboxylase ; inhibition of
this enzyme stops cell division through the blocking
of polyamine synthesis [90]. One of the major
$ 1
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I
0
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Y
'
0 '10-6
10-5
\
10-4
Drug concentration (mol/l)
FIG. 4. Effect of W7 o n phytohaemagglutininstimulated [ 3H]thymidine incorporation into peripheral blood lymphocytes. Results are means k SEM
of triplicate determinations from a single representative experiment. [3H]Thymidine uptake in
the absence of W7 is expressed as 100%. W7 caused
a dose-dependent inhibition of [3H]thymidine
uptake in to stimulated lymphocytes .
characteristics of neoplastic cells compared with
normal cells is their ability to proliferate normally
lin low calcium medium [91]. Some clues now exist
as to how tumour cells may lose this calcium
requirement for division. A tyrosine phosphokinase activity found at the cytoplasmic face of
cell membranes is associated with the oncogene
product of a number of classes of tumour viruses
[89], and it has been reported that calmodulin
itself mediates the ability of this tyrosine phosphokinase activity to promote cell proliferation.
MacManus [92] has described a calcium binding
protein of molecular weight 11 500 daltons, called
oncomodulin, which appears to be specific for
cancer cells. This protein has many of the properties of 'EF hand' proteins and is able t o activate
otherwise calmodulin dependent enzymes such as
cyclic nucleatide phosphodiesterase. Since this
protein may activate at lower calcium concentration than calmodulin, it may not depend on raising
the intracellular concentration of calcium for its
activity. The role of calmodulin in DNA synthesis
and cellular proliferation may be important in the
neoplastic process and might, therefore, have
therapeutic implications for cancer.
non-muscle cells, calcium's effect is probably
mediated by calmodulin. Contractile processes in
smooth muscle and in non-muscle cells are believed
to be myosin-linked and regulated by calcium
dependent myosin light-chain kinase. The enzyme
consists of two proteins, a large polypeptide and a
small regulatory protein which binds in the
presence of calcium to the larger protein and
which is identical with calmodulin. The protein
phosphorylation produced by the kinase ultimately
catalyses the hydrolysis of ATP to release energy
for muscle contraction. A similar mechanism presumably operates for non-muscle contractile proteins in the regulation of cellular motility [93].
Interestingly, calmodulin appears t o be involved
not only in muscle contraction, but also in providing the energy necessary for this process. This is
probably true of both skeletal and smooth muscle.
It has been shown that calmodulin is the delta
subunit of phosphorylase kinase and confers
calcium sensitivity on the enzyme; hence, glycogen
is broken down, providing glucose for energy
metabolism. Complementary to this, calmodulin
activates glycogen synthase kinase which phosphorylates and inactivates glycogen synthase [94]. In
addition, levels of glucose 1,6-bisphosphate, which
is a key regulator of several enzymes of carbohydrate metabolism, may be partially controlled
by calmodulin; the phosphatase which breaks
down glucose 1,6-bisphosphate appears to be
stimulated by calmodulin [95]. Interestingly it has
also been reported that one of the enzymes in the
tricarboxylic acid (Krebs) cycle, succinate dehydrogenase, is regulated by calcium [96]. Thus the
suppression of glycogen synthesis, the stimulation
of glycogen breakdown and the intermediary
metabolism of glucose are, in part, regulated in a
co-ordinated fashion by calcium and calmodulin.
These events, in turn, provide the energy for the
contractile process, which is itself modulated by
calmodulin.
Calmodulin may also have a role in prostaglandin
metabolism; it activates phospholipase A2, leading
to the synthesis in platelets of PGHz and thromboxane. It is also possible that calmodulin may
enhance the activity of cyclo-oxygenase. In addition, there is evidence that calmodulin may be
involved in the synthesis of prostacyclin by the
vascular endothelium. The influence of calmodulin
on prostaglandin metabolism obviously has relevance to the role of platelets and the vascular
endothelium in haemostasis [97,98].
Calmodulin, cellular contractile processes and
iutracellular metabolism
Calmodulin and disease
Troponin C is the major calcium receptor in striated
muscle but in smooth muscle and the filaments of
The studies described in this Review clearly not
only have implications for normal cell function
Gdmodulin and cell function
505
but also potentially for abnormalities that occur
Another condition which is the subject of
in disease. The central role of calmodulin as a
intensive investigation at the moment is essential
mediator of the intracellular calcium signal implies hypertension. There is increasing evidence that
that major abnormalities in structure or expression membrane abnormalities occur in various cell
of calmodulin are likely to be lethal. However, the types in hypertension. Decreased calcium binding
number of receptor proteins and enzymes for to cell membranes and increased cellular calcium
calmodulin and the number of calmodulin binding have been found in essential hypertension in man
proteins of, as yet, unidentified function, make it
as well as in experimental hypertension in animals.
possible that abnormalities might exist amongst Whether these abnormalities in intracellular calcium
these proteins in disease. If this is the case, then are a consequence of extracellular factors affecting
secondary alterations in intracellular calcium or
the sodium pump or intrinsic membrane abnorcalmodulin concentration may occur.
malities has yet to be resolved [105]. Whether
In this connection there are diseases in which calmodulin, through its activation of myosin lightabnormalities of intracellular calcium have been
chain kinase and its effects on smooth muscle
described. Thus calcium levels are reported to be
contraction, is involved in the genesis of hyperelevated in the cells of patients with cystic fibrosis. tension is speculative. It would obviously be of
Reduced activity of the plasma membrane CaZ+/ some interest to know whether intracellular
MgZ'-ATPase in erythrocytes and fibroblasts of
immunoassayable calmodulin and calmodulin
patients with cystic fibrosis have been observed activity is altered in essential hypertension.
and this enzyme is calmodulin activated. Gnegy
The explosion of knowledge in the last decade
et al. [99] have measured calmodulin in cultured
concerning the role of calcium in regulating cell
skin fibroblasts from patients with cystic fibrosis function and the recognition that many calcium
and have reported elevated levels. Such observa- dependent processes are mediated by the calcium
tions are clearly interesting in the light of the
binding protein, calmodulin, seems likely to have
involvement of calmodulin in the secretory process. important pharmacological, therapeutic and clinical
One very important area of interest is the regu- implications.
lation of cell proliferation by calcium and by
cyclic AMP. As has been stated above, calmodulin
levels are elevated in transformed cells and in re- Acknowledgments
generating and neoplastic liver cells. Furthermore,
calmodulin antagonists, when present at the criti- S.T. is a Wellcome Trust Senior Lecturer. We are
cal G1-S transition phase, can prevent cell cycling. grateful to the Wellcome Trust, MRC, SERC and
Yorkshire Cancer Research Campaign for financial
The implications of these findings are, at present,
support.
Table 1 is reproduced with permission
unclear but it is of considerable interest that in
psoriasis, a proliferative disease of the skin, calmo- from a paper by Dr W. L. West in Federation
dulin levels have been reported to be grossly Proceedings (1982), vol. 41. Table 2 is adapted
elevated compared with those of uninvolved skin and reproduced with permission from a paper by
and normal controls [loo]. Another condition in Dr W. Y. Cheung in Federation Proceedings (1982),
which intracellular levels of calmodulin have been vol. 41. Fig. 1 is reproduced with permission from
reported to be increased is in experimental dia- an article by Dr W. Y. Cheung in Scientific Ameribetes; Morley et al. [loll showed that immuno- can (June, 1982). Fig. 3 is reproduced with perassayable calmodulin was increased in several mission from a paper by Ollis et al. (1983) in the
tissues in diabetic mice. The implications of these Journal of Endocrinology (In press).
findings have yet to be established.
In addition to the three conditions described
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