title: Modulation of actin filament sliding by mutations of the SH2 cysteine in Dictyostelium myosin II.
abstract: The cysteine residue called SH2 in the skeletal myosin heavy chain is conserved among various species. Cys 678 in Dictyostelium myosin II is equivalent to SH2 in skeletal myosin. Using the Dictyostelium myosin II heavy chain gene, SH2 was mutated to Gly, Ala, Ser, or Thr. These mutant myosins were expressed in Dictyostelium myosin-null cells. To investigate how these mutations affect the motor functions of myosin, we examined the phenotypes of the transformed cells. We also purified the mutant myosins, and characterized them by measuring the actin-activated MgATPase activity, sliding velocity of actin filaments and force level. All of these mutant myosins complemented the myosin-specific defects of the myosin-null cells. Consistent with these observed phenotypes, all of the purified mutant myosins retained similar actin-activated MgATPase activities and force levels to those of the wild-type myosin (WT). However, the sliding velocities of actin filaments were significantly different (WT > or = Ser > Ala >> Thr > Gly). In particular, the Gly and Thr mutants exhibited a striking decrease in velocity, while the Ser mutant exhibited velocity comparable to that of the wild-type myosin. Thus, mutations of SH2 resulted in uncoupling of ATP hydrolysis and the sliding.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Suzuki
authors.forename: R
authors.initials: R
authors.lastname: Ohkura
authors.forename: S
authors.initials: S
authors.lastname: Sugiura
authors.forename: R
authors.initials: R
authors.lastname: Yasuda
authors.forename: K
authors.initials: K
authors.lastname: Kinoshita
authors.forename: M
authors.initials: M
authors.lastname: Tanokura
authors.forename: K
authors.initials: K
authors.lastname: Sutoh
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D003545:Cysteine / Q000235:genetics*; D004023:Dictyostelium / Q000201:enzymology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000235:genetics*; D010641:Phenotype; D018909:src Homology Domains
chemical_list: D000199:Actins; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D003545:Cysteine