Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology.
Neurabin I, a neuronal actin-binding protein, binds protein phosphatase 1 (PP1) and p70 ribosomal S6 protein kinase (p70S6K), both proteins implicated in cytoskeletal dynamics. We expressed wild-type and mutant neurabins fused to green fluorescent protein in Cos7, HEK293, and hippocampal neurons. Biochemical and cellular studies showed that an N-terminal F-actin-binding domain dictated neurabin I localization at actin cytoskeleton and promoted disassembly of stress fibers. Deletion of the C-terminal coiled-coil and sterile alpha motif domains abolished neurabin I dimerization and induced filopodium extension. Immune complex assays showed that neurabin I recruited an active PP1 via a PP1-docking sequence,(457)KIKF(460). Mutation of the PP1-binding motif or PP1 inhibition by okadaic acid and calyculin A abolished filopodia and restored stress fibers in cells expressing neurabin I. In vitro and in vivo studies suggested that the actin-binding domain attenuated protein kinase A (PKA) phosphorylation of neurabin I. Modification of a major PKA site, serine-461, impaired PP1 binding. Finally, p70S6K was excluded from neurabin I/PP1 complexes and required the displacement of PP1 for recruitment to neurabin I. These studies provided new insights into the assembly and regulation of a neurabin I/PP1 complex that controls actin rearrangement to promote spine development in mammalian neurons.
Oliver
Carey J
CJ
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Terry-Lorenzo
Ryan T
RT
Elliott
Elizabeth
E
Bloomer
Wendy A Christensen
WA
Li
Shi
S
Brautigan
David L
DL
Colbran
Roger J
RJ
Shenolikar
Shirish
S
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D019281:Dimerization; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D054645:Protein Phosphatase 1; D051381:Rats; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D015398:Signal Transduction; D022502:Stress Fibers / Q000378:metabolism / Q000648:ultrastructure; D000099273:Neurabins
D000199:Actins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D010749:Phosphoprotein Phosphatases; D010750:Phosphoproteins; D054645:Protein Phosphatase 1; D019893:Ribosomal Protein S6 Kinases; D000099273:Neurabins