Sphingoid base signaling via Pkh kinases is required for endocytosis in yeast

Abstract

In yeast, sphingoid base synthesis is required for the internalization step of endocytosis and organization of the actin cytoskeleton. We show that overexpression of either one of the two kinases Pkh1p or Pkh2p, that are homologous to mammalian 3-phosphoinositide-dependent kinase-1 (PDK1), can specifically suppress the sphingoid base synthesis requirement for endocytosis. Pkh1p and Pkh2p have an overlapping function because only a mutant with impaired function of both kinases is defective for endocytosis. Pkh1/2p kinases are activated in vitro by nanomolar concentrations of sphingoid base. These results suggest that Pkh1/2p kinases are part of a sphingoid base-mediated signaling pathway that is required for the internalization step of endocytosis. The Pkc1p kinase that is phosphorylated by Pkh1/2p kinases and plays a role in endocytosis was identified as one of the downstream effectors of this signaling cascade.

Fig. 1. Overexpression of PKH1 and PKH2 specifically suppresses the lcb1-100 endocytic defect. (A) Strain RH3809 (lcb1) was transformed with high copy plasmids carrying PKH1, PKH2, SKM1, PKC1 or YCK2 kinases genes (Table I), and the corresponding transformants were assayed for α-factor internalization at 37°C and compared with lcb1-100 cells. (B) The sla2-41 (end4-1) and rvs161 (end6-1) temperature-sensitive mutants (RH1597 and RH2082) were transformed by a high-copy number plasmid bearing PKH1 or PKH2 and assayed for α-factor uptake at 37°C.

Fig. 2. PKH1/2 overexpression suppresses the fluid-phase endocytic defect of the lcb1-100 mutant. Wild-type cells (wt, RH448) and lcb1-100 (lcb1, RH3809) cells carrying a PKH1, PKH2 or CKB2 high-copy number plasmid (lcb1+PKH1, lcb1+PKH2 or lcb1+CKB2) were assayed for lucifer yellow (LY) accumulation in the vacuole at 37°C. The same field of cells viewed by fluorescence (left panel) and by Nomarski optics (right panel) is shown.

Fig. 3. (A) Pkh1p and Pkh2p have redundant function in endocytosis. The single mutant pkh1Δ (RH5413) and pkh2Δ (RH5388) cells were assayed for α-factor uptake at 37°C and compared with the wild-type cells (wt, RH2964). (B) Pkh1/2p kinases are required for endocytosis. Radiolabeled α-factor uptake assays were performed at 24°C (open symbols) or 37°C (closed symbols) on wild-type (RH5411) and pkh-ts (RH5412) strains.

Fig. 4. Pkh1/2p kinases are required for fluid-phase endocytosis. Wild-type (wt, RH5411) and pkh-ts (RH5412) cells were incubated with LY for 1 h at 37°C. To visualize LY uptake, cells were viewed by FITC fluorescence optics (left panel). The same fields of cells were viewed by Nomarski optics (right panel) to visualize the vacuoles.

Fig. 5. Pkh1/2p kinase overexpression specifically suppresses the lcb1-100 actin organization defect. Logarithmic cultures of wild-type cells (wt, RH448), lcb1-100, sla2-41 (end4-1) and rvs161 (end6-1) mutant cells (RH3802, RH1597 and RH2082), with or without the PKH1 and PKH2 plasmids, were grown at 24°C, shifted to 37°C for 2 h, fixed, stained with TRITC–phalloidin and observed by fluorescence (actin) and Nomarski microscopy.

Fig. 6. Phytosphingosine activates the Pkh1/2p kinases. (A) Immunoprecipitated Pkh1-HA₃ was incubated with its substrate Pkc1-HA₃ in the presence of [γ-³²P]ATP and increasing amounts of PHS. Phosphorylated Pkc1-HA₃ was revealed by a Cyclone Storage Phosphor Imager (Packard) after separation on a 7.5% SDS–polyacrylamide gel. (B) The amount of radiolabeled [³²P]Pkc1-HA₃ was quantified in each lane and basal phosphorylation of Pkc1p by Pkh1p without PHS was set as 1. (C and D) The same assay was done using Pkh2-HA₃ as the kinase.

Fig. 7. Pkh2p kinase specifically phosphorylates Pkc1p and is not activated by C6-ceramide. (A) Immunoprecipitated Pkh2-HA₃ was incubated with Pkc1-HA₃ in the presence of [γ-³²P]ATP and increasing amounts of C6-ceramide. Phosphorylated Pkc1-HA₃ was revealed by a Cyclone Storage Phosphor Imager (Packard) after separation on a 7.5% SDS–polyacrylamide gel. (B) The amount of radiolabeled [³²P]Pkc1-HA₃ was quantified in each lane and basal phosphorylation of Pkc1p by Pkh2p without C6-ceramide was set to 1. (C) Immunoprecipitated Pkh2-HA₃ or the kinase-dead mutant pkh2-KR-HA₂ was incubated with Pkc1-HA₃ in the presence of [γ-³²P]ATP and increasing amounts of PHS. Phosphorylated Pkc1-HA₃ was revealed by a Cyclone Storage Phosphor Imager (Packard) after separation on a 7.5% SDS–polyacrylamide gel.