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A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly.

Liang W, Warrick HM, Spudich JA - J. Cell Biol. (1999)

Bottom Line: Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes.These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments.The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.

ABSTRACT
Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes. Here we report a suppressor screen of Dictyostelium myosin II- cells containing 3 x Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

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Examples of expression of myosin II from suppressors. Expression of mutant myosin IIs in the myosin II– cell line HS1 transformed with the following suppressor mutants of 3×Asp myosin II: lane 1, 3×Asp myosin II control; lane 2, D1823Y; lane 3, ΔCOOH terminus1; lane 4, R1880P; lane 5, Δ1999–2004; lane 6, ΔCOOH terminus2; lane 7, R1880P; lane 8, ΔCOOH terminus3; lane 9, ΔCOOH terminus4; lane 10, ΔCOOH terminus5; and lane 11, wild-type myosin II control. The ΔCOOH terminus suppressors were revealed by immunoblotting experiments using the mAb 55, which binds an epitope at the COOH terminus of the tail (Pagh and Gerisch 1986). They failed to bind mAb 55.
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Figure 2: Examples of expression of myosin II from suppressors. Expression of mutant myosin IIs in the myosin II– cell line HS1 transformed with the following suppressor mutants of 3×Asp myosin II: lane 1, 3×Asp myosin II control; lane 2, D1823Y; lane 3, ΔCOOH terminus1; lane 4, R1880P; lane 5, Δ1999–2004; lane 6, ΔCOOH terminus2; lane 7, R1880P; lane 8, ΔCOOH terminus3; lane 9, ΔCOOH terminus4; lane 10, ΔCOOH terminus5; and lane 11, wild-type myosin II control. The ΔCOOH terminus suppressors were revealed by immunoblotting experiments using the mAb 55, which binds an epitope at the COOH terminus of the tail (Pagh and Gerisch 1986). They failed to bind mAb 55.

Mentions: Mutagenesis was performed on a strain of Dictyostelium that had its endogenous mhcA gene deleted (HS1) and contains an extrachromosomal plasmid expressing mhcA–3×Asp myosin II (pBIG-ASP). To check whether the suppressor mutations were intragenic or extragenic, the plasmid from each suppressor was rescued and retransformed into unmutagenized myosin II– cells, and the transformed cells were spread on bacterial lawns. The phenotypes of all the suppressors were reproduced, verifying that all 28 suppressor mutations were intragenic. The characteristics of the suppressors are shown in Table . Typically the expression level of myosins from the suppressors was similar to that from the wild-type and 3×Asp myosin II cells, but the size of the myosins varied (Fig. 2). 7 of 28 of the suppressors were full-length myosin II, 9 were small internal deletions of 1–7 residues, and 12 were truncations from the COOH terminus. There was no correlation between the means of mutagenesis (NQNO or UV) and the sizes of myosin IIs expressed from the suppressors. The sizes of the 12 ΔCOOH terminus suppressor myosin IIs were the same or larger than a previously studied mutant myosin II called ΔC34. ΔC34–myosin II, a truncated Dictyostelium myosin II lacking the 34-kD COOH terminus of the tail (the regulatory domain, residues 1819–2116; see Fig. 3), constitutively assembles into thick filaments, and ΔC34–myosin cells are able to complete the Dictyostelium developmental cycle and form fruiting bodies (O'Halloran and Spudich 1990). Our ΔCOOH terminus myosin II suppressors are likely to be longer variants of the ΔC34–myosin II constitutive assembly phenotype, and we therefore focused on the remaining 16 suppressors.


A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly.

Liang W, Warrick HM, Spudich JA - J. Cell Biol. (1999)

Examples of expression of myosin II from suppressors. Expression of mutant myosin IIs in the myosin II– cell line HS1 transformed with the following suppressor mutants of 3×Asp myosin II: lane 1, 3×Asp myosin II control; lane 2, D1823Y; lane 3, ΔCOOH terminus1; lane 4, R1880P; lane 5, Δ1999–2004; lane 6, ΔCOOH terminus2; lane 7, R1880P; lane 8, ΔCOOH terminus3; lane 9, ΔCOOH terminus4; lane 10, ΔCOOH terminus5; and lane 11, wild-type myosin II control. The ΔCOOH terminus suppressors were revealed by immunoblotting experiments using the mAb 55, which binds an epitope at the COOH terminus of the tail (Pagh and Gerisch 1986). They failed to bind mAb 55.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169343&req=5

Figure 2: Examples of expression of myosin II from suppressors. Expression of mutant myosin IIs in the myosin II– cell line HS1 transformed with the following suppressor mutants of 3×Asp myosin II: lane 1, 3×Asp myosin II control; lane 2, D1823Y; lane 3, ΔCOOH terminus1; lane 4, R1880P; lane 5, Δ1999–2004; lane 6, ΔCOOH terminus2; lane 7, R1880P; lane 8, ΔCOOH terminus3; lane 9, ΔCOOH terminus4; lane 10, ΔCOOH terminus5; and lane 11, wild-type myosin II control. The ΔCOOH terminus suppressors were revealed by immunoblotting experiments using the mAb 55, which binds an epitope at the COOH terminus of the tail (Pagh and Gerisch 1986). They failed to bind mAb 55.
Mentions: Mutagenesis was performed on a strain of Dictyostelium that had its endogenous mhcA gene deleted (HS1) and contains an extrachromosomal plasmid expressing mhcA–3×Asp myosin II (pBIG-ASP). To check whether the suppressor mutations were intragenic or extragenic, the plasmid from each suppressor was rescued and retransformed into unmutagenized myosin II– cells, and the transformed cells were spread on bacterial lawns. The phenotypes of all the suppressors were reproduced, verifying that all 28 suppressor mutations were intragenic. The characteristics of the suppressors are shown in Table . Typically the expression level of myosins from the suppressors was similar to that from the wild-type and 3×Asp myosin II cells, but the size of the myosins varied (Fig. 2). 7 of 28 of the suppressors were full-length myosin II, 9 were small internal deletions of 1–7 residues, and 12 were truncations from the COOH terminus. There was no correlation between the means of mutagenesis (NQNO or UV) and the sizes of myosin IIs expressed from the suppressors. The sizes of the 12 ΔCOOH terminus suppressor myosin IIs were the same or larger than a previously studied mutant myosin II called ΔC34. ΔC34–myosin II, a truncated Dictyostelium myosin II lacking the 34-kD COOH terminus of the tail (the regulatory domain, residues 1819–2116; see Fig. 3), constitutively assembles into thick filaments, and ΔC34–myosin cells are able to complete the Dictyostelium developmental cycle and form fruiting bodies (O'Halloran and Spudich 1990). Our ΔCOOH terminus myosin II suppressors are likely to be longer variants of the ΔC34–myosin II constitutive assembly phenotype, and we therefore focused on the remaining 16 suppressors.

Bottom Line: Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes.These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments.The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.

ABSTRACT
Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes. Here we report a suppressor screen of Dictyostelium myosin II- cells containing 3 x Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

Show MeSH
Related in: MedlinePlus