Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis.
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.
Sekiya-Kawasaki
Mariko
M
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202, USA.
Groen
Aaron Chris
AC
Cope
M Jamie T V
MJ
Kaksonen
Marko
M
Watson
Hadiya A
HA
Zhang
Chao
C
Shokat
Kevan M
KM
Wendland
Beverly
B
McDonald
Kent L
KL
McCaffery
J Michael
JM
Drubin
David G
DG
D000199:Actins / Q000378:metabolism*; D064127:Aurora Kinases; D003599:Cytoskeleton / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011720:Pyrazoles / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D015003:Yeasts / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D000199:Actins; D005656:Fungal Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D011956:Receptors, Cell Surface; C061570:protein kinase N; D020794:Receptor Protein-Tyrosine Kinases; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C