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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.

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Related in: MedlinePlus

Initial characterization of ark1Δ prk1-as mutants. (A) To observe the actin morphology, cells were stained with rhodamine-phalloidin. The range of actin morphologies of ark1Δ prk1-as3 cells treated with 1NA-PP1 for 2 min is shown. A mock-treated cell is also shown. (B) Actin morphology of small-budded ark1Δ prk1-as1 (AS1) and ark1Δ prk1-as3 (AS3) cells was scored as a function of 1NA-PP1 concentration in a blind study (n ≥ 200 for each sample). The cells were treated with the inhibitor for 1 h before fixation. (C and D) ark1Δ PRK1 (WT), ark1Δ prk1-as1, and ark1Δ prk1-as3 cells were cultured without 1NA-PP1 (C), or with the indicated concentration of 1NA-PP1 for 30 min (D), and then processed for anti-Ent1p Western blotting. (E) Ent1 phosphorylation as a function of time in ark1Δ prk1-as1 cells treated with 80 μM 1NA-PP1. (C–E) Phosphorylated (Ent1-P) and unphosphorylated (Ent1) forms of Ent1p are indicated. (F) Actin morphology of small-budded ark1Δ prk1-as3 cells in the presence of 40 μM 1NA-PP1 at indicated time points (n ≥100). (G) Actin morphology of ark1Δ prk1-as3 and ark1Δ prk1-as3 arp2–1. The cells were mock treated or treated with 1NA-PP1 at 25°C for 15 min before fixation. Strains: ark1Δ PRK1, DDY2547; ark1Δ prk1-as1, DDY2595; ark1Δ prk1-as3, DDY2597; ark1Δ prk1-as3 arp2–1, DDY2610. Bars, 5 μm.
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fig1: Initial characterization of ark1Δ prk1-as mutants. (A) To observe the actin morphology, cells were stained with rhodamine-phalloidin. The range of actin morphologies of ark1Δ prk1-as3 cells treated with 1NA-PP1 for 2 min is shown. A mock-treated cell is also shown. (B) Actin morphology of small-budded ark1Δ prk1-as1 (AS1) and ark1Δ prk1-as3 (AS3) cells was scored as a function of 1NA-PP1 concentration in a blind study (n ≥ 200 for each sample). The cells were treated with the inhibitor for 1 h before fixation. (C and D) ark1Δ PRK1 (WT), ark1Δ prk1-as1, and ark1Δ prk1-as3 cells were cultured without 1NA-PP1 (C), or with the indicated concentration of 1NA-PP1 for 30 min (D), and then processed for anti-Ent1p Western blotting. (E) Ent1 phosphorylation as a function of time in ark1Δ prk1-as1 cells treated with 80 μM 1NA-PP1. (C–E) Phosphorylated (Ent1-P) and unphosphorylated (Ent1) forms of Ent1p are indicated. (F) Actin morphology of small-budded ark1Δ prk1-as3 cells in the presence of 40 μM 1NA-PP1 at indicated time points (n ≥100). (G) Actin morphology of ark1Δ prk1-as3 and ark1Δ prk1-as3 arp2–1. The cells were mock treated or treated with 1NA-PP1 at 25°C for 15 min before fixation. Strains: ark1Δ PRK1, DDY2547; ark1Δ prk1-as1, DDY2595; ark1Δ prk1-as3, DDY2597; ark1Δ prk1-as3 arp2–1, DDY2610. Bars, 5 μm.

Mentions: ark1Δ prk1-as1 and ark1Δ prk1-as3 cells showed specific sensitivity to 1NA-PP1. Upon treatment of these mutants with 1NA-PP1, unpolarized actin and actin clumps were observed in a dose-dependent manner (Fig. 1, A and B), indicating that inhibitor treatment mimics the phenotype seen upon loss of Ark1p and Prk1p (Cope et al., 1999). Actin cables appeared to be unaffected by inhibitor addition (Fig. 1 A; +1NA-PP1). As assessed by quantifying the percentage of cells forming actin clumps, the effect of 1NA-PP1 was saturated at 80 μM for ark1Δ prk1-as1, and at 40 μM for ark1Δ prk1-as3 (Fig. 1 B). As a further indication that ark1Δ prk1-as3 cells are more sensitive to the inhibitor, at optimal inhibitor doses, 40 μM for ark1Δ prk1-as3 and 80 μM for ark1Δ prk1-as1, ∼95% of ark1Δ prk1-as3 cells formed actin clumps, whereas ∼80% of ark1Δ prk1-as1 cells formed clumps (Fig. 1 B). The actin cytoskeleton of ark1Δ PRK1 cells was not affected by 40–120 μM 1NA-PP1 (unpublished data).


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)

Initial characterization of ark1Δ prk1-as mutants. (A) To observe the actin morphology, cells were stained with rhodamine-phalloidin. The range of actin morphologies of ark1Δ prk1-as3 cells treated with 1NA-PP1 for 2 min is shown. A mock-treated cell is also shown. (B) Actin morphology of small-budded ark1Δ prk1-as1 (AS1) and ark1Δ prk1-as3 (AS3) cells was scored as a function of 1NA-PP1 concentration in a blind study (n ≥ 200 for each sample). The cells were treated with the inhibitor for 1 h before fixation. (C and D) ark1Δ PRK1 (WT), ark1Δ prk1-as1, and ark1Δ prk1-as3 cells were cultured without 1NA-PP1 (C), or with the indicated concentration of 1NA-PP1 for 30 min (D), and then processed for anti-Ent1p Western blotting. (E) Ent1 phosphorylation as a function of time in ark1Δ prk1-as1 cells treated with 80 μM 1NA-PP1. (C–E) Phosphorylated (Ent1-P) and unphosphorylated (Ent1) forms of Ent1p are indicated. (F) Actin morphology of small-budded ark1Δ prk1-as3 cells in the presence of 40 μM 1NA-PP1 at indicated time points (n ≥100). (G) Actin morphology of ark1Δ prk1-as3 and ark1Δ prk1-as3 arp2–1. The cells were mock treated or treated with 1NA-PP1 at 25°C for 15 min before fixation. Strains: ark1Δ PRK1, DDY2547; ark1Δ prk1-as1, DDY2595; ark1Δ prk1-as3, DDY2597; ark1Δ prk1-as3 arp2–1, DDY2610. Bars, 5 μm.
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Related In: Results  -  Collection

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fig1: Initial characterization of ark1Δ prk1-as mutants. (A) To observe the actin morphology, cells were stained with rhodamine-phalloidin. The range of actin morphologies of ark1Δ prk1-as3 cells treated with 1NA-PP1 for 2 min is shown. A mock-treated cell is also shown. (B) Actin morphology of small-budded ark1Δ prk1-as1 (AS1) and ark1Δ prk1-as3 (AS3) cells was scored as a function of 1NA-PP1 concentration in a blind study (n ≥ 200 for each sample). The cells were treated with the inhibitor for 1 h before fixation. (C and D) ark1Δ PRK1 (WT), ark1Δ prk1-as1, and ark1Δ prk1-as3 cells were cultured without 1NA-PP1 (C), or with the indicated concentration of 1NA-PP1 for 30 min (D), and then processed for anti-Ent1p Western blotting. (E) Ent1 phosphorylation as a function of time in ark1Δ prk1-as1 cells treated with 80 μM 1NA-PP1. (C–E) Phosphorylated (Ent1-P) and unphosphorylated (Ent1) forms of Ent1p are indicated. (F) Actin morphology of small-budded ark1Δ prk1-as3 cells in the presence of 40 μM 1NA-PP1 at indicated time points (n ≥100). (G) Actin morphology of ark1Δ prk1-as3 and ark1Δ prk1-as3 arp2–1. The cells were mock treated or treated with 1NA-PP1 at 25°C for 15 min before fixation. Strains: ark1Δ PRK1, DDY2547; ark1Δ prk1-as1, DDY2595; ark1Δ prk1-as3, DDY2597; ark1Δ prk1-as3 arp2–1, DDY2610. Bars, 5 μm.
Mentions: ark1Δ prk1-as1 and ark1Δ prk1-as3 cells showed specific sensitivity to 1NA-PP1. Upon treatment of these mutants with 1NA-PP1, unpolarized actin and actin clumps were observed in a dose-dependent manner (Fig. 1, A and B), indicating that inhibitor treatment mimics the phenotype seen upon loss of Ark1p and Prk1p (Cope et al., 1999). Actin cables appeared to be unaffected by inhibitor addition (Fig. 1 A; +1NA-PP1). As assessed by quantifying the percentage of cells forming actin clumps, the effect of 1NA-PP1 was saturated at 80 μM for ark1Δ prk1-as1, and at 40 μM for ark1Δ prk1-as3 (Fig. 1 B). As a further indication that ark1Δ prk1-as3 cells are more sensitive to the inhibitor, at optimal inhibitor doses, 40 μM for ark1Δ prk1-as3 and 80 μM for ark1Δ prk1-as1, ∼95% of ark1Δ prk1-as3 cells formed actin clumps, whereas ∼80% of ark1Δ prk1-as1 cells formed clumps (Fig. 1 B). The actin cytoskeleton of ark1Δ PRK1 cells was not affected by 40–120 μM 1NA-PP1 (unpublished data).

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