Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae

Abstract

The Orm family proteins are conserved integral membrane proteins of the endoplasmic reticulum that are key homeostatic regulators of sphingolipid biosynthesis. Orm proteins bind to and inhibit serine:palmitoyl-coenzyme A transferase, the first enzyme in sphingolipid biosynthesis. In Saccharomyces cerevisiae, Orm1 and Orm2 are inactivated by phosphorylation in response to compromised sphingolipid synthesis (e.g., upon addition of inhibitor myriocin), thereby restoring sphingolipid production. We show here that protein kinase Ypk1, one of an essential pair of protein kinases, is responsible for this regulatory modification. Myriocin-induced hyperphosphorylation of Orm1 and Orm2 does not occur in ypk1 cells, and immunopurified Ypk1 phosphorylates Orm1 and Orm2 robustly in vitro exclusively on three residues that are known myriocin-induced sites. Furthermore, the temperature-sensitive growth of ypk1(ts) ypk2 cells is substantially ameliorated by deletion of ORM genes, confirming that a primary physiological role of Ypk1-mediated phosphorylation is to negatively regulate Orm function. Ypk1 immunoprecipitated from myriocin-treated cells displays a higher specific activity for Orm phosphorylation than Ypk1 from untreated cells. To identify the mechanism underlying Ypk1 activation, we systematically tested several candidate factors and found that the target of rapamycin complex 2 (TORC2) kinase plays a key role. In agreement with prior evidence that a TORC2-dependent site in Ypk1(T662) is necessary for cells to exhibit a wild-type level of myriocin resistance, a Ypk1(T662A) mutant displays only weak Orm phosphorylation in vivo and only weak activation in vitro in response to sphingolipid depletion. Additionally, sphingolipid depletion increases phosphorylation of Ypk1 at T662. Thus, Ypk1 is both a sensor and effector of sphingolipid level, and reduction in sphingolipids stimulates Ypk1, at least in part, via TORC2-dependent phosphorylation.

Fig. 1.

Genetic epistasis indicates Ypk action negatively regulates Orm function. (A) Primary structure (Left) and predicted topology (Right) of Orm proteins. Presumptive transmembrane segments (green); candidate Ypk1 sites (red). (B) Alignment of the cytosolic N termini of Orm1 and Orm2. Identities (bold blue); conservative substitutions (bold black); three nested consensus Ypk1 phosphorylation sites (bold red). (C) Absence of Orm1 and Orm2 compensates for Ypk1 deficiency. Serial 10-fold dilutions of strains of the indicated genotypes were spotted on yeast extract peptone dextrose (YPD) plates and photographed after incubation for 2 d at the indicated temperatures.

Fig. 2.

Ypk1 phosphorylates the N terminus of Orm1 exclusively at Ser51, Ser52, and Ser53. (A) GST-Orm1(1–85) or GST-Orm2(1–80), purified from E. coli, were incubated with [γ-³²P]ATP and either Ypk1 or an analog-sensitive (as) variant, Ypk1(L424A), purified from S. cerevisiae, in the absence or presence of 3-MOB-PP1, and the products resolved by SDS/PAGE and analyzed as described in Materials and Methods. (B) As in A, except that ATP was used and the products were analyzed by immunoblotting with anti–phospho-AKT substrate antibody (Cell Signaling Technology, Inc.) and with anti-GST antibody. (C) As in A, except that GST-Orm1(1–85) contained the indicated site-directed mutations.

Fig. 3.

Ypk1 is required for Orm phosphorylation and is activated by sphingolipid depletion. (A) Wild-type (YDB146), ypk1∆ (YDB344), and ypk2∆ (YDB340) cells, each expressing (FLAG)₃-Orm1 from the ORM1 promoter at the ORM1 locus, were grown to midexponential phase in YPD and then treated with myriocin (0.4 μM). At the indicated times, samples were withdrawn, lysates prepared, and the resulting extracts resolved and analyzed with anti-FLAG antibodies as described previously (4). (B) Wild-type (BY4741) cells expressing from the GAL1 promoter either Ypk1-myc or a catalytically inactive (KD) mutant, Ypk1(K376A)-myc, were grown to midexponential phase, and a portion of each culture was then treated with (+) myriocin in methanol (1.25 μM final concentration) or (−) an equal volume of the same solvent only. After 2 h, all of the samples were lysed and Ypk1 was recovered by immunoprecipitation with mouse ascites fluid containing anti–c-Myc mAb 9E10. The resulting immunoprecipitates were then incubated with [γ-³²P]ATP and GST-Orm2(1–80) as described in Materials and Methods.

Fig. 4.

Sphingolipid depletion induces Tor2-dependent phosphorylation of Ypk1. (A) Wild-type cells (BY4741) or an otherwise isogenic tor2^ts mutant expressing either Ypk1^11A-myc (662T) or Ypk1^11A(T662A)-myc (662A) were grown at 26 °C and then treated with (+) myriocin (1.25 μM) or (−) solvent alone. After 2 h, the cells were lysed and the resulting extracts were resolved by phosphate-affinity SDS/PAGE and analyzed by immunoblotting with anti–c-myc mAb 9E10. (B) As in A, except that the cells expressing Ypk1^11A-myc were strain JRY8012 (pdr5∆ snq2∆ yor1∆) to reduce ATP-binding cassette transporter-mediated drug efflux. Cells were grown at 30 °C and treated with Tor inhibitor NVP-BEZ235 in DMSO (2 μM final concentration) or with an equal volume of the same solvent alone (0). When both drugs were present, myriocin and NVP-BEZ235 were added at the same time. (C) Serial 10-fold dilutions of tor2^ts or wild-type (BY4741) cells carrying pRS316 (empty vector) or expressing from the same vector a hyperactive Ypk1 variant, Ypk1(D242A) (pFR273), were spotted on plates lacking (Left) or containing myriocin (0.6 μM) (Right). After incubation for 2 d at the indicated temperatures, the plates were photographed. (D) Wild-type (BY4741) cells carrying pRS316 (vector) or expressing Ypk1(D242A) from the same vector were plated as lawns on plates lacking (0) or containing the indicated final concentration (0.3 or 0.6 μM) of myriocin and then overlaid with sterile filter discs on which was spotted in the same final volume (10 μL) either solvent alone (0) or 1- or 10-μL samples of a stock (10 mM) of NVP-BEZ235 in DMSO. After incubation at 30 °C for 3 d, the plates were photographed.

Fig. 5.

Tor2-dependent phosphorylation of Ypk1 is necessary for optimal Orm phosphorylation. (A) As in Fig. 3A, except that the ypk1∆ cells expressing (FLAG)₃-Orm1 (YDB344) cells also carried plasmids expressing either wild-type Ypk1 (WT) (pAM20) or a Ypk1(T662A) mutant (pFR221). (B) As in Fig. 3B, except the cells (BY4741) expressed from the GAL1 promoter were either Ypk1-myc (pAM54) or Ypk1(T662A)-myc (pFR119). (C) Sphingolipid deficiency stimulates Ypk1-mediated phosphorylation of Orm proteins via TORC2. Apparent sites of action of phosphoprotein phosphatases are also indicated.