Myosin 1G Is an Abundant Class I Myosin in Lymphocytes Whose Localization at the Plasma Membrane Depends on Its Ancient Divergent Pleckstrin Homology (PH) Domain (Myo1PH) (original) (raw)
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Domain structure of a mammalian myosin I beta
Proceedings of the National Academy of Sciences, 1994
We have determined the primary structure of a myosin I (called m myosin I, MMI3) from bovine brain and Identified Its functional domain. The protein was previously purified from brain and adrenal gand. Several constructs were generated and expressed in Escherichia cofl as glutathione S-traferase fusion proteins and the recombinant proteins were recognized by monoclona antibodies that recognize either "head" or "tail" dom of native myosin I. A gel overlay method was used to confirm that dulin binds to the consensus calmodulin-binding sequence in MMIIJ. Binding assays were used to detect interaction with anionic phospholipid vesicles. We conclude that MMI_3 consists of an amino-terminal 80.5-kDa domain that contains the ATPand actin-binding sites, followed by an 8.5-kDa domain with three calmodulin-bindng sequences and a basic 30-kDa carboxylterminal tail segment that binds to anionic phospholipids and membranes.
The Cargo-Binding Domain Regulates Structure and Activity of Myosin 5
Nature, 2006
Myosin 5 is a two-headed motor protein that moves cargoes along actin filaments 1, 2 . Its tail ends in paired globular tail domains (GTDs) thought to bind cargo 3 . At nanomolar calcium, actin-activated ATPase is low and the molecule is folded. Micromolar calcium activates ATPase and the molecule unfolds 3-6 . We now describe the structure of folded myosin and the GTD's role in regulating activity. Electron microscopy shows the two heads lie either side of the tail, contacting the GTDs at a lobe of the motor domain (∼Pro117-Pro137) that contains conserved acidic side chains, suggesting ionic interactions between motor domain and GTD. Myosin 5 heavy meromyosin (HMM), a constitutively active fragment lacking the GTDs, is inhibited and folded by a dimeric GST-GTD fusion protein. Motility assays reveal that at nanomolar calcium HMM moves robustly on actin filaments whereas few myosins bind or move. These results combine to show that with no cargo, the GTDs bind in an intramolecular manner to the motor domains producing an inhibited and compact structure that binds weakly to actin and allows the molecule to recycle towards new cargoes.
Molecular Biology of the Cell, 2000
Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process.
Journal of Biological Chemistry, 2001
The myotonic dystrophy kinase-related kinases RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we uncovered two major substrates, p130 and p85, for MRCK␣-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an ␣-helical C terminus with leucine repeats, and a centrally located conserved motif with the MRCK␣-kinase phosphorylation site. Like MBS130, p85 is specifically associated with protein phosphatase 1␦ (PP1␦), and this requires the N terminus, including the ankyrin repeats. This association is required for the regulation of both the catalytic activities and the assembly of actin cytoskeleton. The N terminus, in association with PP1␦, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The C-terminal effects consist of two independent events that involved both the conserved phosphorylation inhibitory motif and the ␣-helical leucine repeats. The former was able to interact with PP1␦ only in the phosphorylated state and result in inactivation of PP1␦ activity. This provides further evidence that phosphorylation of a myosin binding subunit protein by specific kinases confers conformational changes in a highly conserved region that plays an essential role in the regulation of its catalytic subunit activities.
1995
In an effort to determine diversity and function of mammalian myosin I molecules, we report here the cloning and characterization of myr 3 (third unconventional myosin from rat), a novel mammalian myosin I from rat tissues that is related to myosin I molecules from protozoa. Like the protozoan myosin I molecules, myr 3 consists of a myosin head dOmain, a single light chain binding motif, and a tail region that includes a COOH-terminal SH3 domain. However, myr 3 lacks the regulatory phosphorylation site present in the head domain of protozoan myosin I molecules. Evidence was obtained that the COOH terminus of the tail domain is involved in regulating F-actin binding activity of the NH2-terminal head domain. The light chain of myr 3 was identified as the Ca 2+binding protein calmodulin. Northern blot and immunoblot analyses revealed that myr 3 is expressed in many tissues and cell lines. Immunofluorescence studies with anti-myr 3 antibodies in NRK cells demonstrated that myr 3 is localized in the cytoplasm and in elongated structures at regions of cell-cell contact. These elongated structures contained F-actin and o~-actinin but were devoid of vinculin. Incubation of NRK cells with Con A stimulated the formation of myr 3-containing structures along cell-cell contacts. These results suggest for myr 3 a function mediated by cell-cell contact. M YOSIN I molecules represent a subfamily of the rapidly expanding myosin superfamily. Like conventional muscle myosin (myosin II), they exhibit an NH2-terminal head region, a light chain binding region, and a COOH-terminal tail region. The head region is relatively well conserved in all myosins; it comprises ATP-and actin-binding sites and exhibits actin-activated ATPase activity (Pollard et al., 1991). The head region and the light chain-binding region with the associated light chain(s) are sufficient to produce directed force along actin filaments (Toyoshima et al., 1987). The tail regions of myosin I molecules, in contrast to the tail regions of conventional muscle myosin (myosin II), do not dimerize or form filaments. For some of the myosin I tails, it has been demonstrated that they bind to membranes (Adams and Pollard, 1989; Miyata et al., 1989; Hayden et al., 1990). All myosin I tails share a diagnostic myosin I tail homology motif possibly involved in membrane binding (Btihler et al., 1994). A subgroup of myosin I molecules ("amoeboid") identified in Acanthamoeba castellanii and Dictyostelium discoideum contain in their tail regions a Src homology 3 (SH3) domain.
Proteins: Structure, Function, and Bioinformatics, 2009
Cell motility, such as smooth muscle contraction and cell migration, is controlled by the reversible phosphorylation of the regulatory light chain of myosin II and other cytoskeletal proteins. Mounting evidence suggests that in smooth muscle cells and other types of cells in vertebrates, myosin phosphatase (MP) plays an important role in controlling the phosphorylation of myosin II as well as other cytoskeletal proteins, including ezrin, moesin, and radixin.1 MP is a holoenzyme consisting of a catalytic subunit of a type-1 Ser/Thr phosphatase (PP1C) delta isoform, a myosin phosphatase targeting subunit 1 (MYPT1), and an accessory subunit M21. In this ternary complex, MYPT1 is responsible for regulating the phosphatase activity.1 A recent X-ray crystallographic study revealed an allosteric interaction between PP1C and the N-terminal ankyrin repeat domain of MYPT1 that confers the substrate specificity of the enzyme.2 MP activity is suppressed when Thr 696 or Thr 853 of MYPT1 is phosphorylated by various kinases, such as ROCK, ZIPK, ILK, and PAK.1 , 3 However, it is still unclear how the phosphorylation of MYPT1 inhibits MP activity. The amino acid sequence around Thr 696 of MYPT1 is highly conserved among MYPT1 family members including MYPT2 and MBS85. Therefore, structural insights into the inhibitory domain of MYPT1 are expected to provide new clues to fully elucidate the mechanism that controls phosphatase activity via the phosphorylation of MYPT1 or other family members involved in kinasephosphatase crosstalk in cytoskeletal regulation.
Thermodynamic evidence of non-muscle myosin II–lipid-membrane interaction
Biochemical and Biophysical Research Communications, 2008
A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Nonmuscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles made of DMPG/DMPC at a molar ratio of 1:1 at 10 mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23°C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement.