Abstract
The serine/threonine kinase p21-activated kinase 1 (Pak1) controls the actin cytoskeletal and ruffle formation through mechanisms that are independent of GTPase activity. Here we identify filamin FLNa as a Pak1-interacting protein through a yeast two-hybrid screen using the amino terminus of Pak1 as a bait. FLNa is stimulated by physiological signalling molecules to undergo phosphorylation by Pak1 and to interact and colocalize with endogenous Pak1 in membrane ruffles. The ruffle-forming activity of Pak1 is functional in FLNa-expressing cells but not in FLNa-deficient cells. In FLNa, the Pak1-binding site involves tandem repeat 23 in the carboxyl terminus and phosphorylation takes place on serine 2152. The FLNa-binding site in Pak1 is localized between amino acids 52 and 132 in the conserved Cdc42/Rac-interacting (CRIB) domain; accordingly, FLNa binding to the CRIB domain stimulates Pak1 kinase activity. Our results indicate that FLNa may be essential for Pak1-induced cytoskeletal reorganization and that the two-way regulatory interaction between Pak1 and FLNa may contribute to the local stimulation of Pak1 activity and its targets in cytoskeletal structures.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / metabolism*
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Binding Sites
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Cell Line
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Contractile Proteins / chemistry
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Contractile Proteins / genetics
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Contractile Proteins / metabolism*
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Cytoskeleton / metabolism
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Cytoskeleton / ultrastructure
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Enzyme Activation
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Filamins
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Green Fluorescent Proteins
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Humans
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In Vitro Techniques
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Lim Kinases
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Microfilament Proteins / chemistry
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Microfilament Proteins / genetics
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Microfilament Proteins / metabolism*
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Mitogen-Activated Protein Kinases / metabolism
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Mutation
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Phosphorylation
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Protein Kinases / metabolism
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Protein Serine-Threonine Kinases / chemistry
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Signal Transduction
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Substrate Specificity
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Two-Hybrid System Techniques
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p21-Activated Kinases
Substances
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Actins
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Contractile Proteins
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Filamins
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Luminescent Proteins
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Microfilament Proteins
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Recombinant Fusion Proteins
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Green Fluorescent Proteins
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Protein Kinases
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LIMK1 protein, human
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Lim Kinases
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PAK1 protein, human
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Protein Serine-Threonine Kinases
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p21-Activated Kinases
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Mitogen-Activated Protein Kinases