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Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology.

ABSTRACT: 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.

SUBMITTER: Oliver CJ

PROVIDER: S-EPMC133892 | biostudies-literature | 2002 Jul

REPOSITORIES: biostudies-literature

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Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology.

Oliver Carey J CJ 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

Molecular and cellular biology 20020701 13

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-term ...[more]

PMID: 12052877

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Project description:Phagocytosis is a cellular process crucial for recognition and removal of apoptotic cells and foreign particles, subsequently initiating appropriate immune responses. The process of phagocytosis is highly complex and involves major rearrangements of the cytoskeleton. Due to its complexity and importance for tissue homoeostasis and immune responses, it is tightly regulated. Over the last decade, microRNAs (miRNAs) have emerged as important regulators of biological pathways including the immune response by fine-tuning expression of gene regulatory networks. In order to identify miRNAs implicated in the regulation of phagocytosis, a systematic screening of all currently known, human miRNAs was performed using THP-1 macrophage-like cells and serum-opsonized latex beads. Of the total of 2,566 miRNAs analyzed, several led to significant changes in phagocytosis. Among these, we validated miR-124-5p as a novel regulator of phagocytosis. Transfection with miR-124-5p mimics reduced the number of phagocytic cells as well as the phagocytic activity of phorbol-12-myristate-13-acetate (PMA)-activated THP-1 cells and ex vivo differentiated primary human macrophages. In silico analysis suggested that miR-124-5p targets genes involved in regulation of the actin cytoskeleton. Transcriptional analyses revealed that expression of genes encoding for several subunits of the ARP2/3 complex, a crucial regulator of actin polymerization, is reduced upon transfection of cells with miR-124-5p. Further in silico analyses identified potential binding motifs for miR-124-5p in the mRNAs of these genes. Luciferase reporter assays using these binding motifs indicate that at least two of the genes (ARPC3 and ARPC4) are direct targets of miR-124-5p. Moreover, ARPC3 and ARPC4 protein levels were significantly reduced following miR-124-5p transfection. Collectively, the presented results suggest that miR-124-5p regulates phagocytosis in human macrophages by directly targeting expression of components of the ARP2/3 complex.

Dataset Information

Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology.

Publications

Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology.

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