In both yeast and mammals, endocytic internalization is accompanied by a transient burst of actin polymerization1,2. The yeast protein kinases Prk1p and Ark1p, which are related to the mammalian proteins GAK and AAK1, are key regulators of this process3,4,5,6. 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 organization7,8,9. 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 ark1Δ prk1Δ 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.
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We are extremely grateful to M. Duncan for technical advice and helpful suggestions. We thank B. L. Goode for providing the plasmid for TEV–Myc-tagged protein overexpression; M. Sekiya-Kawasaki for advice and for sharing GFP-tagging oligonucleotides and plasmids; D. Nix for providing purified Prk1p; C. Toret and Y. Sun for technical advice on the α-factor uptake assay; and M. Kaksonen for technical advice on microscopy. We also thank the members of the Drubin/Barnes laboratory for helpful discussions, D. Siekhaus and M. Sekiya-Kawasaki for critical reading of the manuscript, and K. Mizuno for encouragement. This work was supported by the Toyobo Biotechnology Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and the Yamanouchi Foundation for Research on Metabolic Disorders to J.T.; the Uehara Memorial Foundation to J.Y.T.; and National Institutes of Health grants to D.G.D.
Supplementary Figures S1, S2, S3 and Table S1 (PDF 1019 kb)
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