Molecular genetic truncation analysis of filament assembly and phosphorylation domains of Dictyostelium myosin heavy chain.
Conventional myosin ('myosin II') is a major component of the cytoskeleton in a wide variety of eukaryotic cells, ranging from lower amoebae to mammalian fibroblasts and neutrophils. Gene targeting technologies available in the Dictyostelium discoideum system have provided the first genetic proof that this molecular motor protein is essential for normal cytokinesis, capping of cell surface receptors, normal chemotactic cell locomotion and morphogenetic shape changes during development. Although the roles of myosin in a variety of cell functions are becoming clear, the mechanisms that regulate myosin assembly into functional bipolar filaments within cells are poorly understood. Dictyostelium is currently the only system where mutant forms of myosin can be engineered in vitro, then expressed in their native context in cells that are devoid of the wild-type isoform. We have utilized this technology in combination with nested truncation and deletion analysis to map domains of the myosin tail necessary for in vivo and in vitro filament assembly, and for normal myosin heavy chain (MHC) phosphorylation. This analysis defines a region of 35 amino acids within the tail that is critical for filament formation both for purified myosin molecules and for myosin within the in vivo setting. Phosphorylation analysis of these mutants in intact cytoskeletons demonstrates that the carboxy-terminal tip of the myosin heavy chain is required for complete phosphorylation of the myosin tail.
Lee
R J
RJ
Department of Biochemistry, Stanford University School of Medicine, CA.
Egelhoff
T T
TT
Spudich
J A
JA
D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics; D003599:Cytoskeleton / Q000737:chemistry*; D004023:Dictyostelium / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D018390:Gene Targeting; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009218:Myosins / Q000737:chemistry / Q000235:genetics*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary
D005656:Fungal Proteins; D008565:Membrane Proteins; D009218:Myosins