calmodulin-ticb10(322)

322 0962-8924/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. trends in CELL BIOLOGY (Vol. 10) August 2000PII: S0962-8924(00)01800-6

Calcium (as Ca21) is an element that is crucial fornumerous biological functions. In many organisms,the vast majority of Ca21 is complexed with phos-phates to form exo- or endoskeletons that not onlyserve as structural scaffolds but also buffer the levelsof Ca21 within extracellular fluids at ~1023 M. Bycontrast, the resting concentrations of intracellularfree Ca21 (~1027 M) is 104 times lower than that out-side cells, providing the potential for the ready import of Ca21 into cells, where it can act as a secondmessenger.

Various extracellular stimuli promote the move-ment of Ca21 either from outside the cell (viaplasma-membrane Ca21 channels) or from intracellularstores into the intracellular milieu (Fig. 1a). TheCa21 is released in elemental aliquots called sparks,puffs or waves depending on the extent of the intra-cellular area covered. This free Ca21 is only brieflyavailable to act as a cellular signal, however, becauseCa21-binding proteins and Ca21 pumps immediatelycombine to sequester and transport it to intracellularstorage sites or outside the cell.

The short pulses of Ca21 exert specific changes incellular function depending on their route of entryinto the cell, their local sites of action and, finally, bytheir pattern of modulation. The particular mem-brane channel or intracellular receptor responsiblefor the release of Ca21 exerts considerable influenceon the eventual effects of the Ca21 signal1. The modeof cellular entry also influences the site of action of

the Ca21 signal. Hence, separate intracellular loci ororganelles are potentially distinct compartments oflocalized Ca21 signalling2 (Fig. 1a). Therefore, Ca21

signals in the nucleus exert different effects fromthose generated in the cytoplasm or near the plasmamembrane of the same cell3. Additionally, themodulation of the amplitude or frequency of Ca21

spikes (AM and FM, respectively) encodes important signalling information4. This has recently been illustrated for cases in which an optimal frequencyof intracellular Ca21 oscillations is important for theexpression of different genes5.

Calcium-regulated proteins: calmodulinHow do Ca21 signals produce changes in cell func-

tion? The information encoded in transient Ca21

signals is deciphered by various intracellular Ca21-binding proteins that convert the signals into a widevariety of biochemical changes. Some of these proteins, such as protein kinase C, bind to Ca21 andare directly regulated in a Ca21-dependent manner.Other Ca21-binding proteins, however, are inter-mediaries that couple the Ca21 signals to biochemicaland cellular changes (Fig. 1b). Among this lattergroup are a family of proteins that is distinguishedby a structural motif known as the EF hand. An EFhand consists of an N-terminal helix (the E helix)immediately followed by a centrally located, Ca21-coordinating loop and a C-terminal helix (the F helix). The three-dimensional arrangement ofthese domains is reminiscent of the thumb, indexand middle fingers of a hand, hence the name EFhand.

These proteins respond to Ca21 in one of two ways(Fig. 1b). One group (e.g. parvalbumin and calbindin)do not undergo a significant change in confor-mation on binding Ca21 and function as Ca21 buffersor Ca21 transporters. The second group, the Ca21

sensors, undergo a Ca21-induced change in confor-mation6. The most prominent examples of sensorsinclude troponin C (a protein dedicated to regu-lating striated-muscle contraction), the multifunc-tional Ca21 transducer calmodulin (CaM), the S100family of proteins and, most recently, the neuronalmyristoylated proteins such as recoverin7.

The molecular and cellular mechanisms under-lying the ability of a majority of the Ca21-sensor proteins to integrate Ca21 signals into specific cellularresponses are not clearly understood. Much of whatwe do know about the mechanisms that the sensorproteins use to transduce Ca21 signals is based on information gained from CaM, probably the mostintensively studied member of the EF-hand familyof sensors. In the remainder of this article, CaM willtherefore serve as a model or prototype for other potential Ca21 transducers. A review of some of themechanisms responsible for regulating CaM at thesubcellular and molecular levels might reveal valuableclues as to how Ca21-sensor proteins convert Ca21

signals into cellular function.CaM is expressed in all eukaryotic cells where it

participates in signalling pathways that regulatemany crucial processes such as growth, proliferationand movement. It is relatively small (vertebrate CaM

Calmodulin: aprototypical

calcium sensorDavid Chin and Anthony R. Means

Calmodulin is the best studied and prototypical example of the

EF-hand family of Ca21-sensing proteins. Changes in

intracellular Ca21 concentration regulate calmodulin in three

distinct ways. First, at the cellular level, by directing its

subcellular distribution. Second, at the molecular level, by

promoting different modes of association with many target

proteins. Third, by directing a variety of conformational states in

calmodulin that result in target-specific activation. The

calmodulin-dependent regulation of protein kinases illustrates the

potential mechanisms by which Ca21-sensing proteins can

recognize and generate affinity and specificity for effectors in a

Ca21-dependent manner.

The authors are inthe Dept of

Pharmacologyand Cancer

Biology, DukeUniversity Medical

Center, Durham,NC 27710, USA.

E-mail: [email protected];

[email protected]

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TCB 08/00 paste-up 30/6/00 8:54 am Page 322

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trends in CELL BIOLOGY (Vol. 10) August 2000 323

has 148 residues), evolutionarily highly conservedand comprises four EF hands. The first two EFhands combine to form a globular N-terminal domain that is separated by a short flexible linker froma highly homologous C-terminal domain consisting ofEF hands 3 and 4 (Fig. 2).

Ca21 sensors must be able to detect and respond to a biologically relevant range of intracellular freeCa21 concentrations. CaM fits this profile as itsaffinity for Ca21 (Kd 5 5 3 10

27 M to 5 3 1026 M) fallswithin the range of intracellular Ca21 concentrationsexhibited by most cells (1027 M to 1026 M). However,it has additional discrimination for Ca21, as the C-terminal pair of EF hands has a three- to fivefoldhigher affinity for Ca21 than the N-terminal pair ofsites. By contrast, many Ca21-binding proteins witha considerably higher affinity (Kd ,10

27 M) act asbuffers by sequestering excess free Ca21, whereasCa21-binding proteins with a considerably loweraffinity (Kd .10

25 M) could not act as sensors because they are unable to detect the range ofchanges in intracellular free Ca21 concentrationsthat normally occur in cells.

The two domains of CaM adopt differentconformations in the absence or presence of Ca21

(Fig. 2). In the absence of Ca21, the N-terminal domain of the apo-CaM molecule adopts a closedconformation in which the helices in both EFhands are packed together. By contrast, still in the absence of Ca21, the C-terminal domain of apo-CaMadopts a semiopen conformation in which a par-tially exposed hydrophobic patch is accessible tosolvent. This might allow the C-terminal domain ofCaM to interact with some target proteins at restinglevels of intracellular free Ca21 (Ref. 8).

In the event of a transient rise in Ca21, the Ca21

ion is coordinated in each Ca21-binding loop ofCa21CaM by seven, primarily carboxylate, ligands.The binding of Ca21 leads to substantial alterationsin the interhelical angles within the EF hands ineach domain and dramatically changes the two domains of CaM to produce more open confor-mations (Fig. 2). These structural rearrangements inCaM result in the concerted exposure of hydrophobicgroups in a methionine-rich crevice of each domainthat is distinct from the Ca21-binding loops. The ex-posure to solvent of these hydrophobic residues isakin to a Ca21-controlled unfolding of CaM and un-leashes considerable free energy. It is this capacity toconvert the Ca21-binding event into biochemical en-ergy that characterizes the Ca21-sensor proteins andis the basis of their ability to transduce Ca21 signals.

Calmodulin: location, mobility and translocationIs CaM regulated at the subcellular level, and how

is this related to Ca21 signalling? The concentrationand location of CaM do appear to play an importantrole in regulating its biological activity. CaM consti-tutes at least 0.1% of the total protein present incells (1026 M 1025 M) and is expressed at even

trends in Cell Biology

Nucleus

Ca2+ sensors Effectors

(a)

(b)

Ca2+

Ca2+

Ca2+ Ca2+

Ca2+

Ca2+

Amplitudemodulated (AM)

Frequencymodulated (FM)

Ca2+ buffers and transporters

FIGURE 1

(a) Sources of intracellular Ca21 signals. Ca21 enters cells viaextracellular plasma-membrane receptors or from intracellularstores, producing transient local or global changes in itsdistribution. The Ca21 oscillations are modulated in theiramplitudes (AM) or frequencies (FM) and are therefore capableof conveying signalling information in complex ways. (b) EF-hand Ca21-binding proteins are classified as buffers/transportersand sensors. The Ca21 sensors change conformation on bindingCa21 and transduce changes in cell function by regulatingdownstream effectors.


(a) (b)

FIGURE 2

The Ca21-regulated conformational change in calmodulin. Themain chain structure of Ca21-free (apo) CaM (a) andCa214CaM (b) are shown in red with their respective N-terminal domains on top. Methionine side chains are shownin purple to denote the location of potential hydrophobicpockets in each of the two domains. Ca21 binding produceslarge changes in the helices in both domains, resulting in theexposure of several hydrophobic residues.


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324 trends in CELL BIOLOGY (Vol. 10) August 2000

higher levels in rapidly growing cells, especiallythose undergoing cell division and differentiation.The local intracellular availability of CaM is likely tobe biologically significant because various CaM-dependent effectors are regulated over a wide rangeof free CaM concentrations (10212 M 1026 M).

Recent studies of CaM tagged with green-fluor-escent protein (GFP) show that CaM is found through-out the cytosol and nucleus in HeLa cells, although it is concentrated around the mitotic apparatus in cells undergoing mitosis (Fig. 3), especially around the centrioles and the cytoplasmic furrow during

cytokinesis9. Other fluorescently labelled CaMmolecules have provided information on its cellularmobility and location. Experiments with serum-deprived Swiss 3T3 fibroblasts first indicated thatthe majority of CaM was freely diffusible, but theCaM was then immobilized in response to stimulationby serum10. However, other studies on unstimulatedsmooth-muscle cells showed that most CaM isbound, possibly to Ca21-independent binding proteins, at resting concentrations of free Ca21.

In response to a rise in Ca21, CaM exhibits a com-plex pattern of cellular localization, including a significant redistribution from the cytosol to the nu-cleus11. This stimulus-dependent movement of CaMto the nucleus and its activation has also been de-tected in neurons12. CaM has also been seen to accumulate slowly in the nucleus of hormone-treated pancreatic acinar cells13. The mechanism oftranslocation in smooth-muscle cells apparently involves the passive diffusion of CaM into the nucleus, where it might associate with targets in aCa21-dependent manner14.

The synchronization between CaM and Ca21 signalsis also being explored. A direct relationship betweena rise in the levels of intracellular free Ca21 and theCa21-dependent activation of CaM was first observedduring a response to wound healing in fibroblasts15.Ca21 oscillations in the secretory granules of pan-creatic acinar cells have also been correlated with oscillations in the local concentration of CaM13.Recent studies on sea-urchin eggs undergoing mito-sis, however, indicate that the spatial patterns ofCa21 are different from those of Ca21-activatedCaM16. Interestingly, the Ca21-dependent activation

of CaM exhibits a heterogenous distri-bution pattern in the cells that havebeen studied, indicating the presence ofdiscrete populations of CaM. Thesestudies emphasize the importance oftemporal and spatial relationships be-tween Ca21 signals and CaM function.

Calmodulin: regulation of effectorsAn important obstacle to studies on

many Ca21-sensor proteins is the prob-lem of identifying downstream targets.Biochemical and genetic approacheshave recently started to identify targetsfor some of the S100 class of proteins17

and also for members of the myristoyl-ated Ca21 sensors, such as frequenin18.By contrast, CaM has been known forsome time to regulate several classes ofproteins and enzymes in a Ca21-depend-ent manner. The binding of target pro-teins by CaM raises the affinity of CaM for Ca21 by approximately tenfold19 andsensitizes the CaMeffector complex tochanges in Ca21 concentrations. Interest-ingly, many of the most highly charac-terized effectors (e.g. the CaM-dependent adenylyl cyclases, phospho-diesterases, protein kinases and the proteinphosphatase calcineurin) are directly or


(a) (b) (c)

FIGURE 3

Distribution of calmodulin and tubulin in the mitotic spindle. (a) A spindle visualized with an anticalmodulin antibody.

(b) A Nomarski image of a mitotic spindle formed by incubationin a Xenopus extract. (c) A spindle visualized with

an antitubulin antibody.

TABLE I SOME RECENT EXAMPLES OF CALMODULIN-REGULATED PROTEINS

Protein Comments Ref.

Cabin1 Thymocyte transcriptional regulator 45

NAP-22 Neuronal substrate of protein kinase C 46

Striatin Neuronal, associates with phosphatase 2A 47

CAP-19 Neuronal, IQ calmodulin-binding motif 48

EGF-receptor Human, CaM binds at juxtamembrane 49

MLC phosphatase (targeting subunit) Participant in muscle contraction/relaxation 50

Connexin 32 Located at gap junctions 51

ChURP Located in the nucleus 52

High MW protein Cardiac muscle phosphoprotein 53

Beta-2-glycoprotein Membrane-associated protein in kidney 54

Retinal proteins Involved in neuronal synaptic transmission 55

Extracellular proteins Located in animal body fluids 56

Sperm proteins Spermatocyte, acrosome reaction 57

Plant proteins Plasma membrane transporter 58

Yeast proteins Involved in cell growth and division 59

Phosphatidylinositol 3-kinase Component in receptor signalling 60


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indirectly involved in protein phosphorylation.CaM also regulates the activities of the plasma-membrane Ca21 pump, various ion channels, theryanodine receptor and isoforms of the inositol(1,4,5)-trisphosphate receptor. The list of CaM tar-gets is extensive and constantly growing (Table 1).

Are there differences in the mechanisms by whichCaM and other Ca21 transducers regulate their targets? CaM performs a variety of roles, and CaM-binding proteins can be categorized into at least sixclasses based on their modes of regulation in thepresence and absence of Ca21 (Fig. 4). One group ofeffectors, which we designate class A, binds essen-tially irreversibly to CaM irrespective of Ca21. CaMis thus more appropriately considered a subunit ofthese proteins. One example is phosphorylase kinase, an enzyme that requires denaturing condi-tions to dissociate CaM but is activated in the presence of Ca21. Members of a second group of ef-fectors (class B) bind to CaM in the absence of Ca21

(i.e. to the apo-CaM form) but dissociate reversiblyin the presence of Ca21 (Ref. 20). Examples includeproteins such as neuromodulin and neurogranin,which might serve as intracellular reservoirs forCaM at resting concentrations of Ca21 but liberateCa21-activated CaM in response to a transient Ca21

signal.A third group of effectors (class C) includes

smooth-muscle myosin-light-chain kinase (MLCK)and calcineurin. These class-C effectors form low-affinity, inactive complexes with CaM at low con-centrations of Ca21, when CaM is unoccupied orpartially occupied by Ca21 [,2 (mole Ca21) (moleCaM)21]. At high concentrations of Ca21, these tar-gets engage in a high-affinity complex and are acti-vated by CaM21,22. A fourth class of proteins (class D)binds to CaM in the presence of Ca21, but, in thiscase, CaM inhibits their function. This group in-cludes enzymes such as select members of the G-protein-receptor kinases23, as well as the inositol(1,4,5)-trisphosphate receptor type 124.

A fifth group of effectors (class E), such as theCaM-dependent protein kinases I, II and IV, exhibitmore conventional behaviour and are activated byCa21CaM. The class-E targets also exhibit an access-ory form of regulation in which CaM binding pro-motes their regulation (specifically via phosphoryl-ation) by another CaM-regulated kinase (i.e. aCaM-kinase kinase ), which we designate class F. Inthe specific case of the multimeric CaM kinase II,both the substrate and the catalytic subunits requireCaM binding to promote intermolecular autophos-phorylation25. This novel case, in which one CaM-dependent protein (class E) is directly regulated byanother CaM-dependent protein (class F), demon-strates the convergence of different CaM-regulatedpathways and is indicative of CaM-signalling cascades.

The observation that CaM regulates a specific setof proteins yet engages in different types of Ca21-dependent interactions implies that CaM and its targets both exhibit certain complementary featuresthat enable CaM recognition but possess other as-pects that still allow CaM to discriminate between

various classes of effectors. As CaM, like many Ca21

sensors, is a relatively small protein, it must there-fore use multiple interaction surfaces to accomplishthese ends. These interaction sites enable CaM toconvert the energy provided by Ca21 binding intoeffector regulation.

Calmodulineffector coupling: binding andactivation

The Ca21-controlled exposure of hydrophobicgroups in the two domains of CaM releases a con-siderable amount of biochemical energy, which istransduced into two separable effects: a change inthe affinity of CaM for the effector and/or an al-teration in the effectors function. Studies focusing onone group of CaM-regulated enzymes in particular,the CaM-dependent protein kinases, have providedimportant insights into some of the mechanismsunderlying these phenomena. A short peptide of~20 residues that is responsible for bindingCa21CaM, designated a CaM-binding domain, hasbeen identified in many CaM-regulated proteins(Fig. 5a) and in other types of CaM-binding pro-teins26. The crystal structure of CaM kinase I revealsthat the CaM-binding domain directly interactswith and sterically obstructs the putative substrate-binding sites of the inactive enzyme27. Furthermore,the N-terminal part of the CaM-binding sequenceloops away from the enzyme, exposing the hydrophobic side chain of Trp303 to solvent andproviding potential access for Ca21CaM to bind(Fig. 5b). This proposal is supported by experimentsshowing that the mutation of Trp303 to Ser in CaMkinase I significantly lowered the apparent affinityof CaM kinase I for Ca21CaM28.


CaM-A+Low [Ca2+] High [Ca2+]

apoCaM CaM-A

+ A

CaMapoCaM CaM-B

+ B - B

+ B+ C

- C+ D

- D

+ E

- E

+ F

- F

CaM-C+CaM CaM-C

CaM-D-CaM

CaM-E+

CaM-E++

CaM-F+CaM

FIGURE 4

Ca21-dependent functions of various classes of calmodulin-binding (CaM-binding)proteins. CaM and various classes of targets exist in free or bound states. Targetclasses A, B and C are associated with CaM or Ca21-free CaM (apo-CaM) at low(resting) intracellular free Ca21 concentrations (red). When Ca21 concentrations arehigh (green), class B dissociates from CaM, classes D, E and F associate with CaM, classes A, C, E and F are activated by CaM (1), and class D is inactivated by CaM (2).


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A homologous hydrophobic residue is conservedin other CaM kinases (Fig. 5a). In the absence of detailed information on complexes between CaMand its intact effectors, spectroscopic and crystallo-graphic studies of Ca21CaM complexed with pep-tides corresponding to the CaM-binding domains offour CaM kinases including CaM kinase I show ineach case that this conserved hydrophobic residueinteracts exclusively with the methionine-rich hydrophobic pocket in the C-terminal domain ofCa21CaM2932 (Fig. 5c). Recently determined three-dimensional structures of Ca21CaM bound to peptides from the plasma membrane Ca21ATPasepump and a CaM-kinase kinase also reveal additionalmodes of interaction between CaM and these otherCaM-binding peptides33,34. These peptide studies indicate that the C-terminal domain of Ca21CaMmight confer binding energy on the intact enzymes.Indeed, this appears to be the case because comple-mentary mutagenesis experiments on the Metresidues of CaM showed that an evolutionarily in-variant Met124 in the C-terminal domain ofCa21CaM that contacts the conserved hydrophobicresidues in several CaM-binding peptides (Fig. 5c) isnecessary for high-affinity binding and activation ofCaM kinase I as well as for three other CaM-dependentprotein kinases35,36.

In contrast to the C-terminal domain ofCa21CaM, residues in the hydrophobic pocket ofthe N-terminal domain of Ca21CaM perform vary-ing functions with different CaM-dependent kinases.The results from the crystallographic studies showthat hydrophobic residues in the N-terminal do-main of Ca21CaM mainly interact with the C-terminal part of the CaM-binding peptides of smooth-muscle MLCK and CaM kinase II, respectively(Fig. 5a). Progressive C-terminal deletions andchimeric substitutions in the CaM-binding domainof smooth-muscle MLCK showed that the C-terminalhalf of the CaM-binding domains of these enzymestrends in Cell Biology

S K K Q A F N A T A V V R H M R KR K L K G A I L T T M L A T R N F S

R K W

W

Q K T G H A V R A I G R L S SR R W K K N F I A V S A A N R F K K

CaM C-domain CaM N-domain

I L W F R G L N R I Q T Q I R V V NP S W T T V I L V K S M L R K R S F

Interaction site on calmodulin(a)

(b)

(c)

CaM KICaM KIIsmMLCKskMLCKCa2+ATPaseCaM KK

Main chain of Ca2+ - CAM

CAM-binding domainof smMLCK

Conserved tryptophan

Met124 residue

4

FIGURE 5

(a) Alignment of amino acid sequences from selectedcalmodulin-binding (CaM-binding) domains. The enzymes areCaM kinase I (CaM KI), CaM kinase II (CaM KII), smooth- andskeletal-muscle myosin-light-chain kinases (smMLCK andskMLCK), the plasma-membrane Ca21-pumpATPase (Ca21

ATPase) and CaM-kinase kinase (CaM KK). In most cases, ahydrophobic residue (red) from the corresponding peptidesinteracts with the C-terminal domain of Ca21CaM. The boxedresidue in CaM KI is Trp303. The N-terminal domain ofCa21CaM interacts primarily with the C-terminal half of thepeptides. (For additional information on CaM binding domains,see http://calcium.oci.utoronto.ca/) (b) Two crystalstructures showing the main chain of Ca214CaM on the left andCaM kinase I on the right. The N-terminal domain of CaM andthe ATP-binding lobe of CaM kinase I are both positioned on top,with helices red and sheets green. The Trp303 side chain fromthe CaM-binding domain of CaM kinase I (black) extends awayfrom the enzyme in the direction of CaM. (c) Crystal structureshowing the main chain of Ca214CaM (white) in complex withthe helical CaM-binding domain of smooth-muscle myosin-light-chain kinase (green). CaM wraps around the helix so that theconserved Trp of the peptide makes contact with Met124 (red)in the C-terminal domain of CaM.


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are required for Ca21CaM-dependent acti-vation37,38. Furthermore, deletion studies of CaM kinase I show that the C-terminal portion of itsCaM-binding sequence confers high affinity forCa21CaM, as well as CaM-dependent activity39.These results complement those from experimentson CaM showing that the hydrophobic pocket in itsN-terminal domain generates a high-affinity com-plex with CaM kinase II, activates smooth-muscleMLCK and combines the functions of high-affinitybinding and activation of CaM kinase I35,40.

In comparison with unbound Ca21CaM, theCa21CaMpeptide complexes exhibit a dramaticcontraction enabling CaM to wrap around and se-quester the helical CaM-binding peptides (Fig. 5c).Experiments on hydrophilic residues of CaM showthat charged and polar residues are also required toactivate the smooth-muscle MLCK by promotingthe accessibility of substrate to this particular en-zyme. Surprisingly, the hydrophilic residues onCaM responsible for this effect are originally separated from each other on both domains of freeCa21CaM by more than 50 but then form a stable latch less than 5 apart in the Ca21CaMpeptide complex41. These polar groups do notcontact the CaM-binding peptide directly, so theymight exert their effects on the intact protein kinaseby interacting with an area distinct from its CaM-binding domain. Indeed neutron- and X-ray-scattering studies on the kinase domain of skeletal-muscle MLCK indicate that the CaM-binding domain is displaced to one side of the enzyme bythe binding of Ca21CaM42. This event could exposethe substrate-binding site of the enzyme and indicateshow CaM might remove an inhibitory CaM-bindingdomain away from the kinase domain, thus leadingto enzyme activation.

The preceding mechanistic studies show that theregulation of enzymes by Ca21CaM is a highly or-dered, cooperative and complementary process thatcontributes to both the affinity and specificity fortargets. Another surprising outcome is the discoverythat the structures of Ca21CaMpeptide complexesare relevant to their corresponding enzymes. However,in addition to the obvious limitation in the use ofpeptides to study mechanisms of enzyme acti-vation, there are mounting indications that peptidesmight not be entirely suitable for studying otherfunctions of the full-length target protein. For example, Ca21 has a considerably higher affinity forthe CaMMLCK-peptide complex than for the cor-responding CaMMLCK-enzyme complex19. Also,mutations in the CaM-binding domain of smooth-muscle MLCK have a significantly greater effect onCaM binding and activation than the same changeswithin the context of the corresponding CaM-bindingpeptide40. Differences in the Ca21-dependent inter-action of CaM with either the CaM-binding peptideof skeletal-muscle MLCK or the intact enzyme havealso been observed by small-angle scattering43. Oneexplanation for the adaptability and the higheraffinity exhibited by the shorter CaM-binding do-mains is the conformational flexibility inherent inisolated peptides. By contrast, the relatively fixed

conformation of the intact, folded enzymes restrictstheir ability to adapt similarly to structural changeswithin their CaM-binding domains. It is helpful tobear these caveats in mind when peptides are usedto model effector function.

Perspective and conclusionThe extensive characterization of CaM provides a

useful precedent for less-well-understood Ca21 sen-sors. At the subcellular level, the spatial and temporalcoordination between Ca21, CaM and its effectorsare important for channelling all three componentsinto a productive signalling pathway. The ability ofCaM to integrate Ca21 signals into different cellularcontexts by migrating between various compart-ments further underscores this point. At the inter-molecular level, CaM uses different modes of Ca21-dependent interactions, which are responsible for generating high affinity as well as specificity fortargets. At the submolecular level, the Ca21-triggeredexposure of energy-donating groups on CaM is coupledto energy-accepting groups on its targets, leading tochanges in Ca21 binding by CaM as well as in thefunction of its effectors.

Finally, how are these levels of regulating CaM related to each other? It is likely that the mobility ofseparate pools of CaM derives from the different interactions between CaM and its targets. Therefore,some classes of proteins might anchor CaM to spe-cific cellular locations, depending on the stability ofa particular CaMeffector complex in the absence orpresence of a Ca21 signal. The affinity of such com-plexes is likely to be due to complementary interac-tions between sites on the target proteins and siteson CaM that change conformation in response toCa21. The Ca21-dependent interactions not only af-fect the affinity of the complex but also regulate theactivity of effectors. This apparent ability of aCaMeffector complex to decode Ca21 signals hasbeen exemplified in a recent study showing thatCaM participates in converting Ca21 oscillations intochanges in the autonomous enzymatic activity of atleast one target, CaM kinase II44. Additional studieson CaM will lead to a more-complete integration ofits levels of regulation. Meanwhile, it will be inter-esting to see whether any of the mechanisms exhib-ited by CaM will be relevant to other Ca21 sensors.

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Acknowledgements

Our researchcited in thisreview was

funded by NIHgrants HD-07503

and GM-33976to ARM. We

thank themembers of the

Meanslaboratory for

stimulatingdiscussions.


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