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The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast.

deHart AK, Schnell JD, Allen DA, Hicke L - J. Cell Biol. (2002)

Bottom Line: Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination.Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1.The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast alpha-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base-regulated serine-threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum- and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in alpha-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

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Ypk1 is required for α-factor internalization. (A and B) Internalization of 35S-α-factor was measured by the continuous presence protocol at 37°C (except where noted) after growth in YPUAD. ypk1G490R cells are the same as udi5–1 cells. (A) YPK1 (LHY291, •); ypk1G490R (LHY2543 ⋄); ypk1G490R with pYPK1, a centromeric plasmid (LHY2712, ♦); ypk1G490R (LHY2543, ○, at 24°C). (B) YPK1 YPK2 (LHY2632, •); ypk1Δ YPK2 (LHY2536, □); YPK1 ypk2Δ cells (LHY2633, ⋄). (C) Schematic diagrams of Ypk1, Ypk2 (68% identical to Ypk1), and human SGK (50% identical to Ypk1). Residues mutated in this study and their counterparts in Ypk1 homologues are shown. The percent identity of the kinase domains is shown in the gray box. Phosphorylation sites are indicated with an arrow with known kinases noted.
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fig1: Ypk1 is required for α-factor internalization. (A and B) Internalization of 35S-α-factor was measured by the continuous presence protocol at 37°C (except where noted) after growth in YPUAD. ypk1G490R cells are the same as udi5–1 cells. (A) YPK1 (LHY291, •); ypk1G490R (LHY2543 ⋄); ypk1G490R with pYPK1, a centromeric plasmid (LHY2712, ♦); ypk1G490R (LHY2543, ○, at 24°C). (B) YPK1 YPK2 (LHY2632, •); ypk1Δ YPK2 (LHY2536, □); YPK1 ypk2Δ cells (LHY2633, ⋄). (C) Schematic diagrams of Ypk1, Ypk2 (68% identical to Ypk1), and human SGK (50% identical to Ypk1). Residues mutated in this study and their counterparts in Ypk1 homologues are shown. The percent identity of the kinase domains is shown in the gray box. Phosphorylation sites are indicated with an arrow with known kinases noted.

Mentions: Ubiquitination of the Ste2 cytoplasmic tail is required before internalization (Hicke and Riezman, 1996). We performed a screen of ethyl methanesulfonate–mutagenized cells to identify novel proteins involved in ubiquitin-dependent receptor internalization. One mutant, udi5–1 (ubiquitin-dependent internalization), that was defective in α-factor internalization at both 24°C and 37°C (Fig. 1 A), showed reduced growth on YPUAD + 2 mM EGTA. We screened a genomic DNA library for plasmids that rescued this growth defect and identified a plasmid carrying the YPK1 gene. A centromere-based plasmid carrying YPK1 restored the ability of udi5–1 both to grow on YPUAD + 2 mM EGTA (unpublished data) and to internalize α-factor (Fig. 1 A). A ypk1Δ strain had an internalization defect similar to the udi5–1 strain (Fig. 1 B), suggesting that the mutation in the udi5–1 strain was in YPK1. We rescued the ypk1 gene from udi5–1 cells, and found that it had a single point mutation in the coding region for the Ypk1 catalytic domain that changed glycine 490 to arginine. Expression of Ypk1G490R in ypk1Δ cells did not rescue internalization, whereas expression of Ypk1 did (unpublished data). These results demonstrate that UDI5 is allelic to YPK1, and hereafter we refer to udi5–1 as ypk1G490R.


The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast.

deHart AK, Schnell JD, Allen DA, Hicke L - J. Cell Biol. (2002)

Ypk1 is required for α-factor internalization. (A and B) Internalization of 35S-α-factor was measured by the continuous presence protocol at 37°C (except where noted) after growth in YPUAD. ypk1G490R cells are the same as udi5–1 cells. (A) YPK1 (LHY291, •); ypk1G490R (LHY2543 ⋄); ypk1G490R with pYPK1, a centromeric plasmid (LHY2712, ♦); ypk1G490R (LHY2543, ○, at 24°C). (B) YPK1 YPK2 (LHY2632, •); ypk1Δ YPK2 (LHY2536, □); YPK1 ypk2Δ cells (LHY2633, ⋄). (C) Schematic diagrams of Ypk1, Ypk2 (68% identical to Ypk1), and human SGK (50% identical to Ypk1). Residues mutated in this study and their counterparts in Ypk1 homologues are shown. The percent identity of the kinase domains is shown in the gray box. Phosphorylation sites are indicated with an arrow with known kinases noted.
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Related In: Results  -  Collection

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fig1: Ypk1 is required for α-factor internalization. (A and B) Internalization of 35S-α-factor was measured by the continuous presence protocol at 37°C (except where noted) after growth in YPUAD. ypk1G490R cells are the same as udi5–1 cells. (A) YPK1 (LHY291, •); ypk1G490R (LHY2543 ⋄); ypk1G490R with pYPK1, a centromeric plasmid (LHY2712, ♦); ypk1G490R (LHY2543, ○, at 24°C). (B) YPK1 YPK2 (LHY2632, •); ypk1Δ YPK2 (LHY2536, □); YPK1 ypk2Δ cells (LHY2633, ⋄). (C) Schematic diagrams of Ypk1, Ypk2 (68% identical to Ypk1), and human SGK (50% identical to Ypk1). Residues mutated in this study and their counterparts in Ypk1 homologues are shown. The percent identity of the kinase domains is shown in the gray box. Phosphorylation sites are indicated with an arrow with known kinases noted.
Mentions: Ubiquitination of the Ste2 cytoplasmic tail is required before internalization (Hicke and Riezman, 1996). We performed a screen of ethyl methanesulfonate–mutagenized cells to identify novel proteins involved in ubiquitin-dependent receptor internalization. One mutant, udi5–1 (ubiquitin-dependent internalization), that was defective in α-factor internalization at both 24°C and 37°C (Fig. 1 A), showed reduced growth on YPUAD + 2 mM EGTA. We screened a genomic DNA library for plasmids that rescued this growth defect and identified a plasmid carrying the YPK1 gene. A centromere-based plasmid carrying YPK1 restored the ability of udi5–1 both to grow on YPUAD + 2 mM EGTA (unpublished data) and to internalize α-factor (Fig. 1 A). A ypk1Δ strain had an internalization defect similar to the udi5–1 strain (Fig. 1 B), suggesting that the mutation in the udi5–1 strain was in YPK1. We rescued the ypk1 gene from udi5–1 cells, and found that it had a single point mutation in the coding region for the Ypk1 catalytic domain that changed glycine 490 to arginine. Expression of Ypk1G490R in ypk1Δ cells did not rescue internalization, whereas expression of Ypk1 did (unpublished data). These results demonstrate that UDI5 is allelic to YPK1, and hereafter we refer to udi5–1 as ypk1G490R.

Bottom Line: Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination.Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1.The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast alpha-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base-regulated serine-threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum- and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in alpha-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

Show MeSH