Biochemical characterization of a Dictyostelium myosin II heavy-chain phosphatase that promotes filament assembly.
In Dictyostelium cells, myosin II is found as cytosolic nonassembled monomers and cytoskeletal bipolar filaments. It is thought that the phosphorylation state of three threonine residues in the tail of myosin II heavy chain regulates the molecular motor's assembly state and localization. Phosphorylation of the myosin heavy chain at threonine residues 1823, 1833 and 2029 is responsible for maintaining myosin in the nonassembled state, and subsequent 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-chain 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 displays two orders of magnitude higher activity towards myosin phosphorylated on the heavy chains than it does towards myosin phosphorylated on the regulatory light chains, consistent with a role in the control of filament assembly. The purified myosin heavy-chain phosphatase activity promotes bipolar filament 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 reorganization.
Murphy
M B
MB
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106-4970, USA.
Egelhoff
T T
TT
D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D018995:Myosin Heavy Chains / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D054648:Protein Phosphatase 2; D015800:Protozoan Proteins; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D019319:Okadaic Acid; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D043263:Myosin-Light-Chain Phosphatase; D018995:Myosin Heavy Chains