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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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Model for Ark-kinase function in endocytosis. (A) In wild-type cells, Prk1p promotes proper disassembly of the actin-associated endocytic complex. Phosphatase(s) might be responsible for reformation and/or activation of the endocytic complex. Ark1p, a closely related homologue of Prk1p, is likely to perform an overlapping function. (B) When Ark1p and Prk1p are inhibited, vesicles associated with actin filaments and endocytic proteins accumulate. See text for further discussion.
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fig5: Model for Ark-kinase function in endocytosis. (A) In wild-type cells, Prk1p promotes proper disassembly of the actin-associated endocytic complex. Phosphatase(s) might be responsible for reformation and/or activation of the endocytic complex. Ark1p, a closely related homologue of Prk1p, is likely to perform an overlapping function. (B) When Ark1p and Prk1p are inhibited, vesicles associated with actin filaments and endocytic proteins accumulate. See text for further discussion.

Mentions: In this work, we examined the rapid and acute effects of Prk1p kinase inhibition and reactivation by applying a chemical genetics approach. We showed that abnormal actin clumps formed and disappeared within 1 to 2 min of Prk1p inhibition and reactivation, respectively. Further, we showed that the actin clumps contain endocytic proteins and ∼100-nm vesicles. We propose that Prk1p directly regulates the coupling between actin assembly and endocytosis by promoting disassembly and/or inactivation of an early endocytic complex that stimulates actin assembly (Fig. 5 A). When Prk1p is inhibited, this complex is stabilized, and actin assembly continues to be stimulated by endocytic proteins such as the Prk1p target Pan1p and the associated Arp2/3 complex (Zeng and Cai, 1999; Duncan et al., 2001), and/or other targets including Sla1p and Ent1p (Watson et al., 2001; Zeng et al., 2001; Fig. 5 B). In mammalian cells, the μ2 subunit of AP2 is phosphorylated by AAK1 at Thr-156 (ITSQVT156G) (Ricotta et al., 2002). However, the budding yeast AP2 homologue, Apm4p (Huang et al., 1999), is not important for receptor internalization and does not contain potential Prk1p-phosphorylation motifs similar to (L/IxxQxTG). Rather, our genetic experiment showing that Arp2p is required for clump formation supports the idea that Arp2/3-mediated actin assembly is negatively regulated by Prk1p, potentially via phosphorylation of the Arp2/3 activator, Pan1p. Our data also support the proposal that actin participates directly in yeast 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)

Model for Ark-kinase function in endocytosis. (A) In wild-type cells, Prk1p promotes proper disassembly of the actin-associated endocytic complex. Phosphatase(s) might be responsible for reformation and/or activation of the endocytic complex. Ark1p, a closely related homologue of Prk1p, is likely to perform an overlapping function. (B) When Ark1p and Prk1p are inhibited, vesicles associated with actin filaments and endocytic proteins accumulate. See text for further discussion.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Model for Ark-kinase function in endocytosis. (A) In wild-type cells, Prk1p promotes proper disassembly of the actin-associated endocytic complex. Phosphatase(s) might be responsible for reformation and/or activation of the endocytic complex. Ark1p, a closely related homologue of Prk1p, is likely to perform an overlapping function. (B) When Ark1p and Prk1p are inhibited, vesicles associated with actin filaments and endocytic proteins accumulate. See text for further discussion.
Mentions: In this work, we examined the rapid and acute effects of Prk1p kinase inhibition and reactivation by applying a chemical genetics approach. We showed that abnormal actin clumps formed and disappeared within 1 to 2 min of Prk1p inhibition and reactivation, respectively. Further, we showed that the actin clumps contain endocytic proteins and ∼100-nm vesicles. We propose that Prk1p directly regulates the coupling between actin assembly and endocytosis by promoting disassembly and/or inactivation of an early endocytic complex that stimulates actin assembly (Fig. 5 A). When Prk1p is inhibited, this complex is stabilized, and actin assembly continues to be stimulated by endocytic proteins such as the Prk1p target Pan1p and the associated Arp2/3 complex (Zeng and Cai, 1999; Duncan et al., 2001), and/or other targets including Sla1p and Ent1p (Watson et al., 2001; Zeng et al., 2001; Fig. 5 B). In mammalian cells, the μ2 subunit of AP2 is phosphorylated by AAK1 at Thr-156 (ITSQVT156G) (Ricotta et al., 2002). However, the budding yeast AP2 homologue, Apm4p (Huang et al., 1999), is not important for receptor internalization and does not contain potential Prk1p-phosphorylation motifs similar to (L/IxxQxTG). Rather, our genetic experiment showing that Arp2p is required for clump formation supports the idea that Arp2/3-mediated actin assembly is negatively regulated by Prk1p, potentially via phosphorylation of the Arp2/3 activator, Pan1p. Our data also support the proposal that actin participates directly in yeast 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