Regulation of myosin self-assembly: phosphorylation of Dictyostelium heavy chain inhibits formation of thick filaments.

Kuczmarski, Edward R.; Spudich, James A.

doi:10.1073/pnas.77.12.7292

Cited by 245 publications

(149 citation statements)

References 36 publications

(24 reference statements)

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“…The myosin concentrations required for assembly of the RLCunphosphorylated NM2s in the absence of ATP, ∼3-10 nM, are similar to the values of 14 nM reported for Dictyostelium myosin II (22) and ∼5 nM for Acanthamoeba myosin II (23), but only ∼10% of the values previously determined for NM2s purified from thymus and brush border (18). The differences between our data for the three recombinant NM2s and the data for thymus and brush border NM2s are most likely because the earlier values were for the myosin remaining in the supernatant after centrifugation of polymerized myosins at ∼100,000 × g for 20 min, whereas we find it requires centrifugation at ∼400,000 × g for 20 min to sediment the dimers, tetramers, and hexamers that form under polymerization conditions.…”

Section: Discussionsupporting

confidence: 63%

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Liu

Billington

Shu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLCphosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.C lass II myosins, the first of 37 known myosin classes (1, 2) to be discovered and originally thought to be specific for muscle, are now known to occur widely in nonmuscle cells in numerous species. Human nonmuscle cells express three different myosin 2 paralogs: nonmuscle myosins (NM) NM2A, NM2B, and NM2C. Like all class II myosins (3, 4), NM2 monomers are hexapeptides of two identical heavy chains (HC), two essential light chains (ELC), and two regulatory light chains (RLC). The HCs of NM2A, NM2B, and NM2C are encoded by different genes, have 60-80% sequence similarity, and isoforms of each are derived from alternative splicing (4).Each HC has an N-terminal globular motor domain with an ATPase site and an actin-binding site, followed by a lever arm that binds one ELC and one RLC, and a long tail that homodimerizes to form a coiled-coil α-helix. The HC ends with a short C-terminal nonhelical tailpiece (5). Like most class II myosins, NM2 monomers assemble into bipolar filaments by antiparallel association of their elongated coiled-coil helical tails. The filaments of NM2s are appreciably smaller than filaments of muscle myosins, with lengths of about 300 nm, consisting of ∼30 monomers for filaments of NM2A and NM2B and ∼14 monomers for filaments of NM2C (5).As described originally for smooth muscle myosin based on light-scattering data (6-8), filaments of unphosp...

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Section: Discussionsupporting

confidence: 63%

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Liu

Billington

Shu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…This translocation is correlated with a transient increase in the rate of myosin II heavy chain (MHC) 1 as well as light chain phosphorylation (3,4,6). In addition, in vitro studies strongly suggest that MHC phosphorylation plays an important role in the regulation of myosin II filament formation (7)(8)(9)(10). The importance of MHC phosphorylation in the regulation of myosin II in vivo was demonstrated by Egelhoff et al (11).…”

mentioning

confidence: 91%

Dictyostelium Myosin II Is Regulated during Chemotaxis by a Novel Protein Kinase C

Abu-Elneel

Karchi

Ravid

1996

Journal of Biological Chemistry

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When cells of Dictyostelium are starved, they acquire the ability to bind cAMP to specific cell surface receptors and to respond to this signal by chemotaxis. The process of chemotaxis involves phosphorylation and reorganization of myosin II (1-5). In response to cAMP stimulation, myosin II, which exists as thick filaments, translocates to the cortex (5). This translocation is correlated with a transient increase in the rate of myosin II heavy chain (MHC) 1 as well as light chain phosphorylation (3, 4, 6). In addition, in vitro studies strongly suggest that MHC phosphorylation plays an important role in the regulation of myosin II filament formation (7-10). The importance of MHC phosphorylation in the regulation of myosin II in vivo was demonstrated by Egelhoff et al. (11). They found that elimination of the MHC phosphorylation sites allows in vivo contractile activity, but this myosin II shows substantial overassembly. Mimicking the negative charge state of phosphorylated myosin II eliminates filament formation in vitro and renders the myosin II unable to drive any tested contractile event in vivo (11).We previously reported the isolation of a MHC-specific PKC (MHC-PKC) from Dictyostelium that phosphorylates Dictyostelium MHC specifically and is homologous to ␣, ␤, and ␥ subtypes of mammalian PKC (9, 12). This kinase, which is membrane-associated and is expressed during Dictyostelium development, was implicated in the increase in MHC phosphorylation during chemotaxis in this species (9). In vitro phosphorylation of MHC by MHC-PKC results in inhibition of myosin II thick filament formation (9), by inducing the formation of a bent monomer of myosin II whose assembly domain is tied up in an intramolecular interaction that precludes intermolecular interaction, which is necessary for thick filament formation (10). The findings that MHC-PKC is a member of the PKC family and that it regulates myosin II assembly suggest a link between the extracellular chemotactic signal and subsequent intracellular events.A considerable amount of information is now available regarding the regulation of myosin II by MHC phosphorylation, and a picture is beginning to emerge in which the molecular changes in myosin II that are involved in MHC phosphorylation are related to cAMP-induced directed cell movement. Nevertheless, the molecular mechanism by which a cAMP signal is transmitted to myosin II, resulting in myosin II localization and chemotaxis, remains unclear. In an attempt to throw some light on this issue, we studied the role of MHC-PKC in vivo by eliminating and by overexpressing the MHC-PKC protein in Dictyostelium cells. Analysis of these cell lines allowed us to address directly the role of MHC-PKC in vivo and consequently the role of MHC phosphorylation in the regulation of myosin II. The results presented here indicate that MHC-PKC is a key modulator in the regulation of myosin II localization in response to the chemoattractant, cAMP.

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“…Analysis of the expression of the penultimate exon indicates that it is present in transcripts synthe sized in the adult thorax and absent in most other muscles (Bernstein et al 1986;Rozek and Davidson 1986), resulting in the muscle-specific localization of MHC isoforms with different carboxyl termini. We spec ulate that the carboxy-terminal sequence of MHC is critical to the assembly of myosin molecules into thick filaments (Kuczmarski and Spudich 1980;Dibb et al 1985) and that differences in the sequence of the carboxyl termini may result in myosin molecules with alternative as sembly properties. The tissue-specific expression of the other alternatively spliced exons within the MHC gene is poorly characterized.…”

Section: Muscle Diversity and The Generation Of Mhc Isoforms In Drosomentioning

confidence: 99%

Ultrastructural and molecular analyses of homozygous-viable Drosophila melanogaster muscle mutants indicate there is a complex pattern of myosin heavy-chain isoform distribution.

O'Donnell¹,

Collier²,

Mogami³

et al. 1989

Genes Dev.

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We describe the ultrastructural and initial molecular characterization of four homozygous-viable, dominantflightless mutants of Drosophila melanogaster. Genetic mapping indicates that these mutations are inseparable from the known muscle myosin heavy-chain (MHC) allele Mhc^, and each mutation results in a muscle-specific reduction in MHC protein accumulation. The indirect flight muscles (IFMs) of each of these homozygous mutants fail to accumulate MHC, lack thick filaments, and do not display normal cylindrical myofibrils. As opposed to the null phenotype observed in the IFM, normal amounts of MHC accumulate in the leg muscles of three of these mutants, whereas the fourth mutant shows a 45% reduction in leg muscle MHC. The ultrastructure of the tergal depressor of the trochanter muscle (TDT, or jump muscle] is normal in one mutant, completely lacks thick filaments in a second mutant, and displays a reduction of thick filaments in two mutants. The thick filament reduction in this latter class of mutants is limited to the four smaller anterior cells of the TDT, indicating that the TDT is a mixed fiber-type muscle. Because all isoforms of muscle MHC are encoded by alternative splicing of transcripts from a single gene, our results suggest that there is a complex pattern of MHC isoform accumulation in Drosophila, The phenotypes of the homozygous-viable mutants provide evidence for the differential localization of MHC isoforms in different muscles, within the same muscle, and even within a single muscle cell. The mutant characteristics also suggest that the use of some alternative exons is shared among the IFM, TDT, and additional muscles whereas the use of others is unique to the IFM.[Key Words: Drosophila-, muscle mutants; myosin heavy chain; alternative RNA splicing]

show abstract

Regulation of myosin self-assembly: phosphorylation of Dictyostelium heavy chain inhibits formation of thick filaments.

Cited by 245 publications

References 36 publications

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Dictyostelium Myosin II Is Regulated during Chemotaxis by a Novel Protein Kinase C

Ultrastructural and molecular analyses of homozygous-viable Drosophila melanogaster muscle mutants indicate there is a complex pattern of myosin heavy-chain isoform distribution.

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