Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3398-3402, April 1994 Cell Biology Reconstitution of a yeast protein kinase cascade in vitro: Activation of the yeast MEK homologue STE7 by STEll (mating response/mto -tivatd protein kine/MEK knase/Saccharomyces cerevisiae) AARON M. NEIMAN AND IRA HERSKOWITZ* Department of Biochemistry and Biophysics, Program in Genetics, University of California, San Francisco, CA 94143-0448 Contributed by Ira Herskowitz, December 23, 1993 ABSTRACT The mating-factor response pathway of Sac- MATERIALS AND METHODS charomyces cerevswiae employs a set of protein kinases similar to kinases that function in signal transduction pathways of Media, Strains, and Plasmids. Standard media and genetic have the kinases encoded methods were used (16,17). Strains AN1012 (ste7A), AN1016 metazoans. We purified yeast protein (stellA), and mating tester IH1793 have been described (18). by STEII, STE7, and FUS3 as fusions to glutathione S-trans- The plasmids used were constructed as ferase (GST) and reconstituted a kinase ccde in which follows. pRD- STE7-RI was made by cloning an EcoRP fragment coding for STEll phosphorylates and activates STE7, which in turn the carboxyl-terminal 381 amino acids of STE7 into pGEX3 phosphorylates the mitogen-activated protein kinase FUS3. (Pharmacia) and then moved as an Xma 1-HindIH fragment GST-STE11 is active even when purified from cells that have into pRD56 (19). A 1.4-kb BgI II-HindUl fragment of pRD- not been treated with a-factor. This observation raises the STE7-RI was replaced with the same fragment of pYGD7- possibility that STEll activity is governed by an inhibitor A220 (7) to create pRD-STE7-RI-A220. pRD-STE7-RI-V363 which is regulated by pheromone. We also identify a STEll- was made by site-directed mutagenesis (20) ofpRD-STE7-RI dependent phosphorylation site in STE7 which is required for with the oligonucleotide 5'-CTATCGCTGACGTCTTTGT- activity of STE7. Conservation of this site in the mammalian TGGAACG-3'. pRDSTE11-RI was made by replacing the STE7 homoiogue MEK and other STE7 relatives suggests that STE7 sequences of pRD-STE7-RI with an EcoRI-Xho I this may be a regulatory phosphorylation site in all MAP kinase fragment coding for the carboxyl-terminal 606 amino acids of kham. STEll. A 2.2-kb HindIII-Cla I fragment of pRD-STE11-RI was replaced with the same fragment ofpNC245-R444 (15) to Haploid MATa and MATa cells of the budding yeast Sac- create pRD-STEll-RI-R444. A HindIII-Xho I fiagment car- charomyces cerevisiae each secrete small peptide phero- rying the carboxyl-terminal 354 amino acids of STEll was mones, a- and a-factor, respectively, to which the opposite cloned into pRD56 to create pRD-STE11-H3. pRD-STE11- cell type responds (1, 2). The signal transduction pathway ATG was made by first cloning a 1.2-kb Nhe 1-HindIll mediating response to pheromone is initiated by a membrane- fragment of pAIS-STEll (from S. Marcus, Cold Spring bound receptor that is coupled to a heterotrimeric guanine Harbor Laboratory, Cold Spring Harbor, NY) into Xba nucleotide-binding regulatory protein (G protein). Dissocia- I/HindIll-cut pUC18 to create pUC-STE11-NH2 and then tion of the G-protein a subunit from the fry subunit complex moving a 1.2-kb BamHI-HindIII fragment of pUC-STE11- in response to ligand binding allows free fry to trigger NH2 into pRD-STE11-H3. GST-FUS3 was expressed from downstream events (3, 4). The STE5 and STE20 gene prod- pGEX-FUS3, which contains the entire FUS3 coding region ucts are thought to link f3y to three protein kinases, encoded in plasmid pGEX3 (Pharmacia). by STE]], STE7, and FUS3 (4-9). These kinases are struc- Mating Tests and (-Galactosidase Assays. Transformants turally related to mammalian protein kinases: FUS3 is a were patched onto an SD plate selective for the plasmid, then mitogen-activated protein kinase (MAP kinase) (10); STE7 is replica plated to a plate containing galactose as the sole a kinase for FUS3 and is a MEK homologue (11, 12); STEll carbon source. After overnight incubation at 30TC, the is a MEK kinase (MEKK) homologue (13). patches were replica plated to a minimal-medium plate spread Functional studies indicate that these kinases act in the with a lawn of the mating tester strain IH1793 (MATa lysl). (3-Galactosidase assays were performed essentially as de- order STEll -* STE7 -- FUS3. In particular, phosphoryla- tion ofFUS3 in response to a-factor requires both STEJIJ and scribed (18). STE7 (8). Furthermore, constitutively active alleles ofSTE]I Protein Purification and Kinase Assays. STEll and STE7 require STE7 and FUS3 for function This fusion proteins were purified as follows. Cells carrying the (6, 7). putative appropriate plasmid were grown to OD6ro of 1.0 in galactose yeast kinase cascade is analogous to two kinase cascades medium, pelleted, suspended in lysis buffer [20 mM Hepes, proposed to function downstream of various growth factor pH 7.6/200 mM KCI/2 mM EGTA/2 mM EDTA/0.1% receptors in mammalian cells: Raf -- MEK -* MAP kinase Nonidet P-40, 10% (vol/vol) glycerol/i mM phenylmethane- and MEKK -- MEK -- MAP kinase (14). sulfonyl fluoride/l mM benzamidine hydrochloride/i mM Our studies focus on STEll, which has been demonstrated leupeptin], and ground with glass beads. The extract was to have protein kinase activity in immunoprecipitates (15). spun at 12,000 x g for 1 hr. The supernatant was mixed with Physiologically relevant substrates of STEll have not yet 1 ml of glutathione-agarose for 30 min, and then the agarose been defined. Here we show that this yeast kinase cascade was pelleted and poured into a column. The column was can be reconstituted in vitro with purified glutathione S-trans- washed with 40 ml of wash buffer 1 (20 mM Hepes, pH ferase (GST)-STE11, -STE7, and -FUS3 fusion proteins and 7.6/500 mM KCI/0.1% Nonidet P-40/10% glycerol) and 10 ml that STE7 is a substrate of STE11. of wash buffer 2 (20 mM Hepes, pH 7.6/200 mM KCl/10%o The publication costs of this article were defrayed in part by page charge Abbreviations: GST, glutathione S-transferase; MAP kinase, mito- payment. This article must therefore be hereby marked "advertisement" gen-activated protein kinase; MEKK, MEK kinase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 3398 Downloaded by guest on September 27, 2021 Cell Biology: Neiman and Herskowitz Proc. Natl. Acad. Sci. USA 91 (1994) 3399 glycerol). The fusion protein was eluted in 3 ml of elution ste7A buffer (20 mM Hepes, pH 7.6/200 mM KCl/10% glycerol/10 stel 1 A mM glutathione). The eluate was concentrated and ex- GST-STE7 changed into storage buffer (20 mM Hepes, pH 7.6/50 mM GST-STE1 1 KCl/10% glycerol) in a Centricon-30 concentrator (Amicon). OST-STE7-A220 For purification, all STE11 and STE7 fusions were expressed GST-STE1 1 -R444 in strain IH2361 (steSA) without pheromone treatment. The GST-STE7-V363 FUS3 fusion protein was purified from Escherichia coli by a GST similar protocol except that cells were lysed by addition of GST lysozyme to 1 mg/ml. For kinase assays, the appropriate proteins were mixed and brought to a final volume of 20 A in kinase buffer (10 mM Tris-HCl, pH 7.5/10 mM MgC12). FIG. 1. Complementation by GST fusion proteins in mating tests Reactions were started by addition of ATP to a of ste7 and stell mutant strains carrying various GST plasmids. final con- AN1012 (ste7A) and AN1016 (stellA) were transformed with plas- centration of 100 uM plus 10 ,uCi of [y-32P]ATP. Reactions mids pRD56 (a CENARS vector expressing GST under control ofthe were stopped after 20 min by addition of SDS sample buffer. GAL] promoter), pRD-STE7-RI (expressing afusion ofthe GSTgene For quantitation ofkinase activity, samples were electropho- to the wild-type STE7 gene), pRD-STE7-RI-A220 (GST-STE7 car- resed in an SDS/polyacrylamide gel and electrophoretically rying a mutation of lysine-220 to arginine), pRD-STE7-RI-V363 transferred to a nitrocellulose membrane. Intensity of the (GST-STE7 carrying a mutation of threonine-363 to valine), pRD- bands was quantified on a PhosphorImager (Molecular Dy- STEll-RI (expressing a fusion of GST to the wild-type STEHJ), or namics), and the blot was probed with anti-GST antibodies pRD-STEll-RI-R444 (GST-STEll carrying a mutation oflysine-444 (from D. Kellogg, University ofCalifornia, San Francisco) to to arginine). ensure that equal amounts of protein were loaded. If this in vitro system is faithfully recapitulating what Phospho Amino Acid Analysis and Tryptic Digsts. For both occurs in vivo, then the phosphorylated GST-FUS31-353 analyses, GST-STE7134-515 was phosphorylated by GST- protein should now be an active kinase. Activation of FUS3, STE11111-717 in vitro and separated from the reaction mix as as with described above. For phospho amino acid analysis, the all MAP kinases, is associated with phosphorylation protein was then blotted to an Immobilon membrane (Milli- on a threonine and a tyrosine residue (8, 23). Phospho amino pore). The region of the membrane containing STE7 was acid analysis of GST-FUS31-353 phosphorylated in vitro excised, and two-dimensional phospho amino acid analysis demonstrated that it contained equal amounts of phospho- was performed (21). Two-dimensional tryptic phosphopep- threonine and phosphotyrosine but no phosphoserine (data tide mapping was performed as described (21) except that the not shown). Although we have not shown that the phosphor- electrophoresis buffer was acetic acid/formic acid/water, 3:1:16 (vol/vol), and that a Kodak 13255 thin-layer chroma- A tography sheet was used.