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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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Pkh kinases are required for receptor-mediated and fluid-phase endocytosis. (A) Growth of pkh1Δ pkh2Δ cells and pkh1Δ pkh2Δ leu2::PKH1 cells from the same tetrad (LHY2716 and LHY2714, respectively) or pkhts (pkh1D398G pkh2Δ) cells (LHY3030) and an isogenic pkh2Δ strain (LHY3032) on YPUAD at 24°C or 30°C or on YPUAD + 1.2 M sorbitol at 30°C after 4 d. The differences in strain background between the two pkh mutants are likely to account for the difference in suppression of the growth defect on sorbitol medium. (B) The same pkh1Δ pkh2Δ (♦) and pkh1Δ pkh2Δ leu2::PKH1 (⋄) strains as in (A) were grown overnight in YPUAD + 1.2M sorbitol. Internalization of 35S-α-factor was measured by the continuous presence protocol at 30°C in YPUAD + 1.2 M sorbitol. (C) LY localization was assayed for wild-type cells (LHY2762), pkh1Δ cells (LHY2759), pkh2Δ cells (LHY2760), pkh1Δ pkh2Δ cells (LHY2716), and end4Δ cells (LHY37). Cells were grown to early logarithmic phase in YPUAD + 1.2 M sorbitol at 24°C, shifted to 30°C for 15 min, and then incubated with LY at 30°C for 60 min. Images were taken using DIC optics (top) and fluorescence optics (bottom). The pkh1Δ pkh2Δ cells that are brightly stained throughout the whole cell are probably lysed cells. (D) pkh1Δ cells (LHY3031, •), pkh2Δ cells (LHY3032, ▪), and pkhts cells (LHY3030, ♦) were grown overnight in YPUAD. Internalization of 35S-α-factor was measured by the continuous presence protocol at 30°C in YPUAD.
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fig5: Pkh kinases are required for receptor-mediated and fluid-phase endocytosis. (A) Growth of pkh1Δ pkh2Δ cells and pkh1Δ pkh2Δ leu2::PKH1 cells from the same tetrad (LHY2716 and LHY2714, respectively) or pkhts (pkh1D398G pkh2Δ) cells (LHY3030) and an isogenic pkh2Δ strain (LHY3032) on YPUAD at 24°C or 30°C or on YPUAD + 1.2 M sorbitol at 30°C after 4 d. The differences in strain background between the two pkh mutants are likely to account for the difference in suppression of the growth defect on sorbitol medium. (B) The same pkh1Δ pkh2Δ (♦) and pkh1Δ pkh2Δ leu2::PKH1 (⋄) strains as in (A) were grown overnight in YPUAD + 1.2M sorbitol. Internalization of 35S-α-factor was measured by the continuous presence protocol at 30°C in YPUAD + 1.2 M sorbitol. (C) LY localization was assayed for wild-type cells (LHY2762), pkh1Δ cells (LHY2759), pkh2Δ cells (LHY2760), pkh1Δ pkh2Δ cells (LHY2716), and end4Δ cells (LHY37). Cells were grown to early logarithmic phase in YPUAD + 1.2 M sorbitol at 24°C, shifted to 30°C for 15 min, and then incubated with LY at 30°C for 60 min. Images were taken using DIC optics (top) and fluorescence optics (bottom). The pkh1Δ pkh2Δ cells that are brightly stained throughout the whole cell are probably lysed cells. (D) pkh1Δ cells (LHY3031, •), pkh2Δ cells (LHY3032, ▪), and pkhts cells (LHY3030, ♦) were grown overnight in YPUAD. Internalization of 35S-α-factor was measured by the continuous presence protocol at 30°C in YPUAD.

Mentions: Due to the importance of T504, we investigated the role of Pkh1 in endocytosis. Pkh1 shares significant homology with two other yeast kinases, Pkh2 and Pkh3. Functions of these kinases are at least partially redundant because mutations in individual PKH genes are not lethal, whereas a pkh1Δ pkh2Δ mutant is dead or slow growing (Casamayor et al., 1999; Inagaki et al., 1999; see below). We tested the ability of pkh1Δ, pkh2Δ, and pkh3Δ cells to internalize α-factor and deliver LY to the vacuole. None of the single pkh mutants showed a defect in either assay as compared with isogenic wild-type cells (unpublished data). We then created double mutants to examine if Pkh kinases function redundantly in internalization. It has been reported that cells carrying a deletion of both PKH1 and PKH2 are inviable (Casamayor et al., 1999; Inagaki et al., 1999). In our genetic background, pkh1Δ pkh2Δ cells were viable, but grew slowly at 24°C (Fig. 5 A). We found that the slow growth phenotype of pkh1Δ pkh2Δ cells could be suppressed substantially by 1.2 M sorbitol, even at 30°C (Inagaki et al., 1999; Fig. 5 A). pkh1Δ pkh2Δ cells grown in sorbitol displayed a strong defect in α-factor internalization and in accumulation of LY in the vacuole (Fig. 5, B and C). By contrast, pkh1Δ pkh2Δ cells grown in sorbitol were not generally defective in protein transport processes because they efficiently transported carboxypeptidase Y to the vacuole (unpublished data) and they appeared morphologically normal (Fig. 5 C). To support these findings, we obtained a mutant strain that is temperature sensitive for Pkh function (pkh1D398G pkh2Δ) (Inagaki et al., 1999), but which grows normally at 24°C (Fig. 5 A). The pkh1ts strain was severely defective for α-factor internalization at the nonpermissive growth temperature of 30°C (Fig. 5 D). Like ypk1 mutants, pkh mutants showed no defect in Ste2 phosphorylation or ubiquitination (unpublished data). These results indicate that the Pkh family of kinases is required for endocytosis, and suggest that at least one of their roles is to activate Ypk1 by phosphorylating T504.


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

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

Pkh kinases are required for receptor-mediated and fluid-phase endocytosis. (A) Growth of pkh1Δ pkh2Δ cells and pkh1Δ pkh2Δ leu2::PKH1 cells from the same tetrad (LHY2716 and LHY2714, respectively) or pkhts (pkh1D398G pkh2Δ) cells (LHY3030) and an isogenic pkh2Δ strain (LHY3032) on YPUAD at 24°C or 30°C or on YPUAD + 1.2 M sorbitol at 30°C after 4 d. The differences in strain background between the two pkh mutants are likely to account for the difference in suppression of the growth defect on sorbitol medium. (B) The same pkh1Δ pkh2Δ (♦) and pkh1Δ pkh2Δ leu2::PKH1 (⋄) strains as in (A) were grown overnight in YPUAD + 1.2M sorbitol. Internalization of 35S-α-factor was measured by the continuous presence protocol at 30°C in YPUAD + 1.2 M sorbitol. (C) LY localization was assayed for wild-type cells (LHY2762), pkh1Δ cells (LHY2759), pkh2Δ cells (LHY2760), pkh1Δ pkh2Δ cells (LHY2716), and end4Δ cells (LHY37). Cells were grown to early logarithmic phase in YPUAD + 1.2 M sorbitol at 24°C, shifted to 30°C for 15 min, and then incubated with LY at 30°C for 60 min. Images were taken using DIC optics (top) and fluorescence optics (bottom). The pkh1Δ pkh2Δ cells that are brightly stained throughout the whole cell are probably lysed cells. (D) pkh1Δ cells (LHY3031, •), pkh2Δ cells (LHY3032, ▪), and pkhts cells (LHY3030, ♦) were grown overnight in YPUAD. Internalization of 35S-α-factor was measured by the continuous presence protocol at 30°C in YPUAD.
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fig5: Pkh kinases are required for receptor-mediated and fluid-phase endocytosis. (A) Growth of pkh1Δ pkh2Δ cells and pkh1Δ pkh2Δ leu2::PKH1 cells from the same tetrad (LHY2716 and LHY2714, respectively) or pkhts (pkh1D398G pkh2Δ) cells (LHY3030) and an isogenic pkh2Δ strain (LHY3032) on YPUAD at 24°C or 30°C or on YPUAD + 1.2 M sorbitol at 30°C after 4 d. The differences in strain background between the two pkh mutants are likely to account for the difference in suppression of the growth defect on sorbitol medium. (B) The same pkh1Δ pkh2Δ (♦) and pkh1Δ pkh2Δ leu2::PKH1 (⋄) strains as in (A) were grown overnight in YPUAD + 1.2M sorbitol. Internalization of 35S-α-factor was measured by the continuous presence protocol at 30°C in YPUAD + 1.2 M sorbitol. (C) LY localization was assayed for wild-type cells (LHY2762), pkh1Δ cells (LHY2759), pkh2Δ cells (LHY2760), pkh1Δ pkh2Δ cells (LHY2716), and end4Δ cells (LHY37). Cells were grown to early logarithmic phase in YPUAD + 1.2 M sorbitol at 24°C, shifted to 30°C for 15 min, and then incubated with LY at 30°C for 60 min. Images were taken using DIC optics (top) and fluorescence optics (bottom). The pkh1Δ pkh2Δ cells that are brightly stained throughout the whole cell are probably lysed cells. (D) pkh1Δ cells (LHY3031, •), pkh2Δ cells (LHY3032, ▪), and pkhts cells (LHY3030, ♦) were grown overnight in YPUAD. Internalization of 35S-α-factor was measured by the continuous presence protocol at 30°C in YPUAD.
Mentions: Due to the importance of T504, we investigated the role of Pkh1 in endocytosis. Pkh1 shares significant homology with two other yeast kinases, Pkh2 and Pkh3. Functions of these kinases are at least partially redundant because mutations in individual PKH genes are not lethal, whereas a pkh1Δ pkh2Δ mutant is dead or slow growing (Casamayor et al., 1999; Inagaki et al., 1999; see below). We tested the ability of pkh1Δ, pkh2Δ, and pkh3Δ cells to internalize α-factor and deliver LY to the vacuole. None of the single pkh mutants showed a defect in either assay as compared with isogenic wild-type cells (unpublished data). We then created double mutants to examine if Pkh kinases function redundantly in internalization. It has been reported that cells carrying a deletion of both PKH1 and PKH2 are inviable (Casamayor et al., 1999; Inagaki et al., 1999). In our genetic background, pkh1Δ pkh2Δ cells were viable, but grew slowly at 24°C (Fig. 5 A). We found that the slow growth phenotype of pkh1Δ pkh2Δ cells could be suppressed substantially by 1.2 M sorbitol, even at 30°C (Inagaki et al., 1999; Fig. 5 A). pkh1Δ pkh2Δ cells grown in sorbitol displayed a strong defect in α-factor internalization and in accumulation of LY in the vacuole (Fig. 5, B and C). By contrast, pkh1Δ pkh2Δ cells grown in sorbitol were not generally defective in protein transport processes because they efficiently transported carboxypeptidase Y to the vacuole (unpublished data) and they appeared morphologically normal (Fig. 5 C). To support these findings, we obtained a mutant strain that is temperature sensitive for Pkh function (pkh1D398G pkh2Δ) (Inagaki et al., 1999), but which grows normally at 24°C (Fig. 5 A). The pkh1ts strain was severely defective for α-factor internalization at the nonpermissive growth temperature of 30°C (Fig. 5 D). Like ypk1 mutants, pkh mutants showed no defect in Ste2 phosphorylation or ubiquitination (unpublished data). These results indicate that the Pkh family of kinases is required for endocytosis, and suggest that at least one of their roles is to activate Ypk1 by phosphorylating T504.

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
Related in: MedlinePlus