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Published 2 September 2003. doi:10.1083/jcb.200305077
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© The Rockefeller University Press, 0021-9525/2003/9/765 $5.00
The Journal of Cell Biology, Volume 162, Number 5, 765-772

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Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis



Mariko Sekiya-Kawasaki1, Aaron Chris Groen1, M. Jamie T.V. Cope1, Marko Kaksonen1, Hadiya A. Watson4, Chao Zhang3, Kevan M. Shokat3, Beverly Wendland4, Kent L. McDonald2, J. Michael McCaffery5 and David G. Drubin1

1 Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
2 Electron Microscope Laboratory, University of California, Berkeley, Berkeley, CA 94720
3 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143
4 Department of Biology, The Johns Hopkins University, Baltimore, MD 21218
5 Integrated Imaging Center, The Johns Hopkins University, Baltimore, MD 21218

Address correspondence to D.G. Drubin, Dept. of Molecular and Cell Biology, 16 Barker Hall, University of California, Berkeley, Berkeley, CA 94720-3202. Tel.: (510) 642-3692. Fax: (510) 643-0062. email: drubin{at}uclink4.berkeley.edu

We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch–related process, and propose that Prk1p negatively regulates the actin assembly–stimulating activity of endocytic proteins.

Key Words: protein kinases; Eps15; Arp2/3; endocytosis; Saccharomyces cerevisiae

The online version of this article includes supplemental material.

Abbreviations used in this paper: 1NA-PP1, 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine; Ark, actin-regulating kinase.


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