The Sphingoid Long Chain Base Phytosphingosine Activates AGC-type Protein Kinases in Saccharomyces cerevisiae Including Ypk1, Ypk2, and Sch9
2005; Elsevier BV; Volume: 280; Issue: 24 Linguagem: Inglês
10.1074/jbc.m502972200
ISSN1083-351X
AutoresKe Liu, Xiping Zhang, Robert L. Lester, Robert C. Dickson,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe Pkh1 protein kinase of Saccharomyces cerevisiae, a homolog of the mammalian 3-phosphoinositide-dependent kinase (PDK1), regulates downstream AGC-type protein kinases including Ypk1/2 and Pkc1, which control cell wall integrity, growth, and other processes. Phytosphingosine (PHS), a sphingoid long chain base, is hypothesized to be a lipid activator of Pkh1 and thereby controls the activity of Ypk1/2. Here we present biochemical evidence supporting this hypothesis, and in addition we demonstrate that PHS also stimulates autophosphorylation and activation of Ypk1/2. Greatest stimulation of Ypk1/2 phosphorylation and activity are achieved by inclusion of both PHS and Pkh1 in an in vitro kinase reaction. We also demonstrate for the first time that Pkh1 phosphorylates the Sch9 protein kinase in vitro and that such phosphorylation is stimulated by PHS. This is the first biochemical demonstration of Sch9 activators, and the results further support roles for long chain bases in heat stress resistance in addition to implying roles in chronological aging and cell size determination, since Sch9 functions in these processes. Thus, our data support a model in which PHS, rather than simply being an upstream activator of Pkh1, also activates kinases that are downstream targets of Pkh1 including Ypk1/2 and Sch9. The Pkh1 protein kinase of Saccharomyces cerevisiae, a homolog of the mammalian 3-phosphoinositide-dependent kinase (PDK1), regulates downstream AGC-type protein kinases including Ypk1/2 and Pkc1, which control cell wall integrity, growth, and other processes. Phytosphingosine (PHS), a sphingoid long chain base, is hypothesized to be a lipid activator of Pkh1 and thereby controls the activity of Ypk1/2. Here we present biochemical evidence supporting this hypothesis, and in addition we demonstrate that PHS also stimulates autophosphorylation and activation of Ypk1/2. Greatest stimulation of Ypk1/2 phosphorylation and activity are achieved by inclusion of both PHS and Pkh1 in an in vitro kinase reaction. We also demonstrate for the first time that Pkh1 phosphorylates the Sch9 protein kinase in vitro and that such phosphorylation is stimulated by PHS. This is the first biochemical demonstration of Sch9 activators, and the results further support roles for long chain bases in heat stress resistance in addition to implying roles in chronological aging and cell size determination, since Sch9 functions in these processes. Thus, our data support a model in which PHS, rather than simply being an upstream activator of Pkh1, also activates kinases that are downstream targets of Pkh1 including Ypk1/2 and Sch9. Sphingolipids play many vital roles in eucaryotic cells where they are structural components of membranes, act as regulators of signal transduction pathways, and associate with sterols to form lipid rafts (1Ohanian J. Ohanian V. Cell. Mol. Life Sci. 2001; 58: 2053-2068Crossref PubMed Scopus (228) Google Scholar, 2Futerman A.H. Hannun Y.A. EMBO Rep. 2004; 5: 777-782Crossref PubMed Scopus (541) Google Scholar). Sphingosine 1-phosphate and ceramide are the best characterized sphingolipid signaling molecules, with sphingosine 1-phosphate promoting growth and preventing apoptosis and ceramide performing an opposing role by promoting apoptosis (3Spiegel S. Milstien S. Nat. Rev. Mol. Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1768) Google Scholar, 4Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (678) Google Scholar). In contrast to the large body of knowledge for ceramide and sphingosine 1-phosphate, the long chain base (LCB) 1The abbreviations used are: LCB, long chain base; AGC, protein kinases A, G, and C; PHS, phytosphingosine; DHS, dihydrosphingosine; PDK1, 3-phosphoinositide-dependent kinase; KD, kinase dead; PH, pleckstrin homology; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid; PKC, protein kinase C; YPD, yeast extract, peptone, and glucose. sphingosine, derived from breakdown of ceramide and then used to make sphingosine 1-phosphate, is a poorly characterized signaling molecule in mammals (5Cuvillier O. Biochim. Biophys. Acta. 2002; 1585: 153-162Crossref PubMed Scopus (290) Google Scholar). The opposite situation exists in the budding yeast Saccharomyces cerevisiae where very little is known about the signaling functions of ceramide and long chain base phosphates, but signaling functions for the long chain bases phytosphingosine (PHS) and dihydrosphingosine (DHS), the homologs of sphingosine, are being revealed. Here we present evidence that PHS is a signaling molecule that activates the protein kinase Pkh1, a homolog of the mammalian 3-phosphoinositide-dependent protein kinase (PDK1), which then activates downstream protein kinases including Ypk1, Ypk2, and Sch9. Many different growth and survival factors activate PDK1 in mammals so that it phosphorylates and activates downstream protein kinases, several of which are members of the protein kinase A, protein kinase G, and protein kinase C (AGC) kinase family that controls multiple cellular processes (6Mora A. Komander D. van Aalten D.M. Alessi D.R. Semin. Cell Dev. Biol. 2004; 15: 161-170Crossref PubMed Scopus (671) Google Scholar). Homologs of PDK1 are widespread in nature, including two in S. cerevisiae, Pkh1 and Pkh2 (7Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Deletion of either PKH1 or PKH2 has little or no effect on cell growth, but deletion of both is lethal, indicating that they perform one or more essential functions. Pkh1 and Pkh2 share about 50% amino acid identity and about 70% amino acid similarity with human PDK1. In addition, a human PDK1 cDNA complements the growth defect of a yeast pkh1Δ pkh2Δ double deletion mutant, demonstrating functional similarity of the human and yeast PDKs (7Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The best characterized downstream substrate of Pkh1 and Pkh2 is the protein kinase Pkc1 (8Inagaki M. Schmelzle T. Yamaguchi K. Irie K. Hall M.N. Matsumoto K. Mol. Cell. Biol. 1999; 19: 8344-8352Crossref PubMed Scopus (117) Google Scholar), which regulates a mitogen-activated protein kinase module that controls cell wall integrity (9Levin D.E. Bartlett-Heubusch E. J. Cell Biol. 1992; 116: 1221-1229Crossref PubMed Scopus (303) Google Scholar, 10Paravicini G. Cooper M. Friedli L. Smith D.J. Carpentier J.L. Klig L.S. Payton M.A. Mol. Cell. Biol. 1992; 12: 4896-4905Crossref PubMed Scopus (183) Google Scholar). Pkc1 controls many other cellular processes, but the mechanisms are less well characterized (11Schmitz H.P. Heinisch J.J. Curr. Genet. 2003; 43: 245-254Crossref PubMed Scopus (46) Google Scholar). In addition to Pkc1, S. cerevisiae contains three other AGC kinase family members, Ypk1, Ypk2/Ykr2, and Sch9, that have been suggested to be substrates of Pkh1/2 based on the similarity of the amino acid sequence of their activation loop, including the threonine (termed the PDK1 site) phosphorylated in Pkc1 by Pkh1/2 (7Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Ypk1 was first shown both in vitro and in vivo to be a substrate for Pkh1 (7Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Some data support the idea that Pkh1 preferentially phosphorylates Ypk1, whereas Pkh2 phosphorylates Ypk2 (12Roelants F.M. Torrance P.D. Bezman N. Thorner J. Mol. Biol. Cell. 2002; 13: 3005-3028Crossref PubMed Scopus (139) Google Scholar). Ypk1 and its paralog, Ypk2, perform one or more functions necessary for growth, possibly a function in cell wall integrity (12Roelants F.M. Torrance P.D. Bezman N. Thorner J. Mol. Biol. Cell. 2002; 13: 3005-3028Crossref PubMed Scopus (139) Google Scholar, 13Schmelzle T. Helliwell S.B. Hall M.N. Mol. Cell. Biol. 2002; 22: 1329-1339Crossref PubMed Scopus (108) Google Scholar). Human serum- and glucocorticoid-inducible kinase (SGK) is a functional homolog of Ypk1 (7Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). In their seminal studies Casamayor et al. (7Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) noted that 3-phosphoinositides, which are the upstream signal for activating mammalian PDK1, did not stimulate the activity of Pkh1, probably because it lacks a pleckstrin homology (PH) domain, such as is found in PDK1 and Akt/protein kinase B. Thus, the activating signal for Pkh1/2 has remained unknown. The first clue about the nature of the activating signal came from an analysis of multicopy suppressor genes that enabled yeast cells to grow in the presence of an inhibitory concentration of myriocin (14Sun Y. Taniguchi R. Tanoue D. Yamaji T. Takematsu H. Mori K. Fujita T. Kawasaki T. Kozutsumi Y. Mol. Cell. Biol. 2000; 20: 4411-4419Crossref PubMed Scopus (102) Google Scholar), an antibiotic that inhibits serine palmitoyltransferase, the first enzyme in the sphingolipid biosynthetic pathway (15Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 2002; 1583: 13-25Crossref PubMed Scopus (199) Google Scholar). YPK1 was identified as such a suppressor gene, and indirect evidence suggested that its phosphorylation was stimulated by a sphingolipid, possibly by PHS. Because Pkh1 was known to phosphorylate Ypk1 and since 3-phosphoinositides did not stimulate phosphorylation (7Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), Sun et al. (14Sun Y. Taniguchi R. Tanoue D. Yamaji T. Takematsu H. Mori K. Fujita T. Kawasaki T. Kozutsumi Y. Mol. Cell. Biol. 2000; 20: 4411-4419Crossref PubMed Scopus (102) Google Scholar) suggested that yeast use sphingolipids instead of phosphoinositides as activating lipids. The first clue that LCBs were the activating sphingolipid came from studies of a temperature-sensitive strain (lcb1-100) having a block in endocytosis at 37 °C (16Friant S. Lombardi R. Schmelzle T. Hall M.N. Riezman H. EMBO J. 2001; 20: 6783-6792Crossref PubMed Scopus (143) Google Scholar). LCB1 encodes a subunit of serine palmitoyltransferase and at the restrictive temperature an lcb1-100 strain stops making sphingolipids (17Zanolari B. Friant S. Funato K. Sutterlin C. Stevenson B.J. Riezman H. EMBO J. 2000; 19: 2824-2833Crossref PubMed Scopus (210) Google Scholar), and instead of producing a transient increase in LCBs, as occurs in wild type cells during heat shock (18Dickson R.C. Nagiec E.E. Skrzypek M. Tillman P. Wells G.B. Lester R.L. J. Biol. Chem. 1997; 272: 30196-30200Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 19Jenkins G.M. Richards A. Wahl T. Mao C.G. Obeid L. Hannun Y. J. Biol. Chem. 1997; 272: 32566-32572Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), the concentration of LCBs drops rapidly (20Hearn J.D. Lester R.L. Dickson R.C. J. Biol. Chem. 2003; 278: 3679-3686Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The endocytosis defect was found to be suppressed by multiple copies of PKC1, YCK2, PKH1, or PKH2 (16Friant S. Lombardi R. Schmelzle T. Hall M.N. Riezman H. EMBO J. 2001; 20: 6783-6792Crossref PubMed Scopus (143) Google Scholar, 21Friant S. Zanolari B. Riezman H. EMBO J. 2000; 19: 2834-2844Crossref PubMed Scopus (80) Google Scholar, 22deHart A.K. Schnell J.D. Allen D.A. Hicke L. J. Cell Biol. 2002; 156: 241-248Crossref PubMed Scopus (89) Google Scholar). How multiple copies of these genes restore endocytosis is unclear, but the data suggested that the activity of these kinases was stimulated by some sphingolipid, and it was found using immunoprecipitated HA-tagged proteins that LCBs gave a 2–3-fold stimulation in Pkc1 phosphorylation by Pkh1 or Pkh2 (16Friant S. Lombardi R. Schmelzle T. Hall M.N. Riezman H. EMBO J. 2001; 20: 6783-6792Crossref PubMed Scopus (143) Google Scholar). To further our understanding of the signaling pathways and cellular processes regulated by LCBs we set out using purified proteins to directly demonstrate that LCBs stimulate phosphorylation and activation of Ypk1, Ypk2, and Sch9 by Pkh1. In addition to showing that LCBs stimulate Pkh1 activity, we also found that LCBs stimulate autophosphorylation and activation of Ypk1, Ypk2, and Sch9. These results show that rather than acting solely as an upstream activator of signaling pathways regulated by Pkh1, LCBs also act downstream to activate kinase substrates of Pkh1. Our data are the first biochemical demonstration of upstream regulators of Sch9, an important protein kinase, related to mammalian Akt/protein kinase B, that plays poorly defined roles in heat stress resistance (23Morano K.A. Thiele D.J. EMBO J. 1999; 18: 5953-5962Crossref PubMed Scopus (56) Google Scholar, 24Sasaki T. Toh-e A. Kikuchi Y. Mol. Gen. 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Cell. 2004; 15: 5295-5305Crossref PubMed Scopus (124) Google Scholar), homologous recombination in ribosomal gene hot spots (30Prusty R. Keil R.L. Mol. Genet. Genomics. 2004; 272: 264-274Crossref PubMed Scopus (13) Google Scholar), and adaptation to changes in nutrients (31Roosen J. Engelen K. Marchal K. Mathys J. Griffioen G. Cameroni E. Thevelein J.M. De Virgilio C. De Moor B. Winderickx J. Mol. Microbiol. 2005; 55: 862-880Crossref PubMed Scopus (142) Google Scholar). Strains, Plasmids, and Media—Cells were grown in YPD (1% yeast extract, 2% peptone, 2% glucose) or complete synthetic medium containing 0.34% yeast nitrogen base (Difco), 1% ammonium sulfate, 2% glucose, 400 mg/liter serine, 200 mg/liter threonine, 150 mg/liter valine, 30 mg/liter aspartic acid and glutamic acid, 50 mg/liter phenylalanine, 30 mg/liter adenine, isoleucine, and tyrosine, and 20 mg/liter each of arginine, histidine, leucine, lysine, methionine, uracil, and tryptophan. Synthetic medium lacking uracil (SD-Ura) was used to select cells transformed with a plasmid carrying URA3. Solid media contained 2% agar. Six histidine residues were added to the N terminus of proteins (Pkh1, Pkh1-KD, Pkh2, Pkh2-KD, Ypk1, Ypk1-KD, Ypk2, Ypk2-KD, Sch9 and Sch9-KD) by cloning genes into pYES2/NTA (Invitrogen) so that gene expression was driven by the galactose-inducible GAL1 promoter. His6-tagged proteins were expressed in yeast strains RCD224 (MATa leu2-3, 112 ura3-52 trp1 his4 rme1), BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), or BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0). Catalytically inactive (kinase dead, KD) versions of Ypk1 (K376R) (14Sun Y. Taniguchi R. Tanoue D. Yamaji T. Takematsu H. Mori K. Fujita T. Kawasaki T. Kozutsumi Y. Mol. Cell. Biol. 2000; 20: 4411-4419Crossref PubMed Scopus (102) Google Scholar), Ypk2 (K373R), Pkh1 (K154R), and Pkh2 (K208R) (8Inagaki M. Schmelzle T. Yamaguchi K. Irie K. Hall M.N. Matsumoto K. Mol. Cell. Biol. 1999; 19: 8344-8352Crossref PubMed Scopus (117) Google Scholar) and phosphorylation site mutants of Ypk1 (T504A), Ypk1 (T662A), Ypk1 (T405AT662A), Ypk1 (T504D), Ypk1 (T662D), and Ypk1 (T405D T662D) were generated by using the Gene-Editor™ in vitro site-directed mutagenesis system (Promega, Madison, WI). Mutant constructs were verified by DNA sequence analysis. The wild type and an Sch9 (K441R) kinase dead mutant were expressed as N-terminal epitope-tagged proteins using a 3HA-SCH9 allele carried on a plasmid (p426-HA3-SCH9) (23Morano K.A. Thiele D.J. EMBO J. 1999; 18: 5953-5962Crossref PubMed Scopus (56) Google Scholar). Purification of His6-tagged Proteins—Yeast cells transformed with a multicopy vector expressing the desired gene under control of the GAL1 promoter were grown overnight at 30 °C to an A600 nm of 0.4 in 1 liter of S-Ura medium containing 2% sucrose and 0.1% glucose. Protein overproduction was induced by adding galactose to 2% and incubating the culture for 8–10 h. A cell-free protein extract was prepared as described previously (8Inagaki M. Schmelzle T. Yamaguchi K. Irie K. Hall M.N. Matsumoto K. Mol. Cell. Biol. 1999; 19: 8344-8352Crossref PubMed Scopus (117) Google Scholar) and loaded onto a 0.8 × 4-cm Poly-Prep column (Bio-Rad) containing 1 ml of nickel nitrilotriacetic acid-agarose (Qiagen, Valencia, CA). The flow-thru was collected and passed through the column again followed by 5 ml of wash buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 20 mm imidazole, 20% glycerol. His6-tagged proteins were eluted with 3 ml of buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 200 mm imidazole, 20% glycerol) except His6-Pkh2, which was eluted with 3 ml of a slightly different buffer (50 mm Tris-HCl, pH 7.5, 300 mm NaCl, 150 mm imidazole). Six 0.5-ml fractions were collected and analyzed by SDS-PAGE and immunoblotting (32Zhang X. Lester R.L. Dickson R.C. J. Biol. Chem. 2004; 279: 22030-22038Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Three fractions with the highest level of tagged protein were pooled, separated into aliquots, and stored at –80 °C. His6- and 3HA-tagged proteins were also purified by immunoprecipitation from cell-free yeast extracts as described previously (16Friant S. Lombardi R. Schmelzle T. Hall M.N. Riezman H. EMBO J. 2001; 20: 6783-6792Crossref PubMed Scopus (143) Google Scholar). In Vitro Protein Kinase Assay—An appropriate amount of kinase and substrate, determined in preliminary experiments, were combined in 18 μl of phosphorylation buffer and warmed to 30 °C before starting the reaction by adding 2 μl of ATP solution (1 mm ATP, 4 μCi of [γ-32P]ATP). The final component concentrations were 50 mm MOPS, pH 7.5, 1 mm dithiothreitol, 10 mm magnesium acetate and 100 μm ATP. The reaction was stopped by the addition of Laemmli buffer followed by SDS-PAGE and phosphorimage analysis to quantify phosphorylation of substrate. Two-step Ypk1/Ypk2 Kinase Activity Assay—Affinity-purified His6-Ypk1 or His6-Ypk2 (1 μg) were activated by incubation with His6-Pkh1 (50 ng) and 50 μm PHS (stock prepared in 95% EtOH and diluted to a final concentration of 1%) in 200 μl of kinase assay buffer (50 mm MOPS, pH 7.5, 1 mm dithiothreitol, and 10 mm magnesium acetate) plus 100 μm ATP at 30 °C. After 30 min the reactions were chilled on ice, and 1 ml of immunoprecipitation buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40) plus 2 μg of anti-His antibody (Amersham, #27-4710-0) and 10 μl of 50% protein A beads were added to each tube. Tubes were rotated at 4 °C overnight, and the immunoprecipitates were washed 3 times with immunoprecipitation buffer and 2 times with kinase assay buffer before suspension in 50 μl of kinase assay buffer. The kinase activity of Ypk1 or Ypk2 was assayed by adding 30 μl of a kinase buffer with 2.5 μm PKI-tide (Alexis Biochemicals, CA), 1 μm microcystin-LR (Alexis Biochemicals), 10 mm magnesium acetate, 100 μm [γ-32P]ATP (4μCi), and 100 μm Cross-tide (GRPRTSSFAEG, BIOSOURCE Int.) as substrate (7Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). After incubation for 15 min at 30 °C, the reaction mixture was centrifuged for 20 s at top speed in a microcentrifuge, and 20 μl of supernatant fluid was spotted onto phosphocellulose paper (2.5 cm circles, Whatman P81) which was processed as described previously (33Alessi D.R. Cohen P. Ashworth A. Cowley S. Leevers S.J. Marshall C.J. Methods Enzymol. 1995; 255: 279-290Crossref PubMed Scopus (155) Google Scholar). The pellets were treated with Laemmli buffer followed by SDS-PAGE to analyze the amount of Ypk1 or Ypk2 in each reaction. No effort was made to separate Ypk1 and Pkh1 because control reactions showed that Pkh1 did not phosphorylate Cross-tide. One unit of kinase activity is defined as the amount of protein required to catalyze phosphorylation of 1 nmol of Cross-tide/min. Two-step Sch9 Kinase Activity Assay—3HA-Sch9 was isolated by immunoprecipitation using an anti-HA monoclonal antibody (Anti-Rat, Roche Applied Science) and IgG-Sepharose beads (Sigma). In the first step of the reaction, immunoprecipitated 3HA-Sch9 (50 ng) was incubated at 30 °C with 2.5 ng of His6-Pkh1 or His6-Pkh2 in a 20-μl reaction containing or lacking 50 μm PHS (stock prepared in 95% EtOH and diluted to a final concentration of 1%) and kinase assay buffer (final concentrations were 40 mm MOPS, pH 7.5, 1 mm dithiothreitol, and 10 mm magnesium acetate). After 30 min the reactions were centrifuged at 3000 rpm for 3 min in a microcentrifuge at 4 °C. The supernatant was discarded, and the beads were resuspended in 500 μl of kinase buffer followed by centrifugation. This washing procedure was repeated a total of three times. In the second step of the reaction, the resuspended beads were incubated at 30 °C for 30 min in a 20-μl reaction containing 1.5 μg of Lsp1 (the molar ratio of Sch9:Lsp1 was 1:100), 50 μm PHS, or 1% ethanol (final concentration), and ATP (4 μCi of [γ-32P]ATP and 100 μm ATP). The reaction was stopped by the addition of Laemmli buffer followed by SDS-PAGE and phosphorimage analysis to quantify the phosphorylation of Lsp1. Miscellaneous Assays—To quantify purified His6-tagged yeast proteins, serial dilutions were resolved on SDS-PAGE along with dilutions of a BSA standard (Bio-Rad). A scanned image of the Coomassie Brilliant Blue-stained gel was analyzed, and the protein quantities of specific bands were determined by using ImageQuant software. LCBs Stimulate Phosphorylation of Ypk1 and Ypk2—Available published data imply that phosphorylation of Ypk1 and Ypk2 by Pkh1 and Pkh2 is stimulated by LCBs. However, there has been no direct biochemical demonstration of this hypothesis. To determine whether LCBs stimulate Pkh1 and phosphorylate the downstream kinases Ypk1 and Ypk2, we performed in vitro kinase assays using His6-tagged, partially purified protein kinases. We focus primarily on Pkh1, since it has functional overlap with Pkh2 and appears to be more important (12Roelants F.M. Torrance P.D. Bezman N. Thorner J. Mol. Biol. Cell. 2002; 13: 3005-3028Crossref PubMed Scopus (139) Google Scholar). We found that phosphorylation of Ypk1 increased linearly for about 40 min when both PHS and Pkh1 were included in the kinase reaction (Fig. 1A). Less phosphorylation was observed when either PHS or Pkh1 were omitted from the reaction, and the lowest level of phosphorylation was observed when only Ypk1 was present in the reaction. To prove that phosphorylation of Ypk1 was due to Pkh1 and not to a contaminating protein kinase, a Pkh1 kinase dead variant (KD, K154R) was examined and found to give about the same level of phosphorylation as in a kinase reaction lacking Pkh1 but containing Ypk1 and PHS (Fig. 1, compare panels A and B). Weak phosphorylation in the Ypk1-only reaction is either due to autophosphorylation or to a contaminating kinase (Fig. 1A), since it was also seen when Ypk1 was incubated with a Pkh1 kinase dead variant (Fig. 1B). We conclude from these results that PHS produces a 5–8-fold stimulation of Ypk1 phosphorylation by Pkh1. In addition, PHS produces an approximate 2-fold stimulation of Ypk1 autophosphorylation either in the absence of wild type Pkh1 (Fig. 1A) or in the presence of the kinase dead mutant (Fig. 1B). Qualitatively similar results were observed when Ypk2 was used as a substrate for Pkh1 (Fig. 1C). Phosphorylation of Ypk2 was barely stimulated by Pkh1 but was stimulated about 2-fold when PHS was added to the reaction. Adding both PHS and Pkh1 to the reaction gave a further 2-fold stimulation in phosphorylation for a combined 4-fold stimulation. To prove that Pkh1 and not a contaminating protein kinase was phosphorylated Ypk2, a Pkh1 kinase dead variant (KD, K208R) was compared with the wild type enzyme. The reaction containing the kinase dead variant did show some phosphorylation of Ypk2 that was stimulated 2-fold by PHS (Fig. 1D). Weak phosphorylation in the Ypk2 reaction is either due to autophosphorylation or to a contaminating kinase, which seems to be slightly stimulated by PHS. Panels E and F in Fig. 1 show representative phosphorimages of the 32P-labeled Ypk1 and Ypk2 proteins produced in kinase reactions. Reactions containing PHS or Pkh1 show two Ypk1 bands of about equal concentration. In reactions containing both PHS and Pkh1 there is an increase in the amount of the upper Ypk1 band, indicating that this species of Ypk1 protein is more phosphorylated than is the lower, faster migrating species. Ypk2 appears as a broader more diffuse band that becomes more phosphorylated in the reaction containing both PHS and Ypk2. The data shown in panels A–D (Fig. 1) represent values for the lower plus the upper band of Ypk1 and the broad band of Ypk2. Pkh1-mediated phosphorylation of Ypk1 was examined in more detail by varying the concentration and type of LCB added to the kinase reaction. PHS at 50 μm stimulated phosphorylation of Ypk1 slightly better than 10 μm, although 100 μm barely stimulated (Fig. 2A). DHS, lacking the 4-OH group found on PHS, was tested as the biological erythro isomer and the non-biological threo isomer. Surprisingly, the threo isomer stimulated Ypk1 phosphorylation better than the erythro isomer (Fig. 2, compare panels B and C), suggesting that DHS may not be as significant as PHS in activating Ypk1 phosphorylation in vivo. The phosphorylated version of DHS, DHS-1-P, showed no stimulation of phosphorylation and neither did the acylated version of sphingosine, C6-ceramide (Fig. 2, D and E), indicating that the free amino and C1-OH groups on PHS are important for kinase stimulation. Stearylamine, a long chain amine, stimulated phosphorylation, but unlike the LCBs, there was no inhibition of phosphorylation at the highest concentration used. This difference suggests that stearylamine and LCBs work in different ways to stimulate phosphorylation. Finally, sphingosine, the LCB found in mammalian but not fungal sphingolipids, was the best stimulator of Ypk1 phosphorylation (Fig. 2G), giving almost twice the stimulation as 50 μm PHS. LCBs and Pkh1 Activate Ypk1 and Ypk2 in Vitro—We next examined what effects LCBs and Pkh1 have on Ypk1 kinase activity by using the artificial substrate, Cross-tide, used previously to assay activation of Ypk1 by Pkh1 and Pkh2 (7Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 12Roelants F.M. Torrance P.D. Bezman N. Thorner J. Mol. Biol. Cell. 2002; 13: 3005-3028Crossref PubMed Scopus (139) Google Scholar, 32Zhang X. Lester R.L. Dickson R.C. J. Biol. Chem. 2004; 279: 22030-22038Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 34Roelants F.M. Torrance P.D. Thorner J. Microbiology. 2004; 150: 3289-3304Crossref PubMed Scopus (91) Google Scholar). The activation assay was done is two steps. In step one, Ypk1 or Ypk2 was incubated with or without Pkh1 or PHS in the presence of ATP so that Ypk1 or Ypk2 could be activated by phosphorylation. In the second step, Ypk1 or Ypk2 was immunoprecipitated, and the precipitate was used in a kinase assay to quantify the transfer of 32P from [γ-32P]ATP to Cross-tide. Ypk1 was activated 4-fold by either PHS or Pkh1, and inclusion of both in the reaction produced an 8-fold, additive increase in Ypk1 activity (Fig. 3). Activation by Pkh1 required its kinase activity, since the K154R kinase dead variant did not activate Ypk1. It should be noted that Pkh1 was also immunoprecipitated, but control reactions showed that it did not phosphorylate Cross-tide. Ypk2 was activated 2–3-fold by PHS but was not activated by Pkh1 alone. However, when both PHS and Pkh1 were added to the reaction, Ypk1 was activated about 4-fold (Fig. 3). Like Ypk1, activation of Ypk2 required Pkh1 kinase activity, since the K154R kinase dead variant did not activate Ypk2. LCBs, Pkh1, and Pkh2 Stimulate Phosphorylation of Sch9—Based upon the sequence similarity of the PDK1 and PDK2 sites in Ypk1 and Ypk2 to the corresponding regions in Sch9, it was suggested that Sch9 is activated by Pkh1 and Pkh2 (7Casamayor A. Torrance P.D. Kobayashi T. Thorner J. Alessi D.R. Curr. Biol. 1999; 9: 186-197Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). However, this hypothesis has never been tested directly. Because phosphorylation of Ypk1, Ypk2, and Pkc1 by Pkh1 and Pkh2 has been demonstrated to be stimulated by PHS, we also wanted to see if phosphorylation of Sch9 is directly stimulated by PHS or indirectly via stimulation of Pkh1 or Pkh2. To determine whether Sch9 is a substrate of Pkh1 and Pkh2 and if phosphorylation is stimulated by LCBs, we performed in vitro
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