Abstract
In both yeast and mammals, endocytic internalization is accompanied by a transient burst of actin polymerization. The yeast protein kinases Prk1p and Ark1p, which are related to the mammalian proteins GAK and AAK1, are key regulators of this process. However, the molecular mechanism(s) by which they regulate actin assembly at endocytic sites have not yet been determined. The Eps15-like yeast protein Pan1p is a Prk1p substrate that is essential for endocytic internalization and for proper actin organization. Pan1p is an Arp2/3 activator and here we show that this activity is dependent on F-actin binding. Mutation of all 15 Prk1p-targeted threonines in Pan1p to alanines mimicked the ark1Delta prk1Delta phenotype, demonstrating that Pan1p is a key Prk1p target in vivo. Moreover, phosphorylation by Prk1p inhibited the ability of Pan1p to bind to F-actin and to activate the Arp2/3 complex, thereby identifying the endocytic phosphoregulation mechanism of Prk1p. We conclude that Prk1p phosphorylation of Pan1p shuts off Arp2/3-mediated actin polymerization on endocytic vesicles, allowing them to fuse with endosomes.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Actin-Related Protein 2
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Actin-Related Protein 3
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Actins / chemistry
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Actins / metabolism
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Adaptor Proteins, Signal Transducing / metabolism
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Alanine / chemistry
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Amino Acid Motifs
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Amino Acid Sequence
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Animals
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Apoptosis Regulatory Proteins
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Calcium-Binding Proteins / metabolism
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Cytoskeletal Proteins / metabolism*
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DNA / chemistry
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Drosophila melanogaster
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Electrophoresis, Polyacrylamide Gel
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Endocytosis
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Endosomes / metabolism
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Fungal Proteins / chemistry
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Green Fluorescent Proteins / metabolism
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Humans
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Immunoblotting
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Intracellular Signaling Peptides and Proteins
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Microscopy, Fluorescence
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Models, Biological
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Molecular Sequence Data
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Mutation
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Open Reading Frames
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Phenotype
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Phosphoproteins / metabolism
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Phosphorylation
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Protein Kinase C / metabolism
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Protein Structure, Tertiary
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Saccharomyces cerevisiae / metabolism
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Sequence Homology, Amino Acid
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Time Factors
Substances
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ACTR2 protein, human
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ACTR3 protein, human
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Actin-Related Protein 2
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Actin-Related Protein 3
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Actins
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Adaptor Proteins, Signal Transducing
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Apoptosis Regulatory Proteins
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Calcium-Binding Proteins
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Cytoskeletal Proteins
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EPS15 protein, human
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Fungal Proteins
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Intracellular Signaling Peptides and Proteins
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NLRP2 protein, human
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Phosphoproteins
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Green Fluorescent Proteins
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DNA
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protein kinase N
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Protein Kinase C
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Alanine