Copyright 0 1992 by the Genetics Society of America

N-Terminal Mutations Modulate Yeast SNFl Protein Kinase Function Francisco Estruch, MichelleA. Treitel, Xiaolu Yang and Marian Carlson Department of Genetics and Development and Institute of Cancer Research, Columbia University, College of Physicians and Surgeons, New York, New York 10032 Manuscript received August 20, 1991 Accepted for publicationJuly 25, 1992

ABSTRACT The SNFl protein kinase is required for expression of glucose-repressed genes in response to glucose deprivation. The SNF4 protein is physically associated with SNFl and positively affects the kinase activity. We report here the characterization of a dominant mutation, SNFl-G53R, that was isolatedas a suppressor of the requirementfor SNF4. The mutant SNFl-G53R protein is still responsive to SNF4 but has greatly elevated kinase activity in immune complex assays; in contrast, the activityis wild type in a protein blotassay. Deletionof the region N-terminal to the kinase domain (codons 5-52) reduces kinase activity in vitro, but the mutant SNF1-AN kinase is still dependent on SNF4. The N terminus is not required for the regulatory response to glucose. In gel filtration chromatography, the SNF1, SNFl-G53R and SNFI-AN proteins showed different elution profiles, consistent with differential formationof high molecular weight complexes. Taken together, the results suggest that the N terminus positively affects the function of the SNFl kinase and may be involved in interaction with a positive effectorother than SNF4. We also showed that the conserved threonine residue 2 10 in subdomain VIII, which is a phosphorylation sitein other kinases, is essential for SNFl activity. Finally, we present evidencethat when the C terminus is deleted, overexpressionof the SNFl kinase domain is deleterious to the cell.

HE SNFlprotein kinase of Saccharomyces cerevisiae is requiredfor expressionof glucose-repressed genes in response to glucose starvation. Mutations in SNFl cause pleiotropic defects in growth on

T

carbon sources that are less preferred than glucose (e.g., sucrose, galactose, maltose,glycerol,ethanol) and cause defects in sporulation of homozygous diploids (CARLSON, OSMOND and BOTSTEIN198 1 ; NEIGEBORN and CARLSON1984). In addition, snfl mutants are generally unhealthy and defective in other et aspects of cell growth control (THOMPSON-JAEGER al. 1991). SNFl is the same gene as CAT1 (ENTIAN and ZIMMERMANN1982; SCHULLER and ENTIAN1987) and CCRl (CIRIACY1977; DENIS1984). SNFl encodes a 72-kD protein-serine/threoninekinase that is expressed in both glucose-repressed and derepressed cells with a similar pattern of subcellular localization (CELENZAand CARLSON1986). Genetic evidence indicates that the kinase activity is essential for SNFl function (CELENZAand CARLSON1989). There is as yet no evidence that the kinase activity is regulated by glucose availability, and the phosphorylation activity detected in vitro is the same for SNFl kinase derived from glucose-repressedor derepressed cells. However, it remains possible thatthe kinase activity is regulated in vivo. T h e SNF4 gene encodes a 36-kD protein that appears to function as an activator of the SNFl kinase (CELENZAand CARLSON1989; CELENZA,ENG and Genetics 1 3 2 639-650 (November, 1992)

CARLSON1989). SNF4 is the samegene as CAT3 (ENTIANand ZIMMERMANN 1982; SCHULLER and ENTIAN 1988). Mutations in SNF4 cause the same spectrum of pleiotropic phenotypes as do mutations in SNFI, with the exception that the requirement for SNF4 function is less stringent at 23" (NEIGEBORN and CARLSON1984; CELENZA,ENG and CARLSON 1989). T h e SNF4 protein is physically associated with the SNFl protein, as judged by their coimmunoprecipitation (CELENZA, ENG and CARLSON1989) and by genetic studies demonstrating physical interaction in vivo (FIELDSand SONG 1989). SNF4 is required for maximal SNFl kinase activity in vitro, and genetic evidence supports the view that this is also true in vivo (CELENZAand CARLSON1989). T h e SNF4 protein does not appear to regulate the SNFl kinase in response to glucose because increased dosage of SNFl partially compensates for a snf4 deletion,restoring glucose-repressible expression of invertase (CELENZA and CARLSON1989). Deletion of the C-terminal sequence of SNFl appeared to reduce dependence of the SNFl kinase activity on SNF4, suggestingthat SNF4 might counteract an inhibitory function of the C-terminal region. In addition, a point mutation was isolated in the SNFl gene that suppressedthe requirement for SNF4. Here, we report genetic studiesof the SNF 1 protein kinase which addressthefunctional significance of different regions of SNFl and the interactions of the

640

F. Estruch et al.

kinase with other proteins. First, to examine further the interaction between SNFl and SNF4, we sought t o isolate additional mutations in SNFZ that suppress the requirementfor SNFI. A dominant mutation that gewas recovered multiple times was characterized netically, and the activity of the mutant protein was characterizedbiochemically. We deleted the region N-terminal to the kinase catalytic domain and report the properties of the mutant. We also examined the effects of mutationsin a conservedputativephosphorylation site that affects regulation of CAMP-dependentproteinkinase.Finally,weexaminedthe effects of overexpression of different regions of t h e SNFl protein. MATERIALSANDMETHODS Yeast strains and general genetic methods: Strains of S. cerevisiae are listed in Table 1. Standard methods were used for genetic analysis (SHERMAN, FINK and LAWRENCE 1978) and transformation (ITO et al. 1983). Media and methods for scoring carbon source utilization were as described previously (NEIGEBORN and CARLSON 1984). Preparation, manipulation and analysis of DNA: DNA was prepared from yeast by the method of HOFFMAN and WINSTON(1987). Preparation of DNA from bacteria and manipulation of DNAwere carried out by standard methods (MANIATIS,FRITSCH and SAMBROOK 1982). Oligonucleotidedirected mutagenesis was performed using as template the 3.2-kb EcoRI-BamHI fragment containing the SNFl gene subcloned into bacteriophage M 13mpl9(NORRANDER, KEMPE and MESSING 1983) and synthetic oligonucleotides purchased from Research Genetics. Mutations were constructed by using the Bio-Rad Muta-Gene M13 in vitro mutagenesis kit as recommended by the supplier. Sequence analysis was performed by the method of SANGER, NICKLEN and COULSON (1 977). Isolation of mutations in SNFl that suppresssnf4: Plasmid pCElO1, which carries the XhoI-BamHI fragment of SNFl in a derivative of YCp50 lacking the XhoI site (CELENZA and CARLSON 1989), was subjected to hydroxylamine mutagenesis (ROSEand FINK 1987).Mutagenized DNA was used in seven independent transformations of MCY 1853 (snf4-A2 ura3). A total of 25,000 Ura+ transformants were replica-plated tosupplemented synthetic medium lacking uracil and containing 2% raffinose, and the plates were incubated anaerobically in GasPaks (BBL). Putative Raf) transformants were colony purified and retested by spotting cell suspensions. Growth was scored relative to controltransformants carrying pCElOl and thecloned SNF4 gene. Plasmid DNA was isolated from 26 transformants by passage through bacteria HBlO1 and was used to retransform MCY1853. Nine plasmids were identified that conferred a R a p phenotype. The mutation in six of these plasmids was located by using a fragment swapping procedure; for the remaining three plasmids, fragment swapping failed to reconstruct a plasmid conferring a suppressor phenotype. In each case, the DNA was digested with BglII, which cleaves once in SNFl and once in the vector. The resulting fragments were gel purified and ligated to the complementary fragment purified from pCElOl, thereby constructing two new plasmids from each mutant plasmid, each of which was tested for the presence of the mutation by transforming MCY 1853. Five mutations were localized in the N-terminal part of SNFl, and one in the C-terminal region. The six

plasmids were derived from different transformations of mutagenized DNA and therefore containedindependent mutations. The fragment containing each mutation was subcloned into M13 mp19 (NORRANDER, KEMPEand MESSING 1983) andsequenced using as primers a set of synthetic oligonucleotides. Areconstructed plasmid carrying the SNFl-G53R mutation was designated YCpG53R, and one carrying snfl-fS501 was designated YCpfs501. Construction of the SNFl-AN mutation: The oligonucle5’-GTCAACATGAGCAGTAAEGGAACTACotide CAAATCGTC-3’ was used fordirected mutagenesis as described above. The underlined nucleotides flank a deletion of codons 5-52. The EcoRI-BamHI fragment from a recombinant phage containing the deletion was subcloned in pUC19 (YANISCH-PERRON, VIEIRAand MESSING 1985), yielding pUCSNFl AN, and it was thentransferredto YCp50, YEp24 and YIp5 (BOTSTEIN et al. 1979; ROSE et al. 1987), yielding YCpSNFlAN, YEpSNFlAN and YIpSNFl AN, respectively (see Figure 1). It was also transferred to M 13 mpl9 and sequenced using as primer the oligonucleotide 5’-GAGGCTGTTTCAATAATCAT-3’, which startsat nucleotide -82. T o construct the chromosomal SNFl-AN allele, the XhoI-BamHI fragment from pUCSNFl AN was used to transform MCY 1845 (snfl-AlO) with selection for growth on rich medium containing 2% sucrose plus 0.0 1% glucose. Two transformants, both raffinose fermenters, were crossed to MCY1853 (snf4-A2). Tetrad analysis of the diploids showed 2+:2- segregations for raffinose utilization. Eight R a f segregants were analyzed by genomic blot hybridization to identify those carrying the SNFl-AN allele. Construction of snfl-T2lOA,snfl-T2lOD and snflT21OAAC mutations: Oligonucleotides used for directed mutagenesis were 5’-CTTAAAGGCTTCTTGTG-3’ and 5’-CTTAAAGMTTCTTGTGG3-’. The underlined nucleotides represent base substitutions that result in changing Thr-2 10 to Ala or Asp, respectively. In each case, the EcoRIBamHI fragment from a recombinant phage was subcloned in pUC19 to construct pUCT210A and pUCTSlOD, respectively. The fragment was then transferred to YCp50, YEp24 and YIp5,generatingYCpT210A, YEpTSlOA, to YIpT21OA and so forth. It was also transferred M 13mpl9 and sequenced using as primer the oligonucleotide 5’-CACAAAATTGTCCATAGAGA-3‘. T o construct the snfl-T2lOAAC mutation, the HincIIfragmentfrom pUCT21OA containing the mutation was ligated tothe HincII fragment of pEB205 (see below)that contains vector sequence. The BglII-BamHI fragmentfrom the resulting plasmid was used to replace the BglII-BamHI fragment of pCEsnfl-A8 (CELENZA and CARLSON 1989), thereby generating YEpT210AAC, whichlacks the C-terminal HincII fragment of SNFl (Figure 1). Construction of GALlO-SNF1 fusions: Plasmid pCCl07 L. CELENZA, unpublished result) contains the SNFl coding region on a HincII-BamHI partial fragment, with two internal HincII sites, cloned in pUC18 (YANISCH-PERRON, VIEIRAand MESSING 1985). The N-terminal HincII site is at position -3 of SNFl. The ATG in the polylinker of pUCl8 was deleted from pCC107 by digesting with SphI and PstI, filling in the ends with T4 polymerase, and ligating. The HindIII-BamHI fragment (the Hind111 site isin the polylinker) was cloned between the Hind I11 and BclI sites of YEp52, a vector containing the GALlO promoter (BROACH et al. 1983). In the resulting plasmid, p202, the first ATG following the GALlO promoter is the ATGof the SNFl gene. The EcoRI-XbaI fragmentcontainingthe GALlO-SNFl fusion was then subcloned between the EcoRI

u.

Yeast SNFl Protein Kinase

64 1

TABLE 1 List of S. cemmisiae strains Genotype

Strain

MCY 1094 MCY 1389 MCY 1845 MCY 1853 MCY 1860 MCY2075 MCY2 124 MCY2 125 MCY2 126 MCY2127 MCY2137 MCY2 155

ade2-101 MATa ura3-52 SUC2 ura3-52 leu2::HIS3 MATa SUC2 MATa snfl-AI0 ade2-101 ura3-52 SUC2 MATa snf4-A2 his4-539 lys2-801 ura3-52 SUC2 MATa snfl-K84R lys2-801 ura3-52 SUC2 M A T a his4-539 ura3-52 leu2-3,112 pep4::URA3 SUCZ MATa snfl-AI0 ade2-101 ura3-52::YlpSNFI-AN SUC2 MATa snfl-AI0 ade2-I01 ura3-52::YIpT210A SUC2 MATa snfl-A10 ade2-I01 ura3-52::YIpT210D SUCZ SNFI-AhJ MATa ade2-101 ura3-52 SUC2 ura3-52 1euP::YlpGAL-SNF1 MATa AC SUC2 MATa SNFI-AN snf4-A2 ade2-101 his4-539 ura3-52 lys2-801 SUC2

and Sal1 sites of YCp50 (ROSE et al. 1987), yielding pGALSNF1. pCClO7 was digested with PstI and theends were blunted with T 4 polymerase and ligated, thereby placing an ATG in the polylinker of pUC18 in frame with the SNFl coding sequence. The polypeptide encoded by this plasmid, pEB200, includes the sequence Met-Pro-Val-Asn added to the N terminus of SNFl. The N- and C-terminal HincII fragments of SNFl (each 0.9 kb) were separately deleted from pEB200 by the following procedure. pEB200 was partially digested with HincII and fragments 0.9 kb smaller than the intact linear were isolated and ligated, yielding pEB205 and pEB206, whichlack the N- and C-terminal regions, respectively. Each deletion leaves the coding sequence in frame. The HindIII-SmaI fragment from each deleted plasmid was subcloned into the HindIII-NruI fragment of pGAL-SNFl that lacks the SNFl sequence. The resulting plasmids, pGAL-SNF1 AC and pGAL-SNFl AK, carry the N and C terminus of SNFl, respectively, under control of the GAL10 promoter. The EcoRI-BamHI fragment from pGAL-SNF 1AC was subcloned in two steps as a XhoI-Sac11 fragment into pRS305 (SIKORSKI and HIETER 1989) to generateYIpGAL-SNF1AC, which was integrated at the leu2 locus of strain MCY 1389. Preparation of SNF4 antibody: A trpE-SNF4 fusion was constructed by cloning the ClalI fragment ofSNF4 into pATH2 (KOERNER et al. 1991), yielding a fusion to codon 21 of SNF4. The resulting plasmid produced an insoluble hybrid protein of the expected size, 62 kD, in Escherichia coli. The hybrid protein was prepared as described (SCHULTZ,MARSHALL-CARLSON and CARLSON1990)and injected into a New Zealand White male rabbitto raise antibody by standard procedures. The anti-SNF4 antibody was affinity purified using the bacterially produced TrpESNF4 fusion protein as previously described (CELENZA and CARLSON 1989), except that preparation of the immunoglobulin G fraction was omitted. In immunoblot analysis, this antibody reacts with a 34-kD protein (the predicted size for SNF4) that is present in wild type and absent from a snf4A mutant, and also detects SNF4-8-galactosidase (CELENZA, ENGand CARLSON 1989). Kinase assays, coimmunoprecipitation and immunoblot analysis: Immune complex kinase assays, coimmunoprecipitation experiments and immunoblot analysis were carried out as described previously (CELENZA and CARLSON 1989; CELENZA, ENG and CARLSON1989). Protein blot kinase

assays were carried out as previously described (CELENZA and CARLSON 1991). Gel filtration chromatography: For preparation of protein extracts, cells growing exponentially in either YPD or synthetic complete medium were harvested by centrifugation and washed once inice-cold buffer A (20 mM Trisphosphate, pH 6.7, 5 mM EDTA). The cells were resuspended in cold buffer B (20 mM Tris-phosphate, pH 6.7, 5 mM EDTA, 50 mM NaCI, 1 mM dithiothreitol, 2 mM phenymethysulfonyl fluoride, and 1 pg/ml each of aprotinin, leupeptin and pepstatin) and disrupted by vortexing with 0.45-mm glass beads. The cell debris was removed by centrifugation, repeated three times, in a Fisher rnicrocentrifuge 235A for 15 min, and the supernatant was used immediately. These procedures were carried out at 4 Chromatography was performed on a Pharmacia fast protein liquid chromatography (FPLC) system using a Superose 12 HR I0/30 column at room temperature. Protein extract (0.2 ml, 1.5-2 mg total protein) was applied to the column. Proteins were eluted with buffer B at at a flow rate of 0.4 ml/min, and 0.2-ml fractions were collected. For fractions showing detectable absorbance at 280 nm, samples (75 PI) were subjected to10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis using anti-SNF1 serum (CELENZA and CARLSON 1986) and affinity-purified anti-SNF4 antibody. Invertase assays: Cultures were grown to mid-log phase in supplemented synthetic medium (SHERMAN,FINK and LAWRENCE 1978) lacking uracil and containing 2% galactose. Invertase activity was assayed in whole cells as previously described (CELENZA and CARLSON 1984). O .

RESULTS

Isolation of mutations in SNFZ that alleviate the requirement for SNF4: To select for dominant mutations that restore SNFl function in a snf4 deletion mutant,wemutagenized a centromere-containing plasmid carrying the SNFl gene (pCE101) with hydroxylamine. The mutagenized DNA was usedto transform a snf4-A2 mutant strain, and plasmids capable of suppressing the raffinose-fermenting defect were isolated. For six independent mutant plasmids, the mutation was located by swapping fragments (see MATERIALS AND METHODS). Five contained mutations

F. Estruch et al.

642

TABLE 2 Mutations in SNFl that suppress dependence onSNF4 Codon Plasmid

b

positiona change

51,105,106,120,138

acid Codon change

53

GGG

1 CAC

CAC21 CAT

G ~ Y

SNFI-G53R

Arg (His)4

CAC

1

1

A 130

Allele

1

AGG

51

Amino

A

AAA AAA ACG 498

TCLAAA

1

ACA AAAAAAATCThrLys

Lys Thr

Lys Ser Lys

snfl-fs501

1

Lys I l e Stop

Number indicates position of first codon shown.

* T h e termination codon that is frameshifted into the reading frame in plasmid

in the N-terminal half of the gene, and one in the Cterminalregion. The mutations were identified by sequence analysis (Table 2), and their positions within the SNFl coding region are indicated in Figure 1. Five plasmids contained the identical mutation, a G-to-A transition at nucleotide 157,that results in replacement of Gly-53 with Arg. The same mutation, designated SNFl-G53R, was recovered twice previously in a similar selection (CELENZA andCARLSON 1989). The Gly-53 residue is the most N-terminal residue of subdomain I, which is conserved among protein kinases and contains the motif Gly-X-Gly-X-XGly . . . Lys that is part of theATP-binding site (HANKS,QUINN and HUNTER 1988). However, Gly5 3 is not aconserved residue. In oneof these plasmids, 4 of 13 consecutive His codons were also deleted, but 12 can be deleted withouteffect (CELENZA and CARLSON 1989). These five plasmids all conferred a strong raffinose-fermenting phenotype in the snf4 mutant. Plasmid YCpG53R was reconstructedfrom sequenced DNA carrying the SNFl-G53R mutation and was used to transform a snfl mutant strain (MCY 1845). The SNFl-G53R allele conferred growth on raffinose and regulated invertaseexpression (Table 3). The plasmid also restored substantial glucoserepressible invertase expression in a snf4 mutant (Table 3). Sequence analysis of the sixth plasmid revealed a base substitution in codon 498 and anadjacent single base insertion. Because of the frameshift, this allele, designated snfl-fs501, is predicted to encode a truncated product (Table 2). A protein of the expected size was detected by immunoblot analysis (Figure 2B). This mutant plasmid did not suppress snf4 effectively, conferring only a weak raffinose-fermenting phenotype, nor didit complement snfl effectively (see Table 3). These results are consistent with a previousanalysis of a series of C-terminal deletions of SNFl (CELENZA and CARLSON 1989), and this plasmid was not studied extensively.

130 is underlined.

SNFl-GS3R product has elevated kinase activity in immune complex assays:To characterize the protein kinase activity of the SNFl-G53R product, we used an immune complex assay. The SNFl protein was immunoprecipitated from wild-type and mutant extracts using affinity-purified polyclonal anti-SNF1 serum (CELENZA and CARLSON 1986), and theimmunoprecipitates were incubated with [y-"P]ATP. Proteins were separated electrophoretically, and labeled products were detected by autoradiography. Half of the sample was subjected to immunoblot analysis for detection of the SNFl products. The immune complex assay was carried out essentially as described by CELENZA and CARLSON (1 989); however,in this study the control samples containing the snfl-K84R mutant protein, inwhich the conserved lysine of the ATPbinding site is replaced by arginine, yielded no phosphorylated products or only faint bands (see Figure 4). Immune complex assaysof strainscarryingthe S N F l 4 5 3 R allele on YCpG53R showed greatly elevated kinase activity relative to the strains carrying the wild-type SNFl gene (Figure 2A). Autophosphorylation and labeling of coprecipitating proteins were elevated to approximately the same extent. Also, the same coprecipitated proteins were labeled in the mutant and wild-type extracts. Immunoblot analysis of the precipitated proteins showed that theSNFl-G53R and wild-type products were present in the same amounts (Figure 2B). In extracts from a snf4 mutant, the SNFI-G53R proteinalso exhibited higher activity than the wild-type SNFl protein, which is consistent with the phenotype observed in vivo. However, the SNFl-G53R kinase activity in the snf4 mutant was still much lower than that detected in a wild-type extract, indicating that the activity of this mutant kinase still responds to SNF4. Autophosphorylation activity of the SNFl-G53R kinase is not elevated in a protein blot assay: The

Yeast SNFl Protein Kinase allelelplasmid

643 H

c

B

SNFl snfl-A10 R

G+

SNFl-GS3R

-8-

fs501 SNFl-fS501 SNF1-AN

T+A,D

snfl-T210A snfl-TZ10D

rtr

T-bA rtr

snfl-T210AAC

K+R

snf 1 -K84R

P

FIGURE1.-Maps of SNFI plasmids and mutations. Open bar, coding sequence. Stippled region, protein kinase catalytic domain. Asterisks indicate the positions of mutations. The GALlO sequence is not drawn to scale. Restriction sites: B, BamH1; Bg, BgllI; Hc, HincII; R, EcoRI; X , XhoI.

pGAL-SNF1 PGAL-SNF1AC PGAL-SNF1AK

-

GALlO

0.5kb

TABLE 3

autophosphorylation protein thatais on carried out blot:proteins are separated by gel electrophoresis, transferred to a filter, exposed to a denaturant, allowed to renature, and then assayed for autophosphorylation activity (CELENZA and CARLSON1986). Thus, the SNFl product is assayed after electrophoretic separation from any other molecules with which it is normally associated. In this assay, themutant SNFl-G53R product showed the same activity as the wild-type SNFl protein(Figure3A).Immunoblot analysis confirmed that equal amounts of the SNFl and SNFl-G53R products were present on the blot (Figure 3B). Thus, this mutant product exhibits greater activity than thewild-type SNFl kinase in an immunecomplex assay where other associated molecules are present, but exhibits the same activity in a protein blot assay. These results are consistent with the notion that the SNFl-G53R mutation affects the interaction of the SNFl protein kinase with a ligand or another protein(s). However, it remains possible that aberrant or incompleterenaturation of the protein in the blot assay prevented detection of an intramolecular effect on the kinase activity. Deletion of the region N-terminal to the kinase domain affects SNFl function: The SNFl kinase includes 52amino acids located N-terminal tothe kinase domain and adjacent to the site of the SNFIG5?R mutation. To assess the functional role of this region, we deleted codons 5 to 52 by oligonucleotidedirected mutagenesis (see MATERIALS AND METHODS). The mutant allele was designated SNFI-AN. The centromere-containing plasmid YCpSNFl AN andthe multicopy plasmid YEpSNFl AN were each used to transform a snfl-A10 strain (MCYl845), andplasmid YIpSNF1 AN was integrated at the uru? locus (yield"

Invertase activity in strains carrying different SNFl alleles Relevant genotype snjl-A10 snjl-A10 (pCElO1) snjl-A10 (YCpC53R) snjl-A10 (YCpfs501) snf4-A2 (pLN 132) snf4-A2 (pCE10 1) snj#-A2 (YCpC53R) snj4-A2 (YCpfs501) snjl-A10 ura3::YlpSNFl-AN SNFl-AN snjl-A10 ura3::YlpT210A snjl-T2lOA snjl-A10 ura3::YlpT2lOD snjl-T2lOD

Plasmid-borne allele

SNF 1 430 SNFl-G53R snjl-fs501 SNF4 160 SNFl SNFl-G53R fs5Olsnjl

Invertase activity R

D