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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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The conserved phosphorylation sites of Ypk1 are required for α-factor internalization. (A) Extracts of ypk1Δ cells expressing HA-tagged wild-type Ypk1, Ypk1T504A, Ypk1T662A, or Ypk1T504A,T662A . (B) The same cells used in A were grown and assayed as in Fig. 1: ypk1Δ (LHY2536, ▪); YPK1 (LHY2563, ♦); ypk1T504A (LHY2568 X); ypk1T662A (LHY2567, □); ypk1T504A,T662A (LHY2569, ⊠).
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fig4: The conserved phosphorylation sites of Ypk1 are required for α-factor internalization. (A) Extracts of ypk1Δ cells expressing HA-tagged wild-type Ypk1, Ypk1T504A, Ypk1T662A, or Ypk1T504A,T662A . (B) The same cells used in A were grown and assayed as in Fig. 1: ypk1Δ (LHY2536, ▪); YPK1 (LHY2563, ♦); ypk1T504A (LHY2568 X); ypk1T662A (LHY2567, □); ypk1T504A,T662A (LHY2569, ⊠).

Mentions: Ypk1 contains two conserved phosphorylation sites, T504 and T662 (Fig. 1 C). T504 is phosphorylated by Pkh1, a yeast homologue of the phosphoinositide-dependent kinase, PDK1 (Casamayor et al., 1999; Inagaki et al., 1999). To determine if either T504 or T662 is involved in the endocytic function of Ypk1, we mutated these residues to alanine alone or in combination. We integrated the constructs into ypk1Δ cells, identified transformants with equivalent expression levels of epitope-tagged Ypk1 variants (Fig. 4 A), and performed α-factor internalization assays on these cells. Cells expressing Ypk1T662A showed a slight defect in internalization, whereas the cells expressing Ypk1T504A or Ypk1T504A,T662A were more strongly impaired (Fig. 4 B). These results indicate that the conserved phosphorylation sites of Ypk1 are required for efficient internalization. T504 appears to play a critical role in internalization, suggesting that phosphorylation by Pkh1 is important for endocytosis. The modest but significant internalization defect observed with cells expressing Ypk1T662A suggests Ypk1 endocytic activity may also be regulated by phosphorylation at T662.


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

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

The conserved phosphorylation sites of Ypk1 are required for α-factor internalization. (A) Extracts of ypk1Δ cells expressing HA-tagged wild-type Ypk1, Ypk1T504A, Ypk1T662A, or Ypk1T504A,T662A . (B) The same cells used in A were grown and assayed as in Fig. 1: ypk1Δ (LHY2536, ▪); YPK1 (LHY2563, ♦); ypk1T504A (LHY2568 X); ypk1T662A (LHY2567, □); ypk1T504A,T662A (LHY2569, ⊠).
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Related In: Results  -  Collection

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fig4: The conserved phosphorylation sites of Ypk1 are required for α-factor internalization. (A) Extracts of ypk1Δ cells expressing HA-tagged wild-type Ypk1, Ypk1T504A, Ypk1T662A, or Ypk1T504A,T662A . (B) The same cells used in A were grown and assayed as in Fig. 1: ypk1Δ (LHY2536, ▪); YPK1 (LHY2563, ♦); ypk1T504A (LHY2568 X); ypk1T662A (LHY2567, □); ypk1T504A,T662A (LHY2569, ⊠).
Mentions: Ypk1 contains two conserved phosphorylation sites, T504 and T662 (Fig. 1 C). T504 is phosphorylated by Pkh1, a yeast homologue of the phosphoinositide-dependent kinase, PDK1 (Casamayor et al., 1999; Inagaki et al., 1999). To determine if either T504 or T662 is involved in the endocytic function of Ypk1, we mutated these residues to alanine alone or in combination. We integrated the constructs into ypk1Δ cells, identified transformants with equivalent expression levels of epitope-tagged Ypk1 variants (Fig. 4 A), and performed α-factor internalization assays on these cells. Cells expressing Ypk1T662A showed a slight defect in internalization, whereas the cells expressing Ypk1T504A or Ypk1T504A,T662A were more strongly impaired (Fig. 4 B). These results indicate that the conserved phosphorylation sites of Ypk1 are required for efficient internalization. T504 appears to play a critical role in internalization, suggesting that phosphorylation by Pkh1 is important for endocytosis. The modest but significant internalization defect observed with cells expressing Ypk1T662A suggests Ypk1 endocytic activity may also be regulated by phosphorylation at T662.

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