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.
Skip Nav Destination
Article navigation
1 September 2003
Report|
September 02 2003
Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis
Mariko Sekiya-Kawasaki,
Mariko Sekiya-Kawasaki
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
Search for other works by this author on:
Aaron Chris Groen,
Aaron Chris Groen
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
Search for other works by this author on:
M. Jamie T.V. Cope,
M. Jamie T.V. Cope
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
Search for other works by this author on:
Marko Kaksonen,
Marko Kaksonen
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
Search for other works by this author on:
Hadiya A. Watson,
Hadiya A. Watson
4Department of Biology, The Johns Hopkins University, Baltimore, MD 21218
Search for other works by this author on:
Chao Zhang,
Chao Zhang
3Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143
Search for other works by this author on:
Kevan M. Shokat,
Kevan M. Shokat
3Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143
Search for other works by this author on:
Beverly Wendland,
Beverly Wendland
4Department of Biology, The Johns Hopkins University, Baltimore, MD 21218
Search for other works by this author on:
Kent L. McDonald,
Kent L. McDonald
2Electron Microscope Laboratory, University of California, Berkeley, Berkeley, CA 94720
Search for other works by this author on:
J. Michael McCaffery,
J. Michael McCaffery
5Integrated Imaging Center, The Johns Hopkins University, Baltimore, MD 21218
Search for other works by this author on:
David G. Drubin
David G. Drubin
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
Search for other works by this author on:
Mariko Sekiya-Kawasaki
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
Aaron Chris Groen
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
M. Jamie T.V. Cope
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
Marko Kaksonen
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
Hadiya A. Watson
4Department of Biology, The Johns Hopkins University, Baltimore, MD 21218
Chao Zhang
3Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143
Kevan M. Shokat
3Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143
Beverly Wendland
4Department of Biology, The Johns Hopkins University, Baltimore, MD 21218
Kent L. McDonald
2Electron Microscope Laboratory, University of California, Berkeley, Berkeley, CA 94720
J. Michael McCaffery
5Integrated Imaging Center, The Johns Hopkins University, Baltimore, MD 21218
David G. Drubin
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
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: [email protected]
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.
Received:
May 15 2003
Accepted:
July 21 2003
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2003
J Cell Biol (2003) 162 (5): 765–772.
Article history
Received:
May 15 2003
Accepted:
July 21 2003
Citation
Mariko Sekiya-Kawasaki, Aaron Chris Groen, M. Jamie T.V. Cope, Marko Kaksonen, Hadiya A. Watson, Chao Zhang, Kevan M. Shokat, Beverly Wendland, Kent L. McDonald, J. Michael McCaffery, David G. Drubin; Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis . J Cell Biol 1 September 2003; 162 (5): 765–772. doi: https://doi.org/10.1083/jcb.200305077
Download citation file:
Sign in
Don't already have an account? Register
Client Account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.
Sign in via your Institution
Sign in via your InstitutionEmail alerts
Advertisement
Advertisement
