Phosphorylation of smooth muscle myosin: evidence for cooperativity between the myosin heads.

The relationship between the actin-activated adenosinetriphosphatase activity of smooth muscle myosin and the extent of myosin light chain phosphorylation is nonlinear. It is suggested that the phosphorylation of the two heads of smooth muscle myosin is an ordered process and that the two heads are influenced by cooperative interactions.

Differential codes for free Ca(2+)-calmodulin signals in nucleus and cytosol.

BACKGROUND: Many targets of calcium signaling pathways are activated or inhibited by binding the Ca(2+)-liganded form of calmodulin (Ca(2+)-CaM). Here, we test the hypothesis that local Ca(2+)-CaM-regulated signaling processes can be selectively activated by local intracellular differences in free Ca(2+)-CaM concentration. RESULTS: Energy-transfer confocal microscopy of a fluorescent biosensor was used to measure the difference in the concentration of free Ca(2+)-CaM between nucleus and cytoplasm. Strikingly, short receptor-induced calcium spikes produced transient increases in free Ca(2+)-CaM concentration that were of markedly higher amplitude in the cytosol than in the nucleus. In contrast, prolonged increases in calcium led to equalization of the nuclear and cytosolic free Ca(2+)-CaM concentrations over a period of minutes. Photobleaching recovery and translocation measurements with fluorescently labeled CaM showed that equalization is likely to be the result of a diffusion-mediated net translocation of CaM into the nucleus. The driving force for equalization is a higher Ca(2+)-CaM-buffering capacity in the nucleus compared with the cytosol, as the direction of the free Ca(2+)-CaM concentration gradient and of CaM translocation could be reversed by expressing a Ca(2+)-CaM-binding protein at high concentration in the cytosol. CONCLUSIONS: Subcellular differences in the distribution of Ca(2+)-CaM-binding proteins can produce gradients of free Ca(2+)-CaM concentration that result in a net translocation of CaM. This provides a mechanism for dynamically regulating local free Ca(2+)-CaM concentrations, and thus the local activity of Ca(2+)-CaM targets. Free Ca(2+)-CaM signals in the nucleus remain low during brief or low-frequency calcium spikes, whereas high-frequency spikes or persistent increases in calcium cause translocation of CaM from the cytoplasm to the nucleus, resulting in similar concentrations of nuclear and cytosolic free Ca(2+)-CaM.

The EF-hand family of calcium-modulated proteins.

The EF-hand homolog proteins bind calcium (Ca2+) with dissociation constants in the micromolar range and are modulated by stimulus-induced increases in cytosolic free Ca2+. We have grouped over 160 different EF-hand homolog proteins into ten subfamilies and ten unique categories. Except for troponin-C, all subfamilies and unique EF-hand homologs represented in vertebrates can be found in the CNS. In this review, structural and functional characteristics of these proteins are discussed, with special emphasis on the multifunctional regulatory protein, calmodulin. The possible function of bending within the central helix of calmodulin is considered and is illustrated with a model calmodulin--target complex.

Activation of enzymes by calmodulins containing intramolecular cross-links.

We have reacted calmodulins containing cysteines substituted at positions 3 and 146 or 5 and 146 with bismaleimidohexane (BMH) to generate intramolecularly cross-linked proteins termed BMHCM or BMHCM1, respectively. Reactions were also performed with N-ethylmaleimide (NEM) in place of BMH to generate corresponding S-ethylsuccinimidylated proteins termed NEMCM or NEMCM1. The abilities of these proteins to activate plant NAD kinase, erythrocyte Ca(2+)-ATPase and bovine brain calcineurin activities were assessed. The BMH- or NEM-reacted proteins activate calcineurin activity as does control calmodulin. Kact values for Ca(2+)-ATPase activation by BMHCM and BMHCM1 are increased 10-fold relative to the control value, with no corresponding change in Vmax values. Activation of this enzyme by NEMCM or NEMCM1 is not different from the control. In NAD kinase activation experiments BMHCM and BMHCM1 are associated with a 10 to 20-fold increase in Kact values and a 60-75% reduction in Vmax values relative to the control. NEMCM1 is not associated with any apparent changes in NAD kinase activation, however, NEMCM is associated with a 10-fold increase in the Kact value. NEM-reacted calmodulin containing a cysteine only at position 3 is not associated with an increased Kact value, implying that this change is due to interactions between S-(ethylsuccinimido)cysteines at positions 3 and 146. In conclusion, cross-linking and associated distortions in the structure of calmodulin appear to have little or no effect on activation of calcineurin enzyme activity. However, bending in the central helix and/or steric restrictions associated with cross-linking increase significantly the Kact value for Ca(2+)-ATPase and NAD kinase activation, and dramatically reduce maximal activation of NAD kinase activity.

Modulation of myosin filament conformation by physiological levels of divalent cation.

The effect of divalent cation, in particular Mg2+, on the properties of synthetic myosin filaments has been studied; and substantial changes in sedimentation and light scattering demonstrated to occur in the physiological range of free Mg2+. A pre-requisite for these studies has been the definition of a modified method for the preparation of myosin in highly monodisperse filament form, rigorously free from thin filament proteins. The sedimentation coefficient at infinite dilution shows a large increase (169 S to 193 S) in the range 0.2 mM to 3 mM in Mg2+. The anomalous frictional increment found for these filaments is thus substantially reduced. The concentration dependence (ks), however, shows a substantial decrease (470 ml/g to 334 ml/g) in the same range of Mg2+, and the calculated filament molecular weight is virtually unchanged. A change in the filament conformation is thus indicated. This is confirmed by an analysis of the turbidity of the filaments in the centrifuge cell, which shows a similar increase in response to the addition of Mg2+. These effects have been found to be independent of ionic strength (0.07 to 0.11), pH (7.0 to 7.6), the presence of MgATP or the presence of low levels of Ca2+ (approximately 100 microM). These effects studied indicate the action of Mg2+ through a low-affinity binding site (Kd approximately 1.5 X 10(-3) M). We consider that a significant change in crossbridge conformation can adequately explain these changes in physical and enzymic properties. A provisional model is proposed, in which the effect of Mg2+ is to bring the crossbridges into closer proximity to the filament shaft.

Novel fluorescent indicator proteins for monitoring free intracellular Ca2+.

We have recently described a fluorescent indicator protein in which red- and blue-shifted variants of green fluorescent protein are joined by the calmodulin-binding sequence from smooth muscle myosin light chain kinase [Romoser V.A., Hinkle P.M., Persechini A. Detection in living cells of Ca(2+)-dependent changes in the fluorescence of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J Biol Chem 1997; 272: 13270-13274]. The fluorescence emission of this protein at 505 nm (380 nm excitation) is reduced by approximately 65% when (Ca2+)4-calmodulin is bound, with a proportional increase in fluorescence emission at 440 nm. We have found that fusion of an engineered calmodulin, in which the C- and N-terminal EF hand pairs have been exchanged, to the C-terminus of this protein results in a novel indicator that responds directly to changes in the Ca2+ ion concentration, with an apparent Kd value of 100 nM for Ca2+ in the presence of 0.5 mM Mg2+. The affinity of the indicator for Ca2+ can be decreased by altering the amino acid sequence of the calmodulin-binding sequence to weaken its interaction with the intrinsic calmodulin domain. The fluorescence emission of this indicator can be used to monitor physiological changes in the free Ca2+ ion concentration in living cells.

Central helix role in the contraction-relaxation switching mechanisms of permeabilized skeletal and smooth muscles with genetic manipulation of calmodulin.

A prominent common feature of calmodulin and troponin structures is the unusually long central helix which separates the two lobes, each containing two Ca2(+)-binding sites. To study the role of certain highly conserved residues in the helix in the contraction-relaxation switching mechanism in muscle, we measured the Ca2(+)-activated force of permeabilized skeletal and smooth muscles with three genetically manipulated forms of calmodulin. Mutated calmodulin was made to substitute for troponin-C in vertebrate skeletal fiber. The mutants had 1-4 deletions in the conserved cluster (positions 81-84) in the solvent-exposed region of the central helix, which also substantially shortened the helix. The force of the maximally activated fiber was found to be diminished only with the mutant in which the entire cluster Ser-81 to Glu-84 (CaM delta 81-84) was deleted. All such deletions were found to be completely ineffective in blocking the Ca2(+)-switching process in smooth muscle strips. The results show for the first time that at least a part of the highly conserved four-residue cluster in the central helix is critical for the contraction mechanism of striated muscle. Further, the possibility is raised that the reduced length of the central helix may be a determining factor in the Ca2(+)-switching mechanism in fast-twitch muscle. These findings combined with the results on smooth muscle indicate diversity in the structure-function specifications for the central helix of calmodulin for different target proteins.

Calmodulin colocalizes with connexins and plays a direct role in gap junction channel gating.

The direct calmodulin (CaM) role in chemical gating was tested with CaM mutants, expressed in oocytes, and CaM-connexin labeling methods. CaMCC, a CaM mutant with greater Ca-sensitivity obtained by replacing the N-terminal EF hand pair with a duplication of the C-terminal pair, drastically increased the chemical gating sensitivity of Cx32 channels and decreased their Vj sensitivity. This only occurred when CaMCC was expressed before Cx32, suggesting that CaMCC, and by extension CaM, interacts with Cx32 before junction formation. Direct CaM-Cx interaction at junctional and cytoplasmic spots was demonstrated by confocal immunofluorescence microscopy in HeLa cells transfected with Cx32 and in cryosectioned mouse liver. This was confirmed in HeLa cells coexpressing Cx32-GFP (green) and CaM-RFP (red) or Cx32-CFP (cyan) and CaM-YFP (yellow) fusion proteins. Significantly, these cells did not form gap junctions. In contrast, HeLa cells expressing only one of the two fusion proteins (Cx32-GFP, Cx32-CFP, CaM-RFP or CaM-YFP) revealed both junctional and non-junctional fluorescent spots. In these cells, CaM-Cx32 colocalization was demonstrated by secondary immunofluorescent labeling of Cx32 in cells expressing CaM-YFP or CaM in cells expressing Cx32-GFP. CaM-Cx colocalization was further demonstrated at rat liver gap junctions by Freeze-fracture Replica Immunogold Labeling (FRIL).

The relationship between the free concentrations of Ca2+ and Ca2+-calmodulin in intact cells.

Using stably expressed fluorescent indicator proteins, we have determined for the first time the relationship between the free Ca2+ and Ca2+-calmodulin concentrations in intact cells. A similar relationship is obtained when the free Ca2+ concentration is externally buffered or when it is transiently increased in response to a Ca2+-mobilizing agonist. Below a free Ca2+ concentration of 0.2 microM, no Ca2+-calmodulin is detectable. A global maximum free Ca2+-calmodulin concentration of approximately 45 nM is produced when the free Ca2+ concentration exceeds 3 microM, and a half-maximal concentration is produced at a free Ca2+ concentration of 1 microM. Data for fractional saturation of the indicators suggest that the total concentration of calmodulin-binding proteins is approximately 2-fold higher than the total calmodulin concentration. We conclude that high-affinity calmodulin targets (Kd /= 100 nM) occurs only where free Ca2+-calmodulin concentrations can be locally enhanced.

Toward a model of the calmodulin-myosin light-chain kinase complex: implications for calmodulin function.

We have developed a model for the interaction of calmodulin and the presumptive calmodulin binding domain of rabbit skeletal muscle myosin light-chain kinase. In our model there is a bend in the central helix of calmodulin such that hydrophobic patches associated with the pairs of Ca2+ binding sites: I, II and III, IV; face one another. This was accomplished by altering the psi dihedral angle at one residue: Ser-81. We have made the presumptive calmodulin binding peptide alpha-helical over its entire length. In the model, this basic amphiphilic helix fits into a cavity formed by apposition of the two hydrophobic regions of calmodulin. We suggest that this general type of model may help explain calmodulin's ability to regulate the activities of its many different targets. Small changes in the conformation of a nonhelical bend within the central helix would have large effects on the relative positions of the two halves of the molecule. In this way, calmodulin might adapt itself to a wide range of possible calmodulin binding domains. The literature pertaining to the model is discussed. We also discuss the results of our own recent investigations of calmodulin species that have been altered by site-directed mutagenesis.

The central helix of calmodulin functions as a flexible tether.

Using site-directed mutagenesis we have created an altered calmodulin in which Gln-3 and Thr-146 have both been replaced by cysteines. We have reacted this protein with the bifunctional reagent, bismaleimidohexane, forming an intramolecular cross-link between the two cysteines. In the crystal structure of native calmodulin alpha-carbons at positions 3 and 146 are 37 A apart. In the bismaleimidohexane cross-linked protein these atoms can be no more than 19 A apart, and model building studies indicate that there is probably a bend in the central helix of calmodulin. A second modified calmodulin was generated by cleaving the central helix of the cross-linked protein at Lys-77 with trypsin. In this molecule, the two lobes of calmodulin are joined solely by the bismaleimidohexane cross-link, which bridges Cys-3 and Cys-146. Vm and Kact values for activation of myosin light chain kinase activity by the cross-linked and cross-linked/trypsinized proteins are not significantly different from those for the control protein. This result indicates that one role for the central helix may be to serve as a flexible tether between the calmodulin lobes. This is consistent with a model calmodulin-enzyme complex in which the central helix is bent, and the two lobes exert a concerted effect. A detailed model of this type has been proposed for the calmodulin-myosin light chain kinase complex (Persechini, A. and Kretsinger, R.H. (1988) J. Cardiovasc. Pharmacol., in press).

Length-dependence of isometric twitch tension potentiation and myosin phosphorylation in mouse skeletal muscle.

The effect of changes in muscle length on post-tetanic isometric twitch tension potentiation and myosin P-light chain phosphorylation was studied at 23 degrees C in the mouse extensor digitorum longus muscle. The length-tension relationship was determined for the same muscles after a 30 min period of quiescence and between 30 s and 3 min after a 1.5 s tetanus at L0. Isometric twitch tension is increased at all muscle lengths after the tetanus; however, the fractional increase in twitch tension rises from 0.2 at L0 to a maximum of 0.3 at 1.2 L0. The fractional increase in twitch tension measured at any fixed muscle length is constant between 30 s and 3 min post-tetanus. P-light chain phosphorylation remains constant between 30 s and 3 min post-tetanus followed by a slow decline to basal values. Under fixed length conditions, there is linear relationship between the relative magnitude of the twitch tension and the extent of P-light chain phosphorylation. Net myosin phosphorylation measured after a 1.5 s tetanus at 1.23 L0 is 35% less than that obtained under the same conditions at L0. Thus, contraction-induced phosphorylation of P-light chain decreases with increased muscle length and post-tetanic potentiation at a constant level of P-light chain phosphorylation increases with increasing muscle length. These observations may be consistent with alterations in the sarcoplasmic Ca2+ ion transient as the muscle is lengthened.

Activation of myosin light chain kinase and nitric oxide synthase activities by calmodulin fragments.

We have investigated the abilities of calmodulin (CaM) tryptic fragments 1-75 (TRCI) or 78-148 (TRCII) to activate gizzard smooth muscle myosin light chain kinase (gMLCK), rabbit skeletal muscle myosin light chain kinase (skMLCK), and neural nitric oxide synthase (nNOS) activities. Our results indicate for all three enzymes that binding of CaM follows an ordered mechanism wherein the C-terminal lobe, represented by TRCII, binds specifically to a site we designated as A, followed by binding of the N-terminal lobe, represented by TRCI, to a site designated as B. With TRCII and TRCI bound to their respective sites, skMLCK and gMLCK activities are both activated to about 80% of their maximum levels. Occupancy of both sites in the MLCK enzymes by TRCI results in only low levels of enzyme activation; occupancy of both sites by TRCII also results in low levels of gMLCK activity, but activates skMLCK activity to 65% of the maximum level. With TRCI bound at site B and either TRCII or TRCI bound at site A, nNOS activity is 50% of the maximum level. Apparent dissociation constants for TRCII binding to site A and TRCI binding to site B are, respectively; 0.3 and 3 microM (skMLCK); 1.2 and 0.8 microM (gMLCK); 10 nM and 150 microM (nNOS). Our results demonstrate that the CaM lobes can make distinct contributions to binding and/or activation of different CaM-dependent enzymes and that the tethering function of the central helix can be mimicked by sufficiently high concentrations of the CaM fragments. We have modeled tethering as if it stabilizes the CaM-enzyme complex by creating a high effective concentration of the N-terminal lobe. Calculated values for this concentration term indicate essentially identical contributions by the central helix to the observed nanomolar dissociation constants of the three CaM-enzyme complexes examined.

Ordered phosphorylation of the two 20 000 molecular weight light chains of smooth muscle myosin.

The time courses of phosphorylation of the Mr 20 000 light chains by purified myosin light chain kinase plus calmodulin were determined. In confirmation of an earlier report [Persechini, A., & Hartshorne, D. J. (1981) Science (Washington, D.C.) 213, 1383-1385], a steady-state kinetic analysis indicates that the phosphorylation occurs in an ordered manner; i.e., at a phosphorylation level of 0.5 mol of 32P incorporated per mol of bound Mr 20 000 light chain, each myosin molecule would have one phosphorylated head. The kinetic parameters obtained for the phosphorylation of the more reactive myosin head are similar to those determined by using isolated light chains. It is suggested that the ordered, or sequential, phosphorylation, and the different reactivities of the two Mr 20 000 light chains, is the result of preexisting asymmetry of the myosin molecule. Similar patterns of myosin phosphorylation are obtained in both the absence and presence of skeletal muscle actin.

Cooperative behavior of smooth muscle myosin.

Phosphorylation of smooth muscle myosin is thought to be a prerequisite for the activation of Mg2+-ATPase activity by actin. However, this idea is not accepted universally and other possible roles have been suggested either as alternative regulatory mechanisms or as mechanisms acting in addition to myosin phosphorylation. To clarify the situation we studied the effects of myosin phosphorylation on actin-activated ATPase activity by using a defined system where each of the component proteins was purified. Data were collected between 4 and 10 min after the addition of ATP. It was found that phosphorylation alone was sufficient to activate the Mg2+-ATPase activity although the relationship between phosphorylation and ATPase activity was not linear. Phosphorylation of approximately half of the available sites caused relatively little activation (to about 10% of the final ATPase activity), whereas the phosphorylation of the remaining sites elicited a marked activation of ATPase activity. These results raise the possibility that cooperative interactions occur between the two myosin heads. Evidence is also presented to suggest that the pathway of phosphorylation might be sequential, rather than random, again implying cooperativity between the myosin heads.

Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity.

Purified rabbit skeletal muscle myosin is phosphorylated on one type of light-chain subunit (P-light chain) by calmodulin-dependent myosin light chain kinase and dephosphorylated by phosphoprotein phosphatase C. Analyses of the time courses of both phosphorylation and dephosphorylation of skeletal muscle myosin indicated that both reactions, involving at least 90% of the P-light chain, were kinetically homogeneous. These results suggest that phosphorylation and dephosphorylation of rabbit skeletal muscle myosin heads are simple random processes in contrast to the sequential phosphorylation mechanism proposed for myosin from gizzard smooth muscle. We also examined the effect of phosphorylation of rabbit skeletal muscle myosin on the actin-activated ATPase activity. We observed an apparent 2-fold decrease in the Km for actin, from about 6 microM to about 2.5 microM, with no significant effect on the Vmax (1.8s-1) in response to P-light-chain phosphorylation. There was no significant effect of phosphorylation on the ATPase activity of myosin alone (0.045 s-1). ATPase activation could be fully reversed by addition of phosphatase catalytic subunit. The relationship between the extents of P-light-chain phosphorylation and ATPase activation (at 3.5 microM actin and 0.6 microM myosin) was essentially linear. Thus, in contrast to results obtained with myosin from gizzard smooth muscle, these results suggest that cooperative interactions between the myosin heads do not play an important role in the activation process in skeletal muscle. Since the effect of P-light-chain phosphorylation is upon the Km for actin, it would appear to be associated with a significant activation of ATPase activity only at appropriate concentrations of actin and salt.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of myosin phosphorylation on the contractile properties of skinned rabbit skeletal muscle fibers.

We have studied the effect of myosin P-light chain phosphorylation on the isometric tension generated by skinned fibers from rabbit psoas muscle at 0.6 and 10 microM Ca2+. At the lower Ca2+ concentration, which produced 10-20% of the maximal isometric tension obtained at 10 microM Ca2+, addition of purified myosin light chain resulted in a 50% increase in isometric tension which correlated with an increase in P-light chain phosphorylation from 0.10 to 0.80 mol of phosphate/mol of P-light chain. Addition of a phosphoprotein phosphatase reversed the isometric tension response and dephosphorylated P-light chain. At the higher Ca2+ concentration, P-light chain phosphorylation was found to have little effect on isometric tension. Fibers prepared and stored at -20 degrees C in a buffer containing MgATP, KF, and potassium phosphate incorporated 0.80 mol of phosphate/mol of P-light chain. Addition of phosphoprotein phosphatase to these fibers incubated at 0.6 microM Ca2+ caused a reduction in isometric tension and dephosphorylation of the P-light chain. There was no difference before and after phosphorylation of P-light chain in the normalized force-velocity relationship for fibers at the lower Ca2+ concentration, and the extrapolated maximum shortening velocity was 2.2 fiber lengths/s. Our results suggest that in vertebrate skeletal muscle, P-light chain phosphorylation increases the force level at submaximal Ca2+ concentrations, probably by affecting the interaction between the myosin cross-bridge and the thin filament.

Different phosphorylated forms of myosin in contracting tracheal smooth muscle.

Calmodulin-dependent myosin light chain kinase phosphorylates two light chain subunits on each myosin molecule. We have developed a method for measuring nonphosphorylated, monophosphorylated, and diphosphorylated forms of myosin in smooth muscle. Four protein bands were separated in tissue extracts by nondenaturing polyacrylamide gel electrophoresis in the presence of pyrophosphate. Immunoblots demonstrated that three forms (designated M, MP, and MP2) reacted with rabbit antisera prepared against the purified phosphorylatable light chain (P-light chain) from bovine tracheal smooth muscle. Evidence was obtained that M, MP, and MP2 represented nonphosphorylated, monophosphorylated, and diphosphorylated myosin, respectively, and that the other protein band was probably filamin. The formation of different phosphorylated forms of myosin was measured in bovine trachealis strips neurally stimulated from 1.0 to 3.5 s and quick-frozen. There was no detectable MP or MP2 in unstimulated muscles; the extent of P-light chain phosphorylation measured directly was 0.02 +/- 0.01 mol of phosphate/mol of P-light chain. After 2.5-s stimulation, maximal values of 0.63 +/- 0.06 mol of phosphate/mol of P-light chain and 0.40 +/- 0.06 MP2/myosintotal were obtained. During continuous neural stimulation from 1.0 to 3.5 s, the relationship between the extent of P-light chain phosphorylation (measured directly or calculated) and the relative amount of MP2 is consistent with a random phosphorylation process.