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

Sekiya-Kawasaki M, Groen AC, Cope MJ, Kaksonen M, Watson HA, Zhang C, Shokat KM, Wendland B, McDonald KL, McCaffery JM, Drubin DG - J. Cell Biol. (2003)

Bottom Line: Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202, USA.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Defective receptor-mediated endocytosis upon inhibition of Prk1p activity. Internalization of 35S-labeled α-factor was measured. In A and B, the time course of internalization was started after 30 min incubation with 1NA-PP1 or mock treatment. (A) ark1Δ PRK1 (WT) and ark1Δ prk1-as3 (AS3) cells without 1NA-PP1. (B) ark1Δ PRK1 (WT) and ark1Δ prk1-as3 (AS3) cells treated with 1NA-PP1. (C) ark1Δ PRK1(WT) and ark1Δ prk1D159A cells. Error bars represent the SD from at least three experiments. The experiment with ark1Δ PRK1 in B was performed twice. Strains: ark1Δ PRK1, DDY2607; ark1Δ prk1-as3, DDY2608; ark1Δ prk1D159A, DDY2609.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172809&req=5

fig3: Defective receptor-mediated endocytosis upon inhibition of Prk1p activity. Internalization of 35S-labeled α-factor was measured. In A and B, the time course of internalization was started after 30 min incubation with 1NA-PP1 or mock treatment. (A) ark1Δ PRK1 (WT) and ark1Δ prk1-as3 (AS3) cells without 1NA-PP1. (B) ark1Δ PRK1 (WT) and ark1Δ prk1-as3 (AS3) cells treated with 1NA-PP1. (C) ark1Δ PRK1(WT) and ark1Δ prk1D159A cells. Error bars represent the SD from at least three experiments. The experiment with ark1Δ PRK1 in B was performed twice. Strains: ark1Δ PRK1, DDY2607; ark1Δ prk1-as3, DDY2608; ark1Δ prk1D159A, DDY2609.

Mentions: Further, we tested whether Prk1p kinase activity is required for α-factor internalization by its receptor, Ste2p. Because of its high sensitivity to 1NA-PP1, all subsequent analyses used the prk1-as3 allele. Without inhibitor, ark1Δ prk1-as3 cells show internalization kinetics indistinguishable from ark1Δ PRK1 (Fig. 3 A). Treatment of ark1Δ prk1-as3 cells with 1NA-PP1 for 30 min specifically inhibited receptor internalization (Fig. 3 B). Even 120 μM inhibitor did not affect α-factor internalization by ark1Δ PRK1 cells (Fig. 3 B). Additionally, a kinase-dead mutant (ark1Δ prk1D159A) also showed a severe block of receptor internalization (Fig. 3 C). Thus, the inhibition of Prk1p kinase activity profoundly blocks the internalization step of receptor-mediated endocytosis.


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

Sekiya-Kawasaki M, Groen AC, Cope MJ, Kaksonen M, Watson HA, Zhang C, Shokat KM, Wendland B, McDonald KL, McCaffery JM, Drubin DG - J. Cell Biol. (2003)

Defective receptor-mediated endocytosis upon inhibition of Prk1p activity. Internalization of 35S-labeled α-factor was measured. In A and B, the time course of internalization was started after 30 min incubation with 1NA-PP1 or mock treatment. (A) ark1Δ PRK1 (WT) and ark1Δ prk1-as3 (AS3) cells without 1NA-PP1. (B) ark1Δ PRK1 (WT) and ark1Δ prk1-as3 (AS3) cells treated with 1NA-PP1. (C) ark1Δ PRK1(WT) and ark1Δ prk1D159A cells. Error bars represent the SD from at least three experiments. The experiment with ark1Δ PRK1 in B was performed twice. Strains: ark1Δ PRK1, DDY2607; ark1Δ prk1-as3, DDY2608; ark1Δ prk1D159A, DDY2609.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172809&req=5

fig3: Defective receptor-mediated endocytosis upon inhibition of Prk1p activity. Internalization of 35S-labeled α-factor was measured. In A and B, the time course of internalization was started after 30 min incubation with 1NA-PP1 or mock treatment. (A) ark1Δ PRK1 (WT) and ark1Δ prk1-as3 (AS3) cells without 1NA-PP1. (B) ark1Δ PRK1 (WT) and ark1Δ prk1-as3 (AS3) cells treated with 1NA-PP1. (C) ark1Δ PRK1(WT) and ark1Δ prk1D159A cells. Error bars represent the SD from at least three experiments. The experiment with ark1Δ PRK1 in B was performed twice. Strains: ark1Δ PRK1, DDY2607; ark1Δ prk1-as3, DDY2608; ark1Δ prk1D159A, DDY2609.
Mentions: Further, we tested whether Prk1p kinase activity is required for α-factor internalization by its receptor, Ste2p. Because of its high sensitivity to 1NA-PP1, all subsequent analyses used the prk1-as3 allele. Without inhibitor, ark1Δ prk1-as3 cells show internalization kinetics indistinguishable from ark1Δ PRK1 (Fig. 3 A). Treatment of ark1Δ prk1-as3 cells with 1NA-PP1 for 30 min specifically inhibited receptor internalization (Fig. 3 B). Even 120 μM inhibitor did not affect α-factor internalization by ark1Δ PRK1 cells (Fig. 3 B). Additionally, a kinase-dead mutant (ark1Δ prk1D159A) also showed a severe block of receptor internalization (Fig. 3 C). Thus, the inhibition of Prk1p kinase activity profoundly blocks the internalization step of receptor-mediated endocytosis.

Bottom Line: Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus