Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation.
Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyDeltaC1225 and MyDeltaC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.
Liu
X
X
Biomolecular Research Group, National Institute for Advanced Interdisciplinary Research, Higashi 1-1-4 Tsukuba, Ibaraki 305-8562, Japan.
Ito
K
K
Morimoto
S
S
Hikkoshi-Iwane
A
A
Yanagida
T
T
Uyeda
T Q
TQ
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
D000199:Actins; D018994:Myosin Light Chains; D011993:Recombinant Fusion Proteins; D018995:Myosin Heavy Chains