Role of B Regulatory Subunits of Protein Phosphatase Type 2A in Myosin II Assembly Control in Dictyostelium discoideum

Abstract

In Dictyostelium discoideum, myosin II resides predominantly in a soluble pool as the result of phosphorylation of the myosin heavy chain (MHC), and dephosphorylation of the MHC is required for myosin II filament assembly, recruitment to the cytoskeleton, and force production. Protein phosphatase type 2A (PP2A) was identified in earlier studies in Dictyostelium as a key biochemical activity that can drive MHC dephosphorylation. We report here gene targeting and cell biological studies addressing the roles of candidate PP2A B regulatory subunits (phr2aB␣ and phr2aB␤) in myosin II assembly control in vivo. Dictyostelium phr2aB␣-and phr2aB␤-null cells show delayed development, reduction in the assembly of myosin II in cytoskeletal ghost assays, and defects in cytokinesis when grown in suspension compared to parental cell lines. These results demonstrate that the PP2A B subunits phr2aB␣ and phr2aB␤ contribute to myosin II assembly control in vivo, with phr2aB␣ having the predominant role facilitating MHC dephosphorylation to facilitate filament assembly.Myosin II plays critical roles in cytokinesis, cortical tension maintenance, cell migration, and multicellular development in Dictyostelium discoideum cells. In vivo myosin II function requires assembly into bipolar filaments, allowing interaction with cortical actin and force production. In resting Dictyostelium cells, a majority of the cell's myosin II resides in a soluble cytosolic pool, which is rapidly assembled into cytoskeletal bipolar filaments during cell motility in response to chemotactic stimuli (1). Phosphorylation of three threonine residues at positions 1823, 1833, and 2029 of the MHC regulates filament assembly and localization of myosin, with phosphorylation driving filament disassembly (5, 7). Dephosphorylation is necessary for filament assembly in vitro and in vivo, and exchange between the assembled and soluble pools in live cells is remarkably rapid, with a half-life (t 1/2 ) of ϳ7 s in live cells (22,23). A series of three MHC kinases (MHCK A, MHCK B, and MHCK C) were identified in previous work and were demonstrated to mediate filament disassembly in live cells (13,23).Although the MHCKs that mediate in vivo assembly control have been established, protein phosphatases involved in assembly control have not been well studied. Murphy and Egelhoff reported PP2A as a major MHC phosphatase in Dictyostelium in studies using purified myosin II and biochemical fractionation approaches (16). In those studies, PP2A holoenzyme displayed substantially higher activity toward myosin phosphorylated on the heavy chains than it did toward myosin phosphorylated on the regulatory light chains (RLC), supporting the hypothesis that PP2A may have a physiological role in the control of filament assembly (16).The heterotrimeric PP2A holoenzyme is a highly conserved regulatory system in diverse eukaryotes. The core PP2A consists of an ϳ36-kDa catalytic subunit (pho2A) tightly associated with a 65-kDa scaffolding A subunit (pppA), forming an AC dimer which then associate...

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