Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules

Craig, Roger; Smith, Robin C.; Kendrick‐Jones, John

doi:10.1038/302436a0

Cited by 399 publications

(335 citation statements)

References 23 publications

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“…Rac/rho binding may partially open this structure, causing a major conformational change in citron dimer thereby affecting the properties of the scaffold. Activation of a protein involving a conformational change of its coiled-coil region has been established for myosin [25].…”

Section: Modeljor Citron Activation and Comparison To Other Putative mentioning

confidence: 99%

A novel partner for the GTP‐bound forms of rho and rac

et al. 1995

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Ahstract Using the yeast two hybrid system and overlay assays we identified a putative rholrac effector, citron, which interacts with the GTP-bound forms of rho and racl, but not with cdc42. Extensive homologies to known proteins were not observed. This 183 kDa protein contains a C~H2 zinc finger, a PH domain, and a long coiled-coil forming region including 4 leucine zippers and the rholrac binding site. We recently identified three others putative rho effeetors characterized by a common rho binding motif. Citron does not share this motif and displays a distinctive protein organization, thus defining a separate class of rho partners.

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Section: Modeljor Citron Activation and Comparison To Other Putative mentioning

confidence: 99%

A novel partner for the GTP‐bound forms of rho and rac

et al. 1995

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“…It will be interesting to see whether vertebrate smooth muscle and nonmuscle myosins, which adopt the 10S monomer conformation requiring sharp bends at two constant positions within the rod (11,43), have similar proline-containing hinges. For striated muscle myosins, a hinge region has been postulated to exist about one-third of the way down the length of the rod, based on sequence analysis, electron microscopy, protease sensitivity, and biophysical measurements (for review see reference 19).…”

Section: The Hinge and Its Possible Role In Enzymatic Regulationmentioning

confidence: 99%

Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail.

Hammer¹,

Bowers²,

Paterson³

et al. 1987

The Journal of Cell Biology

127

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Abstract. We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequences of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity. M YOSIN II from the soil ameba Acanthamoeba castellanii is composed of a pair of 175-kD heavy chains and two pairs of light chains (17 and 17.5 kD) (26). Electron microscopy reveals that these subunits are arranged into a structure similar to that of other myosins, i.e., a highly asymmetric molecule possessing two globular heads and an extended rodlike tail (25, 34). The tail mediates the self-assembly of myosin II molecules into small bipolar filaments containing 16-40 monomers depending on the polymerization conditions (34). The myosin II actin-activated MgZ+-ATPase activity, which resides in the globular head (1), is regulated by phosphorylation of the heavy chain (9). Unphosphorylated myosin II is activated 40-fold by F-actin while myosin II, which has three phosphates incorporated into each heavy chain by a specific kinase, is not actin activatable. Interestingly, the phosphorylation sites reside within a "o30-residue nonhelical tailpiece at the very carboxyl-terminal end of the rodlike tail (10), nearly 100 nm away from the catalytic site. Recent evidence indicates that heavy chain phosphorylation exerts its effects on ATPase activity by altering the conformation of the small bipolar iliaments formed by myosin II (23,24), alt...

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“…For rotary shadowing, 100-300-pl aliquots of protein solutions (myosin rod or myosin-antibody complexes) were diluted 1 : 1 with glycerol (final myosin concentration 0.1 -0.2 mg/ml), mixed and sprayed onto freshly cleaved mica, using a glass atomiser connected to a hand-operated rubber bulb [44]. The mica was rotary shadowed at an angle of 5 -6", as decribed by Burke and Shotton [45].…”

Section: Electron Microscopymentioning

confidence: 99%

Studies on the structure and conformation of brush border myosin using monoclonal antibodies

Citi

Kendrick‐Jones

1987

European Journal of Biochemistry

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We have produced and characterised five monoclonal antibodies against myosin isolated from chicken intestinal epithelial brush border cells. The binding sites of the antibodies on the rod portion of brush border myosin were localised using rotary shadowing/electron microscopy of myosin-antibody complexes. Two antibodies were shown to bind to the tip of the myosin tail, two antibodies to sites about two thirds down the length of the rod, and one antibody about one third down the length of the rod. Brush border myosin was digested with papain, trypsin and a-chymotrypsin, and the myosin fragments obtained were analysed by western blots with the monoclonal antibodies and polyclonal antiserum, and by gel overlay with '251-labelled light chains. Using this approach we were able to identify and map the protease cleavage sites and thus characterise the proteolytic substructure of brush border myosin. Solid-phase assays, western blots and immunofluorescence were used to study the cross-reactivity of these monoclonal antibodies against a variety of myosins from different species and cell types, to assess the immunological relatedness between brush border myosin and homologous molecules present in different tissues and species. Finally, we used a competitive solid-phase assay to measure the 'relative affinities' of the antibodies towards the three possible conformational states of brush border myosin, i. e. filament, extended monomer and folded monomer.Antibodies have been widely used to study the distribution and organisation of contractile proteins in muscle and nonmuscle cells, using immunofluorescence and immunoelectron microscopy techniques [l -51, and to clarify their roles in vivo, using microinjection techniques [6 -81. Antibodies are also convenient tools to study the structural homologies between similar proteins, when sequence data are not available [9], as shown by studies on the polymorphism of skeletal and cardiac myosins in specific fiber types, and at different developmental stages [lo-151. Studies on invertebrate myosins (from Acanthamoeba and Dictyostelium cells) and vertebrate smooth muscle and skeletal myosins [16 -241 have shown that monoclonal antibodies are ideal probes to investigate the relationship between myosin structure and mechanochemical function.In the present study, we describe the production and characterisation of five monoclonal antibodies to the rod portion of myosin purified from a vertebrate nonmuscle tissue, i.e. chicken epithelial brush border cells. One goal of this study was to determine the immunological relatedness between brush border myosin and myosins present in different tissues and species. Secondly, we used these monoclonal antibodies to investigate the structural organisation of brush border myosin. As shown by studies on brain myosin, suitable markers, such as the heavy-chain phosphorylation site and the nucleotide and actin binding sites, can be used to identify Correspondence to S. Citi, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, England CB2 2QHAbbrevi...

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Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules

Cited by 399 publications

References 23 publications

A novel partner for the GTP‐bound forms of rho and rac

A novel partner for the GTP‐bound forms of rho and rac

Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail.

Studies on the structure and conformation of brush border myosin using monoclonal antibodies

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