Biochemical characterization of a Dictyostelium myosin II heavy-chain phosphatase that promotes filament assembly

In Dictyostelium cells, myosin II is found as cytosolic nonassembled monome rs and cytoskeletal bipolar filaments. It is thought that the phosphorylati on state of three threonine residues in the tail of myosin II heavy chain r egulates the molecular motor's assembly state and localization. Phosphoryla tion of the myosin heavy chain at threonine residues 1823, 1833 and 2029 is responsible for maintaining myosin in the nonassembled state, and subseque nt dephosphorylation of these residues is a prerequisite for assembly into the cytoskeleton. We report here the characterization of myosin heavy-chain phosphatase activities in Dictyostelium utilizing myosin II phosphorylated by myosin heavy-chain kinase A as a substrate. One of the myosin heavy-cha in phosphatase activities was identified as protein phosphatase 2A and the purified holoenzyme was composed of a 37-kDa catalytic subunit, a 65-kDa A subunit and a 55-kDa B subunit, The protein phosphatase 2A holoenzyme displ ays two orders of magnitude higher activity towards myosin phosphorylated o n the heavy chains than it does towards myosin phosphorylated on the regula tory light chains, consistent with a role in the control of filament assemb ly. The purified myosin heavy-chain phosphatase activity promotes bipolar f ilament assembly in vitro via dephosphorylation of the myosin heavy chain. This system should provide a valuable model for studying the regulation and localization of protein phosphatase 2A in the context of cytoskeletal reor ganization.