The tumor-sensitive calmodulin-like protein is a specific light chain of human unconventional myosin X

Human calmodulin-like protein (CLP) is an epithelial-specific Ca(2+)-binding protein whose expression is strongly down-regulated in cancers. Like calmodulin, CLP is thought to regulate cellular processes via Ca(2+)-dependent interactions with specific target proteins. Using gel overlays, we identified a approximately 210-kDa protein binding specifically and in a Ca(2+)-dependent manner to CLP, but not to calmodulin. Yeast two-hybrid screening yielded a CLP-interacting clone encoding the three light chain binding IQ motifs of human "unconventional" myosin X. Pull-down experiments showed CLP binding to the IQ domain to be direct and Ca(2+)-dependent. CLP interacted strongly with IQ motif 3 (K(d) approximately 0.5 nm) as determined by surface plasmon resonance. Epitope-tagged myosin X was localized preferentially at the cell periphery in MCF-7 cells, and CLP colocalized with myosin X in these cells. Myosin X was able to coprecipitate CLP and, to a lesser extent, calmodulin from transfected COS-1 cells, indicating that CLP is a specific light chain of myosin X in vivo. Because unconventional myosins participate in cellular processes ranging from membrane trafficking to signaling and cell motility, myosin X is an attractive CLP target. Altered myosin X regulation in (tumor) cells lacking CLP may have as yet unknown consequences for cell growth and differentiation.     

Kinetic analyses of a truncated mammalian myosin I suggest a novel isomerization event preceding nucleotide binding

MI(1IQ) is a complex of calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, rat myr-1 (130-kDa myosin I or MI(130)). We have determined the transient kinetic parameters that dictate the ATP hydrolysis cycle of mammalian myosin I by examining the properties of MI(1IQ). Transient kinetics reveal that the affinity of MI(1IQ) for actin is 12 nm. The ATP-induced dissociation of actin-MI(1IQ) is biphasic. The fast phase is dependent upon [ATP], whereas the slow phase is not; both phases show a Ca(2+) sensitivity. The fast phase is eliminated by the addition of ADP, 10 micrometer being required for half-saturation of the effect in the presence of Ca(2+) and 3 micrometer ADP in the absence of Ca(2+). The slow phase shares the same rate constant as ADP release (8 and 3 s(-)(1) in the presence and absence of Ca(2+), respectively), but cannot be eliminated by decreasing [ADP]. We interpret these results to suggest that actin-myosin I exists in two forms in equilibrium, one of which is unable to bind nucleotide. These results also indicate that the absence of the COOH-terminal 5 calmodulin binding domains of myr-1 do not influence the kinetic properties of MI(130) and that the Ca(2+) sensitivity of the kinetics are in all likelihood due to Ca(2+) binding to the first IQ domain.     

Rat myr 4 defines a novel subclass of myosin I: identification,distribution, localization,and mapping of calmodulin-binding sites with differential calcium sensitivity

We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I''s. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F- actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.     

Calmodulin bridging of IQ motifs in myosin-V

Ca(2+)-saturated calmodulin binds to double-length IQ lever-arm sequences from murine myosin-V, forming a 1:1 "bridging" complex with very high affinity, (K9d)<10 pM for double motifs, IQ34, IQ45 and IQ56). Such a 1:1 complex involves interaction of one calmodulin (CaM) molecule with two adjacent IQ-motifs, providing a molecular mechanism for the observed Ca(2+)-dependent CaM dissociation from the IQ-region. Structural considerations suggest that formation of the 1:1 complex requires a severe distortion of the lever-arm, potentially regulating functional motility. This would be consistent with a recent report of diverse, irregular shapes of the lever arm of myosin-V induced by the presence of Ca(2+).     

Truncation of a mammalian myosin I results in loss of Ca2+-sensitive motility

MYR-1, a mammalian class I myosin, consisting of a heavy chain and 4-6 associated calmodulins, is represented by the 130-kDa myosin I (or MI(130)) from rat liver. MI(130) translocates actin filaments in vitro in a Ca(2+)-regulated manner. A decrease in motility observed at higher Ca(2+) concentrations has been attributed to calmodulin dissociation. To investigate mammalian myosin I regulation, we have coexpressed in baculovirus calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic "motor" domain and the first calmodulin-binding IQ domain of rat myr-1; we refer to this truncated molecule here as MI(1IQ). Association of calmodulin to MI(1IQ) is Ca(2+)-insensitive. MI(1IQ) translocates actin filaments in vitro at a rate resembling MI(130), but unlike MI(130), does not exhibit sensitivity to 0.1-100 micrometer Ca(2+). In addition to demonstrating successful expression of a functional truncated mammalian myosin I in vitro, these results indicate that: 1) Ca(2+)-induced calmodulin dissociation from MI(130) in the presence of actin is not from the first IQ domain, 2) velocity is not affected by the length of the IQ region, and 3) the Ca(2+) sensitivity of actin translocation exhibited by MI(130) involves 1 or more of the other 5 IQ domains and/or the carboxyl tail.     

Calcium, nucleotide, and actin affect the interaction of mammalian Myo1c with its light chain calmodulin

To investigate the interaction of mammalian class I myosin, Myo1c, with its light chain calmodulin, we expressed (with calmodulin) truncation mutants consisting of the Myo1c motor domain followed by 0-4 presumed calmodulin-binding (IQ) domains (Myo1c (0IQ)-Myo1c (4IQ)). The amount of calmodulin associating with the Myo1c heavy chain increased with increasing number of IQ domains from Myo1c (0IQ) to Myo1c (3IQ). No calmodulin beyond that associated with Myo1c (3IQ) was found with Myo1c (4IQ) despite its availability, showing that Myo1c binds three molecules of calmodulin with no evidence of a fourth IQ domain. Unlike Myo1c (0IQ), the basal ATPase activity of Myo1c (1IQ) was >10-fold higher in Ca (2+) vs EGTA +/- exogenous calmodulin, showing that regulation is by Ca (2+) binding to calmodulin on the first IQ domain. The K m and V max of the actin-activated Mg (2+)-ATPase activity were largely independent of the number of IQ domains present and moderately affected by Ca (2+). In binding assays, some calmodulin pelleted with Myo1c heavy chain when actin was present, but a considerable fraction remained in the supernatant, suggesting that calmodulin is displaced most likely from the second IQ domain. The Myo1c heavy chain associated with actin in a nucleotide-dependent fashion. In ATP a smaller proportion of calmodulin pelleted with the heavy chain, suggesting that Myo1c undergoes nucleotide-dependent conformational changes that affect the affinity of calmodulin for the heavy chain. The studies support a model in which Myo1c in the inner ear is regulated by both Ca (2+) and nucleotide, which exert their effects on motor activity through the light-chain-binding region.     

Time-resolved fluorescence anisotropy studies show domain-specific interactions of calmodulin with IQ target sequences of myosin V

Single cysteine mutants of calmodulin, Cam(S38C) and Cam(N111C), have been specifically labelled with Alexa488 maleimide to study the effects of calcium on the structural dynamics of calmodulin complexed with IQ3, IQ4 and IQ34 target peptide motifs of mouse unconventional myosin-V. Using phase fluorometry, the time-resolved anisotropy shows well-separated global and segmental correlation times. The calcium-sensitive global motion of either calmodulin domain can be independently monitored in domain-specific interactions of either apo- or Ca(4).calmodulin with IQ3 or IQ4 peptides. C-domain interactions predominate, and apo-N-domain interactions are unexpectedly weak. The 1:1 complex of Ca(4).calmodulin with IQ34 behaves as a compact globular species. The results demonstrate novel dynamic aspects of calmodulin-IQ interactions relating to the calcium regulation of motility of unconventional myosin.     

Calmodulin bound to the first IQ motif is responsible for calcium-dependent regulation of myosin 5a

Myosin 5a is as yet the best-characterized unconventional myosin motor involved in transport of organelles along actin filaments. It is well-established that myosin 5a is regulated by its tail in a Ca(2+)-dependent manner. The fact that the actin-activated ATPase activity of myosin 5a is stimulated by micromolar concentrations of Ca(2+) and that calmodulin (CaM) binds to IQ motifs of the myosin 5a heavy chain indicates that Ca(2+) regulates myosin 5a function via bound CaM. However, it is not known which IQ motif and bound CaM are responsible for the Ca(2+)-dependent regulation and how the head-tail interaction is affected by Ca(2+). Here, we found that the CaM in the first IQ motif (IQ1) is responsible for Ca(2+) regulation of myosin 5a. In addition, we demonstrate that the C-lobe fragment of CaM in IQ1 is necessary for mediating Ca(2+) regulation of myosin 5a, suggesting that the C-lobe fragment of CaM in IQ1 participates in the interaction between the head and the tail. We propose that Ca(2+) induces a conformational change of the C-lobe of CaM in IQ1 and prevents interaction between the head and the tail, thus activating motor function.     

Enhancement by Mg2+ of domain specificity in Ca2+-dependent interactions of calmodulin with target sequences

Mg2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca2+-dependent target interactions. The binding of Mg2+ does, however, produce significant spectroscopic changes in residues located in the Ca2+-binding loops, and the Mg-calmodulin complex is significantly different from apo-calmodulin in loop conformation. Direct measurement of Mg2+ binding constants, and the effects of Mg2+ on Ca2+ binding to calmodulin, are consistent with specific binding of Mg2+, in competition with Ca2+. Mg2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg2+ has a direct role in conferring stability on both domains of apo-calmodulin. Apo-calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with Kd approximately 70-90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg2+, in marked contrast to the strong enhancement of peptide affinity induced by Ca2+. Under conditions of stimulation and increased [Ca2+], Mg2+ has a role in directing the mode of initial target binding preferentially to the C-domain of calmodulin, due to the opposite relative affinities for binding of Ca2+ and Mg2+ to the two domains. Mg2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca2+ threshold for enzyme activation and increases the importance of a partially Ca2+-saturated calmodulin-target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca2+-dependent target interactions.     

Protein Ser/Thr phosphatases PPEF interact with calmodulin

Regulation of protein dephosphorylation by cytoplasmic Ca(2+) levels and calmodulin (CaM) is well established and considered to be mediated solely by calcineurin. Yet, recent identification of protein phosphatases with EF-hand domains (PPEF/rdgC) point to the existence of another group of Ca(2+)-dependent protein phosphatases. We have recently hypothesised that PPEF/rdgC phosphatases might possess CaM-binding sites of the IQ-type in their N-terminal domains. We now employed yeast two-hybrid system and surface plasmon resonance (SPR) to test this hypothesis. We found that entire human PPEF2 interacts with CaM in the in vivo tests and that its N-terminal domain binds to CaM in a Ca(2+)-dependent manner with nanomolar affinity in vitro. The fragments corresponding to the second exons of PPEF1 and PPEF2, containing the IQ motifs, are sufficient for specific Ca(2+)-dependent interaction with CaM both in vivo and in vitro. These findings demonstrate the existence of mammalian CaM-binding protein Ser/Thr phosphatases distinct from calcineurin and suggest that the activity of PPEF phosphatases may be controlled by Ca(2+) in a dual way: via C-terminal Ca(2+)-binding domain and via interaction of the N-terminal domain with CaM.     

Kinetic characterization of a monomeric unconventional myosin V construct.

An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 microM (37 degrees C), and the velocity of actin movement was approximately 0.3 micrometer/s (30 degrees C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to approximately 70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s(-1)), and ATP hydrolysis occurs at approximately 200 s(-1). The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.     

In calmodulin-IQ domain complexes, the Ca(2+)-free and Ca(2+)-bound forms of the calmodulin C-lobe direct the N-lobe to different binding sites

We have investigated the roles played by the calmodulin (CaM) N- and C-lobes in establishing the conformations of CaM-IQ domain complexes in different Ca(2+)-free and Ca(2+)-bound states. Our results indicate a dominant role for the C-lobe in these complexes. When the C-lobe is Ca(2+)-free, it directs the N-lobe to a binding site within the IQ domain consensus sequence. It appears that the N-lobe must be Ca(2+)-free to interact productively with this site. When the C-lobe is Ca(2+)-bound, it directs the N-lobe to a site upstream of the consensus sequence, and it appears that the N-lobe must be Ca(2+)-bound to interact productively with this site. A model for switching in CaM-IQ domain complexes is presented in which the N-lobe adopts bound and extended positions that depend on the status of the Ca(2+)-binding sites in each CaM lobe and the compositions of the two N-lobe binding sites. Ca(2+)-dependent changes in the conformation of the bound C-lobe that appear to be responsible for directed N-lobe binding are also identified. Changes in the equilibria between extended and bound N-lobe positions may control bridging interactions in which the extended N-lobe is bound to another CaM-binding domain. Ca(2+)-dependent control of bridging interactions with CaM has been implicated in the regulation of ion channel and unconventional myosin activities.     

Ca(2+)-dependent regulation of the motor activity of myosin V

Mouse myosin V constructs were produced that consisted of the myosin motor domain plus either one IQ motif (M5IQ1), two IQ motifs (M5IQ2), a complete set of six IQ motifs (SHM5), or the complete IQ motifs plus the coiled-coil domain (thus permitting formation of a double-headed structure, DHM5) and expressed in Sf9 cells. The actin-activated ATPase activity of all constructs except M5IQ1 was inhibited above pCa 5, but this inhibition was completely reversed by addition of exogenous calmodulin. At the same Ca(2+) concentration, 2 mol of calmodulin from SHM5 and DHM5 or 1 mol of calmodulin from M5IQ2 were dissociated, suggesting that the inhibition of the ATPase activity is due to dissociation of calmodulin from the heavy chain. However, the motility activity of DHM5 and M5IQ2 was completely inhibited at pCa 6, where no dissociation of calmodulin was detected. Inhibition of the motility activity was not reversed by the addition of exogenous calmodulin. These results indicate that inhibition of the motility is due to conformational changes of calmodulin upon the Ca(2+) binding to the high affinity site but is not due to dissociation of calmodulin from the heavy chain.     

Ca(2+)-dependent inhibition of actin-activated myosin ATPase activity by S100C (S100A11), a novel member of the S100 protein family

S100C (S100A11, calgizzarin) inhibits the actin-activated myosin Mg(2+)-ATPase activity of smooth muscle in a dose-dependent manner: its half-maximal effect occurs at a S100C/actin molar ratio of 0.05 and its maximal effect occurs at a ratio of 0.20. Furthermore, S100C was found to bind to actin with a stoichiometry of 1:6-7 in the presence of Ca(2+), with an affinity of 1 x 10(-6) M determined by cosedimentation assays. Other Ca(2+)-binding proteins such as S100A1, S100A2, S100B, and calmodulin did not inhibit actin-activated myosin Mg(2+)-ATPase activity. Calmodulin, S100A1, and S100B reversed the inhibitory effect of calponin in a Ca(2+)-dependent manner, S100A2 had no effect, and S100C had additional inhibitory effects. The results suggest that S100C might be involved in the regulation of actin-activated myosin Mg(2+)-ATPase activity through its Ca(2+)-dependent interaction with actin filaments.     

Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum

Calcium binding protein 40 (CBP40) is a Ca(2+)-binding protein abundant in the plasmodia of Physarum polycephalum. CBP40 consists four EF-hand domains in the COOH-terminal half and a putative alpha-helix domain in the NH(2)-terminal half. We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca(2+)-binding properties. Recombinant proteins of CBP40 bound 4 mol of Ca(2+) with much higher affinity (pCa(1/2) = 6.5) than that of calmodulin. When residues 1-196 of the alpha-helix domain were deleted, the affinity for Ca(2+) decreased to pCa(1/2) = 4.6. A chimeric calmodulin was generated by conjugating the alpha-helix domain of CBP40 with calmodulin. The affinity of Ca(2+) for the chimeric calmodulin was higher than that for calmodulin, suggesting that the alpha-helix domain is responsible for the high affinity of CBP40 for Ca(2+). CBP40 forms large aggregates reversibly in a Ca(2+)-dependent manner. A mutant protein with a deletion of NH(2)-terminal 32 residues, however, could not aggregate, indicating the importance of these residues for the aggregation. The aggregation occurs above micromolar levels of Ca(2+) concentration, so it may only occur when CBP40 is secreted out of the plasmodial cells.     

Chlamydomonas outer arm dynein alters conformation in response to Ca2+

We have previously shown that Ca(2+) directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the beta and gamma heavy chains (HCs). The gamma HC-associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca(2+) with K(Ca) = 3 x 10(-5) M in vitro, suggesting it may act as a Ca(2+) sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and gamma HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the gamma heavy chain. In vitro experiments indicate that LC4 undergoes a Ca(2+)-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca(2+). The sedimentation profile of the gamma HC subunit changed subtly upon Ca(2+) addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated gamma subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca(2+) addition. We propose that Ca(2+)-dependent conformational change of LC4 has a direct effect on the stem domain of the gamma HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm.     

Phosphorylation of brush border myosin I by protein kinase C is regulated by Ca(2+)-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation.

Brush border myosin I from chicken intestine is phosphorylated in vitro by chicken intestinal epithelial cell protein kinase C. Phosphorylation on serine and threonine to a maximum of 0.93 mol of P/mol of myosin I occurs within an approximately 20 kDa region at the end of the COOH-terminal tail of the 119-kDa heavy chain. The effects of Ca2+ on myosin I phosphorylation by protein kinase C are complex, with up to 4-fold stimulation occurring at 0.5-3 microM Ca2+, and up to 80% inhibition occurring at 3-320 microM Ca2+. Phosphorylation required that brush border myosin I be in its phosphatidylserine vesicle-bound state. Previously unknown Ca2+ stimulation of brush border myosin I binding to phosphatidylserine vesicles was found to coincide with Ca2+ stimulation of phosphorylation. A myosin I proteolytic fragment lacking approximately 20 kDa of its tail retained Ca(2+)-stimulated binding, but showed reduced Ca(2+)-independent binding. Ca(2+)-dependent phosphatidylserine binding is apparently due to the concomitant phosphatidylserine-promoted, Ca(2+)-induced dissociation of up to three of the four calmodulin light chains from myosin I. Four highly basic putative calmodulin-binding sites in the Ca(2+)-dependent phosphatidylserine binding region of the heavy chain were identified based on the similarity in their sequence to the calmodulin- and phosphatidylserine-binding site of neuromodulin. Calmodulin dissociation is now shown to occur in the low micromolar Ca2+ concentration range and may regulate the association of brush border myosin I with membranes and its phosphorylation by protein kinase C.     

Unique features in the C-terminal domain provide caltractin with target specificity

Caltractin (centrin) is a member of the calmodulin (CaM) superfamily of EF-hand calcium-binding proteins. It is an essential component of the centrosomal structures in a wide range of organisms. Caltractin and calmodulin apparently function in distinct calcium signaling pathways despite substantial sequence similarity. In an effort to understand the structural basis for such differences, the high-resolution three-dimensional solution structure of the complex between the Ca(2+)-activated C-terminal domain of Chlamydomonas reinhardtii caltractin (CRC-C) and a 19 residue peptide fragment comprising the putative cdc31p-binding region of Kar1p (K(19)) has been determined by multi-dimensional heteronuclear NMR spectroscopy. Formation of the complex is calcium-dependent and is stabilized by extensive interactions between CRC-C and three key hydrophobic anchors (Trp10, Leu13 and Leu14) in the peptide as well as favorable electrostatic interactions at the protein-peptide interface. In-depth comparisons have been made to the structure of the complex of Ca(2+)-activated calmodulin and R(20), the CaM-binding domain of smooth muscle myosin light-chain kinase. Although the overall structures of CRC and CaM domains in their respective complexes are very similar, differences in critical regions in the sequences of these proteins and their targets lead to clear differences in the complementarity of their respective binding surfaces. These subtle differences reveal the structural basis for the Ca(2+)-dependent regulation of distinct cellular signaling events by CRC and CaM.     

Brain myosin-V, a calmodulin-carrying myosin, binds to calmodulin-dependent protein kinase II and activates its kinase activity.

Myosin-V, an unconventional myosin, has two notable structural features: (i) a regulatory neck domain having six IQ motifs that bind calmodulin and light chains, and (ii) a structurally distinct tail domain likely responsible for its specific intracellular interactions. Myosin-V copurifies with synaptic vesicles via its tail domain, which also is a substrate for calmodulin-dependent protein kinase II. We demonstrate here that myosin-V coimmunoprecipitates with CaM-kinase II from a Triton X-100-solubilized fraction of isolated nerve terminals. The purified proteins also coimmunoprecipitate from dilute solutions and bind in overlay experiments on Western blots. The binding region on myosin-V was mapped to its proximal and medial tail domains. Autophosphorylated CaM-kinase II binds to the tail domain of myosin-V with an apparent Kd of 7.7 nM. Surprisingly, myosin-V activates CaM-kinase II activity in a Ca2+-dependent manner, without the need for additional CaM. The apparent activation constants for the autophosphorylation of CaM-kinase II were 10 and 26 nM, respectively, for myosin-V versus CaM. The maximum incorporation of 32P into CaM-kinase II activated by myosin-V was twice that for CaM, suggesting that myosin-V binding to CaM-kinase II entails alterations in kinetic and/or phosphorylation site parameters. These data suggest that myosin-V, a calmodulin-carrying myosin, binds to and delivers CaM to CaM-kinase II, a calmodulin-dependent enzyme.     

Ca2+-sensitive inactivation and facilitation of L-type Ca2+ channels both depend on specific amino acid residues in a consensus calmodulin-binding motif in the(alpha)1C subunit

L-type Ca(2+) channels are unusual in displaying two opposing forms of autoregulatory feedback, Ca(2+)-dependent inactivation and facilitation. Previous studies suggest that both involve direct interactions between calmodulin (CaM) and a consensus CaM-binding sequence (IQ motif) in the C terminus of the channel's alpha(1C) subunit. Here we report the functional effects of an extensive series of modifications of the IQ motif aimed at dissecting the structural determinants of the different forms of modulation. Although the combined substitution by alanine at five key positions (Ile(1624), Gln(1625), Phe(1628), Arg(1629), and Lys(1630)) abolished all Ca(2+) dependence, corresponding single alanine replacements behaved similarly to the wild-type channel (77wt) in four of five cases. The mutant I1624A stood out in displaying little or no Ca(2+)-dependent inactivation, but clear Ca(2+)- and frequency-dependent facilitation. An even more pronounced tilt in favor of facilitation was seen with the double mutant I1624A/Q1625A: overt facilitation was observed even during a single depolarizing pulse, as confirmed by two-pulse experiments. Replacement of Ile(1624) by 13 other amino acids produced graded and distinct patterns of change in the two forms of modulation. The extent of Ca(2+)-dependent facilitation was monotonically correlated with the affinity of CaM for the mutant IQ motif, determined in peptide binding experiments in vitro. Ca(2+)-dependent inactivation also depended on strong CaM binding to the IQ motif, but showed an additional requirement for a bulky, hydrophobic side chain at position 1624. Abolition of Ca(2+)-dependent modulation by IQ motif modifications mimicked and occluded the effects of overexpressing a dominant-negative CaM mutant.