REVIEWS ~ C l l J M HAS BEEN known for some time to be critically important for activating smooth-muscle contraction, exerting its effect by stimulating the phosphorylation of myosin and the concomitant activation of the myosin Mg2"ATPase by actinL Myosin light-chain kirtle (MLCK),the enzyme responsible for myosin phospho~lation, requires Ca2" and calmodulin (CAM, the ubiquitous and multifunctionai Ca2÷-binding protein) for activity. Myosin lightchain phosphatases (MLCPs), which reverse the functional effects of myosin phosphorylation, have also been isolated from diverse smooth-muscle tissues (for example, see Refs 3, 4). The mechanism shown in Fig. 1 describes the molecular events involved in contraction and relaxation of smooth muscle. Stimulation (neuronal or hormonal) of the smoothmuscle cell results in an increase in the intracellular Ca2÷concentration, [Ca2']i, from approximately 140riM in the resting cell to 500-700nM ~ef. 5). Ca2+ enters the sarcoplasm from the extracellular space via voltage-gated or receptor-operated Caz* channels, or from the sarcoplasmic reticulum ($R) via lnositol 1,4,5-trisphosphate receptor/Ca2'-release channels or ryanodine receptor/Ca2÷.release channels. As a consequence of its elevated
The biochemical basis of the regulation of smooth-muscle contraction Bruce G. Allen and Michael P. Walsh The primary signal for smooth-muscle contraction is an increase in sarcoplasmic free Ca2* concentration ([Ca2*]~). This triggers activation of calmodulin-dependent myosin light-chain kinase, which catalyses myosin phosphorylation, thereby activating crossbridge cycling and the development of force or contraction of the muscle cell. Restoration of resting [Ca2*]~ deactivates the kinase; myosin is dephosphorylated by myosin light-chain phosphatase and the muscle relaxes. Recent evidence suggests that other signal-transduction pathways can modulate the contractile state of a smooth-muscle cell by affecting specific steps in the myosin phosphorylation-dephosphorylation mechanism. concentration, Ca2+ binds to CaM (four Ca2. ions per molecule of CAM), inducing a conformationai change that exposes hydrophobic sites for interaction with a number of target proteins, including MLCK. The resultant ternary complex, (Ca~')4-CaM-MLCK, representing the active form of MLCK,catalyses transfer
Relaxation 4Ca~* + CaM _
CaMM.C c 4Ca2"
) LCP ADP'~ "~ Myosin-P
AIP ~ ~ A D P
Contraction Rgure1 Myosin phosphorylation-dephosphorylationas the primary mechanism of regulation of smooth-muscle contraction. CaM, calmodulin; MLCK, myosin light-chain kinase; MLCP, myosinlight-chainphosphatase.Reproducedfrom Ref. 9 with permission. 362
of the terminal phosphoryl group of MgATP2" to Serl9 on each of the two 20kDa light chains of myosin (see below). This simple phosphorylation reactior,, triggers the cycling of myosin crossbridges along the actin filaments, with the development of force or contraction of the muscle. This process is reflected in vitro by activation of the myosin Mg2".ATPase activity by actln. Relaxation of the muscle follows the restoration of resting [Ca2"]j by extrusion of Ca2. from the cell by a sarcolemmal Ca2" pump or a Na'-Ca2÷ exchanger, or pumping of Ca2÷into the SR by a Ca2" pump in the SR membrane6. MLCKis then rapidly inactivated by the dissociation of CaM, and myosin is dephosphorylated by MLCP(s). The most obvious way of controlling smooth-muscle contraction, therefore, is by regulation of [Ca2*]i. There are a variety of extracellular signals that trigger an increase in [Ca2~]i,thereby eliciting a contractile response (for example, a~-adrenergic agonists and membranedepolarizing neurotransmitters), or that induce relaxation via a decrease in [Ca2~]j (for example, [5-adrenergic agonists, atrial natriuretic factor and nitric oxide). These aspects have been reviewed extensively elsewhere7 and will not be considered further in this article. Instead, we will concentrate primarily on the central importance © 1994,ElsevierScienceLid 0968-0004/94/$07.00
REVIEWS of myosin phosphorylation and dephosphorylation in smooth-muscle contraction (see Refs 8, 9).
Myosinphosphorylatlon The physiological substrate of MLCK, smooth-muscle myosin II, is a hexamer, composed of two heavy chains (-230 kDa each) and two pairs of light chains of 20 and 17kDa (LC20and LC17, Fig. 2). MLCK phosphorylates Serl9 of LC20, which allows smooth-muscle contraction. This phosphorylation occurs by a random mechanism, rather than by an ordered mechanism in which phosphorylation of the second head would require prior phosphorylation of the first head of all myosin molecules. Activation of myosin requires phosphorylation of only one head, consistent with numerous studies with demembranated muscle strips in which low levels of myosin phosphorylation were sufficient to induce maximal force development (see, for example, Ref. 11). Myosin il acts as an actin-activated Mg2÷-ATPase, the amino-terminal domains of the heavy chains containing an ATP-hydrolysis site and an actin-binding site. Unphosphorylated LC20 in myosin is thought to inhibit the actin-activated myosin Mg2'-ATPase. Phosphorylation of LC20 by MLCK induces a conformational change in the neck region of myosin, i.e. near the head-tail junction, so that the myosin heads become more mobile and extended from a constrained conformation; this results in activation (deinhibition) of the myosin Mg~'-ATPase. In support of this hypothesis, smooth-muscle S1, unlike intact myosin or heavy meromyosin 0tMM), is not regulated by phosphorylation. Both amino- and carboxy-terminal domains of LC20 are located at the head-tall junction of myosin. The
amino-terminal domain of smoothmuscle LC20is rich in basic amino acids. Replacement of LC20in smooth-muscle myosin by truncated forms of LC20indicated that the amino-terminal 12 residues could be removed without loss of phosphorylation dependence of actin-activated Mg2÷-ATPase activity. However, the actin-activated Mg2+ATPase activity of HMM containing LC20 beginning at Alal7 was independent of phosphorylation, suggesting that the sequence ]3Arg-Pro-Gln-Arg~ is essential for regulation TM. Carboxy-terminal
Head-tail junction Rod-like tail
02 LMM (110 nm)
HMM (45 nm)
Figure 2 Schematic representation of smooth-muscle myosin II. Myosin is composed of two heavy chains and two pairs of light chains, The heavychains (-230 kDa each) form an a-helical coiled-coil structure (the rod-like tail) and contribute most of the mass of the two globular heads, which also contain the light chuins [one of 20 kDa (LC2o)and one of 17 kDa (LC17) associated with each head]. LC2ocontains the site of phosphorylation by MLCK(indicated by P). Myosin can be divided into functional domains by limited proteolysis owing to the presenceof protease-sensitivesites in the hinge regionand at the head-tailjunction, Light meromyosin (LMM) representsthe region of interaction between myosin moleculesto form filaments, and heavymeromyosin(HMM)representsthe crossbridge. HMM can be cleaved to subfragments: $2 protrudes from the body of the thick filament and $1 (which represents a single globular head) contains the sites of ATP hydrolysis and actin binding. The recent determination of the high-resolution crystallographic structure of skeletal muscle myosin $1 indicates that the regulatory light chain (equivalentto LC20)is located at the end of $1 distal from the nucleotide-binding and actin-binding sites, i.e. near the head-tall junction.
truncations have shown that the carboxy.terminal helix in subdomaln 4 of LC20is essenticl for myosin regulation TM. Myosin containing a chimeric LC20composed of the phosphorylated aminoterminal half of smooth-muscle LC20 and the carboxy-terminal half of the skeletal-muscle regulatory light chain neither exhibits actin-activated Mg2*ATPase activity nor promotes the movement of actin filaments over an immobilized myosin substrate in the in vitro motility assay]4, Interactions between the carboxy-terminal domain of LC20 and the heavy chain, therefore, are primarily responsible for regulation by LC20. Kinetic studies have shown that phosphorylation most probably affects the step in the actomyosin ATPase cycle that involves the release of Pi from the myosin heads following the hydrolysis of ATP.
Regulationof MLCK As discussed above, the apoenzyme of MLCK is essentially inactive (<0.1% maximal activity), whereas the (Ca2÷)cCaM-MLCK complex represents
the fully active enzyme. Structural analysis and functional characterization of MLCK, proteolytic fragments of MLCK, synthetic peptides corresponding to putative functional domains, and deletion and site-specific mutants of the kinase have shed considerable light on the molecular mechanism of regulation of this enzyme. The domain structure of chicken-gizzard MLCK is depicted in Fig. 3. The enzyme has a catalytic domain, a CaM-binding site, an autoinhibitory (pseudosubstrate) site, an actin-binding site (which localizes the enzyme to the contractile machinery and accounts for its retention in permeabilized cell and tissue preparations) and an important regulatory site of phosphorylation by Ca2"-CaMdependent protein kinase II (CaMK II). The catalytic domain (Gly526-Trp773) begins with the consensus ATP-binding sequence, GxGxxGxl6K.Smooth-muscle B. G. Allen and M. P. Walsh are at the MRC Group in SignalTransduction,Departmentof Medical Biochemistry, Universityof Calgary, 3330 Hospital Dr. N.W.,Calgary,Alberta, CanadaT2N 4N1.
T,~ :L9- SEPTEMBER 1994 Regulatory domain /
GSGKFGxlsK / ) ATP-binding s i t ~ / / , /
K,~ ~ .
/A'A=oinhibitory domain \ SKDRMKKYMARRKWQKTGHAVRAIGR LS(~ CaM-binding domain Figure 3 Domain structure of myosin light-chain kinase (MLCK). The locations of the actin-binding domain, the ATP-bindingsite, the catalytic domain, the regulatory domain (composed of ovedappingautoinhibitory and calmodulin-bindingdomains), the site of phosphorylationby Ca2*-calmodulin-dependent protein kinase II (circled), and the unc-I and unc-II domains are shown. Numberingis basedon the deducedamino acid sequenceof chicken-gizzardMLCKis.
MLCKcontains two types of structural motif, each approximately 100 amino acid~ in len~h, referred to as unc-I and unc-ll, which are found in the unc-22 gene product (twitchin) of the nematode
/ ~ ' / /
Caenorhabditis elegans and in the giant skeletal-muscle protein titin. Smoothmuscle MLCKcontains one unc-I and three unc-ll motifs. These motifs are not found in MLCKsof mammalian skeletal
CaM-binding C site \ \
64 kDa(inactive; unaffected by Ca2+-CaM) ~ Trypsin
61 kDa (constitutively active)
Rgme4 The mechanism of activation of myosin light-chain kinase by Ca2+--calmodulinor tryptic digestion. The pseudosubstrate (autoinhibitory) domain (Ser787-Va1807) overlaps the calmodulin-bindingdomain (Ala796-Leu813).Reproducedfrom Ref. 9 with permission. 364
muscle or Dictyostelium discoideum, and their function is unlmown. Electrostatic interactions between acidic sidechalns within the catalytic core of MLCK (specifically Glu600, Glu681, AspT19, Glu723 and Asp734) and basic sidechains near Serl9 of LC20(specifically /u'gl6, Argl3, Lysl2, Lysll and Arg4) have been implicated in the enzymesubstrate interaction~6. The CaM-binding site (~a79~ Leu813), like that in many other CaM target proteins, has a predicted basic, amphiphilic ~-helical structure. A s~nthetic peptide corresponding to this domain binds to CaM in the presence of Ca2+ and inhibits CaM-mediated activation of MLCKby competing with the enzyme for CaM. The crystal and solution structures of CaM with bound peptide, corresponding to the CaMbinding domain of smooth and skeletal muscle MLCI(s, respectively, have been determined by X-ray crystalIography 17 and nuclear magnetic resonance spectroscopy~8. A particularly important regulatory region of MLCK- the autoinhibitory (or pseudosubstrate) domain - partially overlaps with the CaM-binding domain. The sequence of this domain (Ser787-ValS07) resembles the amino acid sequence around Serl9 of LC20, particularly with respect to the distribution of basic residues 16. On the basis of this sequence homolo~, Kemp and his colleagues 19 proposed the pseudosubstrate prototope hypothesis 0ntrasteric model), according to which the apoenzyme of MLCKis inactive because the polypeptide chain is folded so that the pseudosubstrate domain is bound to the myosin-binding site, thereby preventing access to the myosin su~ strate. Binding of (Ca2")4~aM to MLCK induces a conformational change that involves removal of the pseudosubstrate domain from the myosinbinding site, allowing myosin binding and phosphoryl transfer to occur. This mechanism is depicted schematically in Fig. 4, which also describes those properties of defined tryptic fragments of MLCK that support this activation mechanism. PartiM proteolysis with trypsin generates initially a 64 Id)a fragment ~hr283--Mg808), which contains the catal~ic site and the autoinhibitory domain, but has lost part of the CaMbinding site. This fragment is inactive because the myosin-binding site is
TIBS 19 - SEPTEMBER 1994
blocked by the pseudosubstrate domain, [ ] Ligand and cannot be activated by CaM since it Extracellular is incapable of binding (CaZ÷)4--CaM. space 1 Further digestion with trypsin to generate a 61 kJ)a fragment (Thr283-Lys779) Sarcolemma removes the autoinhibitory domain and relieves autoinhibition; this fragment is Sarcoplasm AA therefore constitutively active. The catalytic core of MLCKhas been modelled on the basis of the crystal structure of the homologous c.~uMPMLCP dependent protein Idnase, and the pseudosubstrate sequence fits easily Contraction • Myosin-P Myosin . Relaxation ~thin the myosin-binding site, indicating that We intrasteric model for regulation of MLCK by intramolecular MLCK competitive inhibition is structurally High[Ca2*]i (~ = CaM (~) • CaMK II /C) plausible ~e. This mechanism has been v challenged, however, on the basis of the MLCK-P properties of a variety of MLCKmutants (see, for example, Ref. 20) and it is likely that the autoinhibitory domain Rgure Mechanisms of altered Ca2* sensitivity of myosin phosphorylation. Ca2+ sensitization: e.z~tendsbeyond the region homologous extracellular signals such as at-adrenergic and muscarinic agonists may act through recepto LC20and can bind residues that intertors (R) that are coupled via a G protein (G) to phospholipase A2 (PLA2). Ligand occupancy act with LC20and residues that do not. of the receptor activates PLA2, which hydrolyses primarily phosphatidylcholine (PT.dCho)to Using the numbering of chicken-gizzard generate arachidonic acid (AA). AA inhibits myosin light-chain phosphatase (MLCP) and MLCKIs, Glu600, GIu603, GIu608, thereby unmasks myosin light-chain kinase (MLCK) activity, resulting in myosin phosphorylation and muscle contraction. Ca2+ desensitization: if [Ca2+] increases sufficiently to actiAsp609, GIu61I, GIn616, Glu644 and vate Ca2+-calmodulin~Jependentprotein kinase II (CaMK II), this kinase phospherylates Asp746 (all located within the catalytic MLCK, lowering its affinity for Ca2+-calmodulin, thereby inhibiting MLCK activity at a given core) have been implicated in binding [Ca2+]i and favouring relaxation of the muscle owing to myosin dephosphorylation by MLCP. to the autoinhibitory domain2°. Of these residues, only Glu600 and Glu644 appear to be important for binding to detachment of attached, dephos- ists. When [Ca2'], was clamped at pCa phorylated crossbridges. Subsequently, 6.3 (sufficient to induce only partial LC20(Ref, 20). examples of contractions occurring contraction), additional force was Other mechanismsof regulationof without phosphorylation of myosin, induced by phenylephrine. Further smooth-musclecontraction increases in myosin phosphorylation analysis of this response has led to the It is clear that [Ca2~]~is the primary without a change in [Ca2*]i or force suggestion of the following putative sigregulator of the contractile state of development, relaxation accompanied nal transduction pathway~2 (Fig. 5). smooth muscle, since it dictates the by a decrease in [CaZ']i but no dephos- Ligand occupancy of the c~L-adrenergic level of myosin phosphorylation. It is phorylation of myosin, and changes in receptor activates phospholipase A2 via also evident from a wide range of Ca2÷ sensitivity of contraction have a G protein, triggering phospholipid physiological studies, however, that this attracted much attention, and consider- hydrolysis (primarily phosphatidylmechanism cannot explain all the con- able efforts are being devoted to eluci- choline) to generate arachidonic acid. tractile properties of smooth muscle, dating the biochemical bases of such This lipid messenger, possibly assisted by a carrier protein, translocates to the and that other signal-transduction phenomena. myofilaments, where it inhibits MLCP pathways can impinge on specific steps (bound to the myosin filaments) by in the central pathway depicted in Ca2÷sensitizationand desensitization Fig. I. In particular, interest in this area Pharmacomechanical coupling mech- causing dissociation of the catalytic has been driven by several instances of anisms, i.e. mechanisms that do not subunit (37kDa) from the myosindissociation of force and myosin phos- involve a change in membrane poten- binding regulatory subunits (130 and phorylation, originally described by tial, can contract or relax smooth 20 kDa). MLCPinhibition unmasks basal Murphy and his colleagues2L They muscle strips without a change in MLCKactivity, which may reside within observed a correlation between myosin [Ca2"]v One way in which this can oc- MLCKitself or in a distinct kinase that phosphorylation and maximum velocity cur is via sensitization or desensitization also phosphorylates Serl9 of LC~0. of shortening as force was developed of myosin phosphorylation to Caz+. Consequently, myosin phosphorylation following stimulation of the muscle, but For example, vascular smooth-muscle occurs without a change in [Ca2÷]iand tension was maintained during pro- strips permeabilized with Staphylo- the muscle contracts. Ca2" desensitization of contraction longed stimulation while myosin was coccus aureus s-toxin retain receptor dephosphorylated (the 'latch' state). coupling, i.e. they contract in response can occur if the cell is exposed to high This may be due to a slow rate of to a~-adrenergic and muscarinic agon- [Ca2"]i. The most likely mechanism to
 Agonist Extracellular space
Rgure 6 Ca2+-independentcontraction induced by activation of Ca2+-independentprotein kinase C. A component of the at-adrenergic agonist-induced contraction of ferret-aortic smooth muscle may involve coupling of the receptor (R), probably via a heterotrimeric G protein (G), to the effector enzyme phosphatidylcholine-specific phospholipase C (PtdCho-PLC). Ligand occupancy of the receptor activates PtdCho-PLC, res,~,lting in hydrolysis of PtdCho to generate diacylglycerol (DG). In the absence of a [Ca2+]i transient, DG specifically activates Ca2÷-independe~tisoenzymes of PKC, in this instance PKC-e.Ca2÷-dependentand Ca2+-independentPKC isoenzymes can be activated directly by phorbol esters. Two possible mechanisms whereby activation of PKC-~ could lead to contraction are shown: direct phosphorylation of calponin (CAP) and indirect phosphorylation of caldesmon (CAD)via the mitogen-activated protein kinase (MAPK) pathway, both of which are predicted to alleviate inhibition of crossbridge cycling, thereby inducing a slow, sustained contraction. Also shown is an alternative pathway for the production of DG via phospholipase D (PtdCho-PLD)and phosphatidate phosphohydrolase (Pt.dOI-I.PHA). explain this effect involves activation of CaMK II (Ref. 23; Fig. 5). This kinase is less sensitive to Ca2" than is MLCK, hence the requirement for high [Ca~'], (probably approaching l p.M). CaMK Ii catalyses phosphorylation of MLCKat Ser815, resulting In an increase in the concentration of (Ca2")4-CaM required for half.maximal activity, i.e. a decrease in the Ca2" sensitivity of myosin phosphorylation.
Ca2°-Independeatcontraction Turnout-promoting phorbol esters, which are activators of protein kinase C ~KC), induce slowly developing, sustained contractions in smooth-muscle strips from a variety of sources. In some cases, phorbol ester treatment increases [Ca2"],, leading to myosin phosphorylation and contraction. In other cases, however, such as in ferretaortic smooth muscle, phorbol-esterinduced contractions do not involve a change in [Ca2"]~or myosin phosphorylation. Studies with permeabilized single cells have indicated that this response is Caz" independent and probably involves activation of a Ca2"-independent isoenzyme of PKC, specifically the e-isoenzyme~4.25.
Figure 6 depicts a putative signal. transduction pathway' to explain the Ca:'-independent contractile response to phorbol esters and physiological signals, such as a;adrenergic agonists, in some smooth.muscle systems. Ligand occupancy of appropriate receptors triggers activation, via G proteins, of phosphatidylcholine-specific phospholipase C or D, resulting in the production of diacylglycerol without a change in [£a2"]i.Consequently, specific activation of Ca-%independent PKC isoenzymes, in this case PKC-~, is achieved. To date, little is known about events downstream of PKC-~activation leading to the contractile response. However, as discussed below, the thinfilament-associated proteins calponin and caldesmon have been implicated as direct or indirect downsL--~am targets, respectively, of PKC.
Regulation of crossbddge cycling by thin-filament-associated proteins Potential regulatory proteins associated with actin filaments in smooth muscle have been identified and characterized in recent years. Although smooth-muscle thin filaments do not contain troponin, the trimeric complex
responsible for Ca2" regulation of skeletal- and cardiac-muscle contraction, the proteins calponin and caldesmon have been localized to the thin filaments of smooth muscle in situ.
Calponln Calponin is a 34 kDa, smooth-musclespecific protein present at the same molar concentration as tropomyosin (i.e. 1 calponin: 1 tropomyosin: 7 actins), which colocalizes with actin and tropomyosin in isolated smooth-muscle cells 26. Purified calponin binds to actin with a dissociation constant (Kd) of 4.6x10"~M, and inhibits the actin-
activated Mg2"-ATPaseactivity of phosphorylated smooth-muscle myosin by up to 80%. Actin binding and ATPase inhibition are alleviated by phosphorylation of calponln by PKC or CaMK II, and restored following dephosphorylation by a type 2A protein phosphatase. Although calponin phosphorylation has been detected in intact smooth-muscle strips in response to contractile stimuli2;, this remains a controversial issue28. Calponin also binds to tropomyosin and Ca2"-CaM, but the physiological relevance of these interactio~s is unclear.
TIBS 19 - SEPTEMBER 1994 Based on these and other properties of calponin, we have proposed that this protein is involved in regulating the crossbridge cycling rate in smooth muscle. In the resting cell at low [Ca2"]l, myosin is dephosphorylated, crossbridges are detached from actin and the muscle cell is relaxed. Under these conditions, calponin is also dephosphorylated, in which case it is associated with the thin filaments and may inhibit a low crossbridge cycling rate owing to a low basal level of myosin phosphorylation. Stimulation of the muscle, resulting in an increase in [Ca-~ ]i, activates myosin phosphorylaUon and triggers crossbridge cycling. If calponin remains associated with the thin filament, however, this crossbridge cycling rate is inhibited. Inhibition is alleviated by phosphorylation of calponin, causing its dissociation from the actin filament, and is restored by dephosphorylation. Since MLCK, CaMK II and PKC have different sensitivities to Ca2+, the cell can very precisely regulate the ratio of dephosphorylated:phosphorylated myosin and dephosphorylated: phosphorylated calponin, thereby achieving very precise regulation of the crossbridge cycling rate. The presence of distinct regulatory mechanisms that have
(3) caldesmon inhibits the movement of actin filaments over a substrate of immobilized, phosphorylated myosin in the in vitro motility assay; (4) addition of caldesmon to demembranated smooth-muscle strips causes a rightward shift in the force-LC20phosphorylation relationship3°; and (5) a synthetic peptide that displaces endogenous caldesmon from permeabilized ferretaortic cells induces contraction at pCa7 (Ref. 31). The abundance of caidesrnon ranges from 1 caldesmon: 205 actin monomers in tonic vascular smooth muscles to 1 caldesmon:22-28 actin monomers in phasic smooth muscles. The smooth-muscle protein is an 87 kDa elongated (74 × 1.9 nm) molecule that can be divided into three domains: an amino-terminal myosin-binding domain, a central e-helical repeat domain and a carboxy-terminal domain that binds actin, tropomyosin and CaM (the latter in a Ca2"-dependent manner). The physiological significance, if any, of the interactions of caldesmon with tropomyosin and CaM is unknown. On the other hand, the acUn interaction is the basis for inhibition of the actomyosin ATPase; the myosin interaction suggests that caldesmon may function to crosslink actin and myosin filaments, different sensitivities to Ca2" provides and this has been demonstrated in the smooth muscle with the degree of vitro. The central repeat domain, which flexibility and adaptability required for is abseqt from nonmuscle caldesmon, normal physiological function. may serve as a spacer between the In the context of Ca2'-lndependent actin- and myosin-binding sites. Recent evidence has implicated contractions In which PKC-~ has been implicated, it is possible that specific the mitogen-activated protein kinase activation of PKC-~ in the absence of (MAPK) cascade in regulation of cala [Ca21Ltransient results in direct phos- desmon. Caldesmon is phosphorylated phorylation of calponin (Fig. 6). This by MAPK in vitro and at the same sites would trigger its dissociation from the in intact canine-aortic strips treated thin filament, thereby alleviating its with phorbol ester. The cascade shown inhibition of crossbridge cycling, which in Fig. 6 was proposed to explain these could then proceed at a slow rate, observations32. In support of this signalowing to the low resting level of myosin transduction pathway, PKC, Ras, Raf, phosphorylation. This model is consist- MAP kinase kinase (MEK), MAPK and ent with the slow rate of the contractile caldesmon have all been identified in response to phorbol ester treatment of aortic smooth muscle. Interestingly, intact smooth-muscle strips or single PKC-~ and MAPK both transiocate fron~ the cytosol to the sarcolemma in cells. response to phenylephrine; PKC-¢ remains associated with the sarcoCaldesmon Caldesmon 29, which is expressed in iemma, whereas MAPK redistributes smooth-muscle and nonmuscle cells, to the cytosol coincident with conhas also been implicated in the regu- traction ~3. The effects of caldesmon lation of crossbridge cycling: (1) phosphorylation by MAPKhave not yet caldesmon is located on the actin fila- been investigated thoroughly, but the ment in situ; (2) the isolated protein prediction would be that the affinity inhibits actomyosin ATPase activity; for actin is reduced, leading to loss of
inhibition of the actomyosin ATPase and contraction. Again, in the context of CaZ÷-independent contractions referred to earlier, it is conceivable that activation of PKC-~ could trigger the MAPK pathway, leading to contraction via the phosphorylation of caldesmon. The physiological response to activation of PKC-~ may therefore result from both direct phosphorylation of calponin and indirect phosphorylation of caldesmon via the MAPKpathway. Acknowledgements Work in the authors' laboratory is supported by grants from the Medical Research Council of Canada and the Alberta Heart and Stroke Foundation. B. G. A. is a Fellow of the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR), and M. P. W. is an AHFMR Medical Scientist. The authors are very grateful to G. Garnett and n Doell for expert secretarial assistance.
References 1 Sohieszek, A. (1977) in The Biochemistry of Smooth Muscle (Stephens, N, L., ed,), pp. 413-443, University Park Press 2 Daiorowska,R.. Sherry, J. M. F,, Aromatorio, D. K. and Hartshorne, D. J. (1978) Biochemistry 17, 253-258 3 Pate, M. D. (1985) Adv, Prot. Phosphatases 1, 367-382 4 Alesst, D. et al. (1992) Eur../. Biochem, 210, 1023-1035 5 Williams, D, A. and Fay, F. S. (1986) Am, J, Physiol, 250, C779-C791 6 Missiaen, L. et aL (1992) Pharmac. Ther, 56, 191-231 7 Welsh, M. P, Can, J. Physiol. Pharmacol. (in press) 8 Hartshorne, D. J. (1987) in Physio/o~ of the Gastrointestinal Tract (2rid ednl (Johnson, L. R., ed.), pp. 423-482, Raven Press 9 Walsh, M. P. (1991) Biochem. Ce//Biol. 69, 771-800 10 Rayment, I. eta/. (1993) Science 261, 50-57 11 Hoar, P. E., Kerrick, W. G. L. and Cassidy, P. S. (1979) Science 204, 503-506 12 Ikebe, M. and Morita, J. (1991) J. Biol. Chem. 266, 21339-21342 13 Rowe, 1".and Kendrick-Jones,J. (1993) EMBOJ. 12, 4877--4884 14 Trybus, K. M. and Chatman, T. A. (1993) J. Biol. Chem. 268, 4412-4419 15 Olson, N. J. et ~1. (1990) Proc. Nat/Acad. Bci. USA 87, 2284-2288 16 Knighton, D. R. et at. (1992) Science 258, 130-135 17 Meador, W. E., Means, A. R. and Quiocho, F. A. (1992) Science 257, 1251-1255 18 Ikura. M. et el. (1992) Science 256, 632-638 19 Pearson, R. B. et al. (1988) Science 241, 970-973 20 Gallagher, P. J. et el, (1993) J. Biol. Chem. 268, 26578-26582
REVIEWS 21 Dillon,P. E, Aksoy,M. 0., Driska, S, P. and Murphy,R. A, (1981) Science 211, 495-497 22 Gong,M. C. et al. (1992) J. Biol. Chem. 267, 21492-21498 23 Tansey,M. G, et al. (1992) J. Biol. Chem. 267, 12511-12516 24 Collins, '/. M., Walsh, M. P. and Morgan,K. G. (1992) Am. 1 Physiol. 262, H754-H762
TRANSLOCATION OF PREPROITANS across biological membranes is a fundamental process of intracellular protein trafficking. Most proteins that reside in cell organelles are synthesized in the cytosol and must then be transported across the organelle membranes to reach their functional destinationL The preproteins carry signal sequences, also termed leader sequences or targeting sequences, which are recognized by organelle-specific receptors. Proteinconducting channels in the membranes are assumed to permit the selective passage of the polypeptide chains 2,3. In most cases, the translocation process requires the input of energy, ATP or energization of the membrane4. Preprotein-binding proteins inside the organelles are involved in ensuring unidirectional translocation5,6. Recently, much effort has been focused on identifying and characterizing the components of the protein-conductlng channels in the membranes. Mitochondria are surrounded by an outer and an lmler membrane, across which several hundred different proteins have to be imported 7`s, The principles of mitochondrial import for a protein transported into the mitochondrial matrix are depicted in Rg. 1. Many preproteins carry positively charged signal sequences (presequences) at their amino termini, which are recognized by receptors on the mitochondrial surface. A general insertion pore (GIP) permits the passage of the preproteins across the outer membrane. Upon trans]ocation through the mitochondrial N. Pfanner is at the Biochemisches Institut, Universit~t Freiburg, Hermann-Herder-Strasse7, D-79104 Freiburg, Germany: E, A. Craig is at the Department of 8iomolecular Chemistry, University of Wisconsin-Madison, Madison, Wl 53706, USA; and M. Meljer is at the Institute for Molecular Cell Biology, BioCentrum Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands.
Andrea,J. E. and Walsh, M. P. (1992) Hypertension 20, 585-595 26 Winder,S. J. and Walsh, M. P. (1993) Cellular Signalling 5, 677-686 27 ~nder, S.J. etai. (1993)Biochem.1 296,827-836 28 B~r~ny,M. and B~r~ny,K. (1993) Biochim. Biophys. Acta 1179, 229-233 29 Sobue,K. and Sellers,J. R. (1991) .l. Biol. Chem. 266, 12115-12118 25
Pfltzer,G., Zeugner,C., Troschka,M. and Chalovich,J. (1993) P(oc. Nall Acad. ScL USA 90, 5904-5908 31 Katsuyarna,H., Wang,C-L.A. and Morgan,K. G. (1992) J. BioL Chem. ;..67,14555-14558 32 Adam, L. P. and Hathaway,D. R. (1993) FEBS Lett. 322, 56--60 33 Khalil, R. and Morgan,K. G. (1993) Am. J. Physiol. 265, C406-C411 30
The protein import machinery of the mitochondrial inner membrane Nikolaus Pfanner, Elizabeth A. Craig and Michiel Meijer Mitochondria import most of their proteins from the cytosol. Although considerable information is available on the import machineries of the mitochondrial outer membrane and matrix, until recently little was known about the machinery of the inner membrane, Recent studies have identified three mitochondrial inner membrane proteins (MIMs) as essential components of the import machinery. MIM17 and MIM23 seem to form part of a channel, while MIM44, in cooperation with the heat.shock pro. rein Hsp70, binds the preproteins in transit. The electrical membrane potential and ATP are needed to drive protein translocation through the MIM import machinery,
inner-membrane import machinery (MIM), the presequence is proteolytically removed in the matrix and the proteins are folded into their functional forms. In the past five years several components of the mitochondrial import apparatus have been identified 5-11, including: (1) c~osolic cofactors, particularly chaperone components; (2) mitochondrial outer membrane (MOM) proteins, including two import receptors and the components of the GIP; (3) the matrixprocessing peptidase; and (4) the matrix heat-shock proteins HspT0, Hsp60 and their partners. In addition, specialized enzymes for processing or prosthetic modification of preproteins have been identified. However, until the recent identification of three essential MIM proteins, very little was known about the proteinimport machinery of the mitochondrial
inner membrane. It is essential that an electrochemical proton gradient is maintained across the inner membrane, even during the translocation of polypeptide chains. The translocation channel must thus permit the passage of hundreds of different polypeptide chains, but at the same time be selective enough to prevent nonspecific leak. age of ions.
Independenttmnspett machineriesIn mitochondrlalouter and innermembranes Preproteins in transit across the
mitochondrial membranes can be found as intermediates spanning outer and inner membranes at so-called translocation contact sites ~2,~3.An earlier view of preprotein import into mitochondria suggested that the protein import machineries of both the inner and the outer membranes were stably © 1994,ElsevierScienceLtd 0968-0004/94/$07.00