Genetically identified protein kinases in yeast

[k•EVIEWS P r o t e i n phosphorylation is an important form of posttranslational modification. Protein kinases regulate a number of critical cell processes, including transcription and translation, the cell cycle, exit from the mitotic cycle into meiosis, and progression through meiosis. Protein phosphorylation is a key player in many diverse cellular responses to the extracellular environment, and protein kinases are among the mediators of signal transduction in many pathways. The significance of protein phosphorylation to normal cellular metabolism can be inferred from cases where phosphorylation patterns go awry: many oncoproteins are protein kinases. Protein phosphorylation has been the subject of experimental investigation for decades ~. From a biochemical perspective, the transfer of phosphate from a nucleotide triphosphate to a protein substrate can be precisely measured, and standardized assays have been developed. Over the past decade, molecular techniques have been successfully used to identify and isolate genes for protein kinases, and in smaller eukaryotes, analysis of protein phosphorylation is

Genetically identified protein kinases in yeast I: transcription, translation, transport and mating MERL F. HOEKSTRA,ANTHONYJ. DEMAGGIOAND NAMRITADHIILON Studies from a wide array of different fields using Saccharomyces cerevisiae as an experimental organism have uncovered protein phosphorylation as a recurrent theme in the regulation of diverse cellular activities. Protein kinases in yeast regulate a variety of processes; this article discusses several genetically identified protein kinases and the roles that these kinases play in ceU growth and development. based on genetic studies plus a good definition of the structure of protein kinase genes 2,3. In the bakers' and brewers' yeast, Saccharomvces cerevisiae, an amazing array of more than 30 protein kinases have been identified (Table 1). The methods used to identify these protein kinases ranged from the complementation of mutant phenotypes to anti-phosphotyrosine antibody screening 4, and mutations in the genes for these protein kinases result in a broad variety of phenotypes. Because the identification and understanding of protein phosphorylation in genetically amenable smaller eukaryotes has advanced rapidly, we discuss the biology of protein phosphorylation as it relates to certain cellular processes. In particular, we discuss a number of genetically identified protein kinases that were first discovered on the basis of changes in cellular phenotype in mutants, and are now known to play a role in the life cycle of budding yeast or in various aspects of nutritional adaptation (Fig. 1). In this article we focus on the processes of transcription, translation, transport and mating. In a second article, to be published in the September issue of T/G, we deal with DNA metabolism and progression through meiosis. We shall not discuss in detail the cell cycle regulatory CDC28 kinase, nor Scbizosaccharornycespombe and its protein kinases, since these topics have been examined recently s 7.

Transcription, translation and transport Protein phosphorylation has emerged as a primary mechanism for regulating eukaryotic transcription, translation and protein sorting. Several genetically identified protein kinases, such as SNF1 and GCN2, are involved in gene expression. The SNF1 protein kinase was identified in a screen for defects in sugar metabolism, and the GCN2 kinase was identified among mutants that control amino acid biosynthesis. The product of the ~,79S15 gene is a protein kinase associated with protein transport. VPSI5 was identified among mutants with a defect in protein sorting; vps15 mutants mislocalize vacuolar hydrolases to the cell surface. Transcriptional regulation lhe sucrose nonfermentation ( SNF) protein kinase Seven unlinked SUC loci control sucrose utilization; cells caruing any suc mutation cannot produce

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invertase, the enzyme responsible for the hydrolysis of sucrose into glucose and fructose. Yeast normally produce two forms of invertase: a constitutive, intracellular form and an inducible, secreted form. The two invertases are both the product of the SUC2 gene but are produced from distinct, differentially transcribed mRNAs. A longer SUC2 message generates the secreted invertase and is repressed lO0-1000-fold in response to glucose, while a shorter SUC2 message encodes the intracellular invertase. The glucose-repressible mess~tge is not found in snfl mutants s, and the extracellular form of invertase, which is primarily responsible for sucrose utilization, is not produced. Since snfl mutants are pleiotropic, in that they are affected in their utilization of all sugars subject to catabolite repression, SNF1 might indirectly regulate the expression of glucoserepressible genes. The SNF1 gene is thought to encode a protein kinase because of its gene structure and the results of in vitro SNFl-dependent protein kinase assays 9. SNF1 is essential for the release of glucose-repressible genes, like the SUC2 invertase, from glucose repression; expression of a heterologous gene under the control of SUC2 regulatory sequences requires SNF1. SNF1 therefore positively regulates transcription of ghtcose-repressible genes 9. The SUC2 expression defect in snfl cells can be overcome by suppressor mutations in the SSN6 gene. Both ssn6 mutants and ssn6 snfl double mutants constitutively express secreted invertase, suggesting that SSN6 may be a regulator for inducible SUC2 expression, and that SNF1 alleviates repression of the SUC2 transcript via the SSN6 gene product. These data also suggest that SSN6 is regulated by SNFl-mediated phosphorylation. However, not all pleiotropic effects of a snfl mutant are suppressed by mutations in SSN6, indicating that the SNF1 protein kinase probably has other substrates. A further layer of complexity is added to SNFl-mediated transcriptional regulation by the analysis of other SNF genes. A second SNT gene, SNF4, is also required for the derepression of glucose-repressed genes. The SNF4 product co-immunoprecipitates with the SNF1 kinase and SNF4 is required for maximum SNF1 protein kinase activity t°. These data suggest that SNF4 protein acts as a positive effector of SNF1 protein kinase activity. Translational regulation the general control nonderepressed (GCN) protein kinase When yeast are starved for a single amino acid, the expression of 30-40 unlinked genes in 10 amino acid biosynthetic pathways is increased. This process inw)lves -

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0 /'7GO Relationship between several genetically identified protein kinases, the budding yeast life cycle, and nutritional responses. S. cerevisiae cells produce polypeptide hormones called mating facu)rs. The pher~m~nc, lead to G 1 arrest in opposing mating types: R factor arrests a cells and a factor arrests 0t cells. Several protein kinases (STET, STE11, F175,3and KSS1) are involved in tl~e pheromone response pathx~:~yand in parl l:~)ediatc signal transduction. Mating factor-treated cells produce a cellular morp]loh~g} known as 'schmoos'. Schmoos can fuse to form diploid z}gotes. Atlet zygote formation, a nonmating diploid can make several growth decisions. The diploid, like its haploid parents, can grow vegetatively. 5,everal protein kinases regulate specific intracelMar processes during vegetative growth The £\TI, GGV2 and PH085 genes encode kinases that regulate factors involved in nutritional responses, while the VPSI5 gene regulates protein sorting through intracellular compartments. During mitotic grov~th, a number of genetically identified kinases (CI)C28, CDCT, DBF2, HRR25 and MCKI) regulate correct cell cycle progression. Mutations that inactivate these kinases disrupt the cell cycle. Finally, the alternative pathway, to vegetative growth that a yeast diploid can choose is to enter the meiotic ditlcrentiati;~*a program and sporulate. This decision is based upon mating type and nutritional signals and requires the cyclic nucleotide-dependent kinascs (TPKs) and the SMEI and MCK1 kinase for ent U into meiosis and the (~1~(CDC28 and HRR25 protein kinases for progression through meiosis (Artwork by Jamie Simon.)

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A. Non-starvation Conditions

B. Amino Acid Starvation

FIGKi The role of the GCN2 protein kinase in translational regulation of GCN4. (A) The GCN4 mRNAcontains four additional upstream small open reading frames that ffmction in GCN4translational control. Under nonstarving conditions, translation of the GCN4 protein is repressed by the GCD proteins and these proteins can be considered negative-actingfactors for translation. The GCN2 protein has two domains: an amino-terminal protein kinase domain and a carboxy-terminal domain similar to histidyl-tRNAsynthetases (indicated by a clover leaf). Levels of uncharged tRNAsin the cell might be monitored by the carboxy-terminal domain of the GCN2 protein kinase. (B) Under conditions of amino acid starvation, levels of the GCN4protein are dramatically increased. The aminoacyl-tRNAsynthetase domain of GCN2 might monitor the level of uncharged tRNAto trigger derepression of the general control response and in this view would be a primary sensor of amino acid starvation. Consistent with this suggestion, a significant portion of the GCN2 protein kinase is found associated with the 60S ribosomal subunit. In concert with the action of GCN2, the GCNI and GCN3 positive regulators alter the behaviour of ribosomes during translation of the GCN4 upstream ORFs and allow GCN4 protein to be synthesized. During starvation, the net result of these complex interactions is the synthesis of GCN4 transcription trans-activator protein, which leads to the elevated transcription of a large number of GCN4-controlledgenes. another protein kinase, GCN2, which indirectly increases expression of the amino acid biosynthetic genes ll. The stimulation of the expression of biosynthetic genes is initiated by a derepression of basal GCN4 mRNA translation, and the GCN4 trans-activator protein subsequently activates transcription of the biosynthetic genes. The GCN2 protein kinase is required for translational derepression of GCN4 mRNA (Fig. 2). Mutations in GCN2 that alter conserved residues found in protein kinase catalytic domains 2 destroy GCN4 regulation. Derepression of GCN4 also requires four small upstream open reading frames (uORFs) in the 5' untranslated region of the GCN4 message, and negative control of GCN4 is mediated by the general control derepressed (GCD) genes. Mutations in GCD genes lead to constitutive GCN4 derepression, even in a gcn2 mutant background 12. The GCD proteins may be substrates for the GCN2 kinase and phg)sphorylation might partially inactivate these factors. In this model, phosphorylation would alter translation initiation so that the

uORFs in GCN4 are ignored and translation is correctly initiated at the GCN4 start codon. Phosphorylation by GCN2 would partially inactivate GCD factors and alter translation initiation at the uORFs so that ribosomes traverse the uORFs and correctly initiate at the GCN4 start codon. Although the exact mechanism by which amino acid starvation causes the GCN2 protein kinase to derepress GCN4 translation remains unknown, the structure of the kinase suggests how amino acid levels might be monitored by GCN2 n. The carboxy-terminal 530 residues in the GCN2 protein kinase are homologous to histidyl-tRNA synthetases. Mutations in this region of GCN2 inactivate the regulatory functions of the kinase. Under starvation conditions the tRNA synthetase domain might monitor uncharged tRNA concentrations and subsequently activate the protein kinase domain. One possible substrate for GCN2 protein is the translation factor elF-2. In mammalian systems, phosphorylation of the 0t subunit of elF-2 decreases translation initiation. Mutations affecting either the ~ or [3 subunits of yeast elF-2 can stimulate GCN4 translation by a GCN2independent mechanism. GCN2 therefore provides an example of a protein kinase that may link aminoacyMRNA synthesis to the phosphorylation of elF-2.

Protein transport regulation the vacuolar protein sorting ( VPS) kinase Protein kinases are involved in routing proteins to their final destinations. Specifically, among the vacuolar protein sorting (vps) mutants, VPS15 encodes a protein kinase 13. The vps mutants were identified as proficient in protein glycosylation and secretion, but they mislocalized vacuolar enzymes to the cell surface. The VPS15 protein kinase is involved in the regulation of targeting vacuolar proteins. Cell fractionation experiments indicate that the VPS15 protein is located at the cytoplasmic face of either the Golgi apparatus or a transport intermediate between the Golgi and the vacuole. How a cytoplasmic protein kinase is able to influence protein transport events within the secretory pathway is uncertain. However, protein phosphorylation may act as a switch at potential branch points within the secretory pathway to determine the fate of a protein.

Mating responses Cell type in yeast is controlled by the mating-type (MAT) locus (reviewed in Ref. 14). When yeast cells of opposite mating types (called MATa and MAT00 are mixed, a complex series of events occurs, including mating factor-induced signal transduction, G1 arrest, haploid-specific gene expression, and cell fusion. Mutations affecting mating responses have revealed four

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protein kinases (see Fig. 3). The sterile (ste) mutants were identified on the basis of their inability to arrest cell division in response to ~ mating factor, and two STE genes e n c o d e protein kinases. The sst mutations confer supersensitivity to mating factors, and a dosed e p e n d e n t suppressor of one of these mutants encodes the KSS1 protein kinase. Finally, mutants that cannot p r o c e e d through cell fusion define a third class of mating mutations, among which are mutations in the FUS3 protein kinase.

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STErile kinases The STE genes orchestrate a c o m p l e x series of steps involved in the signal transduction from binding mating p h e r o m o n e to cell division arrest and preparation for mating. A hierarchy .within the signalling pathway exists a m o n g the various STE genes FIG~ and includes genes for the A network for S. cerevisiaeprotein kinases associated with mating. Mating in yeast involves p h e r o m o n e receptors (STE2 morphological and developmental changes that are triggered by alterations in gene and STE3), components of a expression and by post-translational regulation through protein phosphoulation. In d~c tripartite G protein (STE4, scheme shown here, the ~ factor mating pheromone binds to a transmembrane receptor STE18 and GPA1), transcription protein (STE2). The cytoplasmic tail of the receptor has constitutive and pheromoneactivators (STE12) and protein inducible phosphorylation sites (P). Binding of 0~factor activates the tripartite G protein kinases (STE71~ and STEllm). CGPAI-STE4-STE18) leading to G1 arrest. During the arrest, a morphological change occurs and yeast become 'schmoo' forms. After factor binding, expression of the mating pathway These protein kinases act in genes is subsequently induced by two protein kinases, STE7 and STE11, and a transcription concert with the STE12 tranactivator, STE12. STE12 is a phosphoprotein that binds to PILE,transcription elements and scription factor to regulate exactivates the transcription of pheromone-responsive genes (PRGs). Although STE7, STE11 pression of haploid-specific and STE12 are components of the mating pathway, direct phosphorylation of STE12 by STE" genes. or STE11 kinases has not been proven. After prolonged exposure to 0t factor, an adaptation Complex genetic interresponse occurs and yeast cells can recover from the G1 arrest. The adaptation to ~ factor is actions are seen b e t w e e n the mediated by another protein kinase, KSSI, which is also involved in pheromone signalling. STE7 and STEll protein while the recovery from 0t factor arrest is mediated in part by SST2. After recovering from G 1 kinases and other mating rearrest, the cells progress past 'start' and proceed through the mitotic cycle. Inhibition of sponse components. For mitotic cell cycle is accomplished in part by the FITS3protein kinase, which negatively regulates the CLN3 cyclin. example, null mutations in the G protein 0~ subunit gene lead pathway. However, these are not redundant functions. to nonviable cells, owing to a constitutive p h e r o m o n e Overexpression of STE7 in a stell mutant, and response and cell cycle arrest. These mutations can be vice versa, does not suppress either mutant. A s u p p r e s s e d by mutations that inactivate either the STEll-dependent protein kinase activity has been STE7 or STEll kinases or the STE12 transcription demonstrated in vivo 16 and, although no specific factor, because these functions act d o w n s t r e a m of the substrates have been identified, antit)ody against G protein. This suppression suggests that cell cycle STE11 co-precipitates a 78 kDa phosphoprotein. If arrest and transcription induction is mediated by STEZ STEll and STE12. Furthermore, overexpression of pp78 is a physiological substrate for STEll, it may STE12 in a ste7or stell mutant increases the low level be a new c o m p o n e n t of the signalling pathway, since the mobility of pp78 differs from the predicted sizes transcription of haploid-specific genes to nearly wildfor currently k n o w n STE components. Other possible type levels r . However, the mating frequency of these candidates for phosphorylation by STElt include cells still does not reach wild-type levels, indicating the STE5, STE7 and STE12 gene products, which are that the two kinases also act elsewhere in the p a t h w a y phosphoproteins in vivo, or the SST2 genc product alter G protein activation and STE12 activation. (below') which has a molecular mass of approximately STE7 and STEll both e n c o d e protein kinases that a p p e a r to act at the same point in the mating 78 kDa. TIG AUGUST1991 VOL. 7 XO. 8

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KSSing a n d FUSion kinases After the initial pheromone response and G1 arrest, cells adapt to the pheromone and no longer respond to mating factor stimuli, resuming cell division. Mutant cells that lack the cytoplasmic tail of the cz factor receptor (the cytoplasmic tail contains pheromoneinduced protein phosphorylation sites) are more sensitive to 0~ factor and recover slowly from G1 arrest, although they are capable of signal transduction. A similar G1 recovery defect is seen in sst2 mutants that are supersensitive to mating factor and remain arrested at low doses of 0t factor. Overexpression of the KSS1 protein kinase gene suppresses these sst2 and ste2 0t factor recovery defects ~. Given that the cytoplasmic tail of the 0~ factor receptor is phosphorylated and that a genetic association exists between STE2 and KSS1, it seemed reasonable to suggest that the KSS1 protein was the receptor kinase. However, this hypothesis is not borne out: kssl mutants are not hypersensitive to mating factor. The KSS1 protein kinase is not essential for the pheromone response but is involved in some aspect of mating and/or adaptation. After arresting their cell cycles in G1 in response to mating pheromone, cells fuse to form diploids. The f u s l - f u s 3 mutants were identified as proficient in pheromone-lnduced gene expression and cell pairing but incapable of fusing. These mutants define a cell fusion step in mating that comes after pheromone response and cell agglutination. After treatment with 0t factor, f u s 3 mutants are defective at inhibiting vegetative growth but retain the ability to induce conjugation. Also, increased FUS3 expression enhances wild-type pheromone sensitivity 19, Coupled with the decreased sensitivity of ./i4s3 mutants to pheromone, these results suggest that the FUS3 putative protein kinase negatively regulates mitosis during mating. The KSSI and FUS3 kinases show similarity to the CDC28 protein kinase. This similarity, however, does not indicate functional redundancy. Neither KSS1 nor FUS3 can complement cdc28 mutants and the phenotypes offus3 and kssl null alleles are different - a kssl null allele has little effect on mating whereas mating in a fi*s3 null mutant is moderately reduced. Also, sequence comparisons do not place KSS1 or FUS3 in the same protein kinase subfamily as CDC28 2,3. However, KSS1 and FUS3 kinases do appear to form a link with CDC28 through their genetic interaction with a cyclin ~,19. The CLN3 gene product is a G1 cyclin that is thought to regulate CDC28 activity. Loss of CLN3 results in a 2-3-fold increase in 0t factor sensitivity and a prolonged G1 phase. Like KSS1 overproduction, a hyperactive cln3 allele shows reduced pheromone response and can suppress sst2 Since kssl mutants with a hyperactive cln3 allele also suppress sst2, KSS1 may positively regulate CLN3 Is. FUS3 kinase, on the other hand, may have an antagonistic role in CLN3 regulation. A cln3 null allele suppresses the G1 arrest defect of f u s 3 mutants and also restores their ~ factor sensitivity TM. While single kssl or fi~s3 null mutants do not have a dramatic effect on mating, a kssl fi*s3 double mutant is sterile (G. Cole, pers. commun,). Perhaps KSS1 and Fus~ kinases have overlapping specificities in maintaining basal levels of signalling or in activating signalling processes.

Perspectives We have described several S. cerevisiae protein kinases involved in cell growth and development that were first identified by mutant phenotypes. Protein kinases appear to be the enzyme of choice for regulating myriad cellular processes. The vast majority of yeast protein kinases that have been identified to date appear to belong to the protein serine/threonine kinase family. While protein tyrosine kinase activity in S. cerevisiae has been recently reported 4,2°, the number of protein tyrosine kinases identified in yeast remains low. This is perhaps not surprising since many protein tyrosine kinases in more complex eukaryotes are receptors and signalling molecules for growth factors and hormones, functions that are perhaps superfluous to single cell growth. The analysis of the phenotypes arising as a result of disabled protein kinases has allowed the characterization of specific pathways in which these protein kinases function. In this review, we have concentrated on a subset of the known protein kinases to highlight how these kinases interact with diverse activities to exert their regulatory influences. In this respect, yeasts have been particularly valuable organisms for studying protein kinases because the powerful genetic approaches afforded by yeast allow precise dissection of the complex functions regulated by protein phosphorylation.

Acknowledgements We thank those people w h o have commented on this

review and especially thank Gary Cole for helpful discussions and Jamie Simon for Fig. 1. We also thank Steve Hanks and Tony Hunter for sharing their protein kinase alignments. Because of the rapid pace at which yeast protein kinases are being identified we have undoubtedly missed several. We apologize to those whose work is not cited. Work in the authors' lab is supported by the NIH, NCI, the Mellon Foundation, and the Lucille P, Markey Charitable Trust. M.F.H. is a Lucille P. Markey Scholar in Biomedical Sciences.

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16 Rhodes, N., Connell, L. and Errede, B. (1990) Genes Det,. 4, 1862-1874 17 Dolan, J.W. and Fields, S. (1990) Genes Dev. 4, 492-502 18 Courchesne, W.E., Kunisawa, R. and Thorner, J. (1989) Cel158, 1107-1119 19 Elion, E.A., Grisafi, P.L. and Fink, G.R. (1990) Cell60, 649~664 20 Dailey, D. el al. (1990) Mol. Cell. Biol. 10, 62444~256 21 Maurer, R. (1988) De~2i7, 469-474

22 Shiro, J. and Hieter, P. (1991) Genes Dev. 5, 549-560 23 Neigeborn, L. and Mitchell, A.P. (1991) Genes Del'. 5, 533-548 IM.F. HOrKSTRa, A.J. DEMAGGIO AND N. DmzzoN AREm THE.] MOLECULAR BIOLOGY AND VIROLOGY LABORATORY~ THE SALK t INSTITUTE FOR BIOLOGICAL STUDIES~ 1 0 0 1 0 N. TORREY PINES

IRoa~ La JOLLA, CA 92186-5800, USA. i

I n recent years a 'remalkable catalog of mechanisms has b e e n identified by which eukaryotic cells e x p a n d their g e n o m e coding capacity b e y o n d the linear blocks of nucleotide sequence originally thought to encode individual proteins. These coding strategies not only create diversity by increasing the n u m b e r of proteins e n c o d e d but also provide a means by which to regulate the expression of these proteins. Nowhere is this more apparent than with eukaryotic viruses. In most cases they have provided the prototypic example of molecular mechanisms that serve to maximize coding potentials. The compactness of the viral g e n o m e may be an important factor contributing to the success of the virus as a cellular parasite. The viruses with RNA g e n o m e s have used many different forms of gene expression, and the influenza viruses provide some illuminating examples of g e n o m e diversity. Although the functions of some of the influenza virus gene products described below are not yet known, the variety of mechanisms used for their synthesis provides a paradigm of successful exploitation of a genome.

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Diversity of coding strategies in influenza viruses ROBERT A. LAMB AND CURT M. HORVATH Influenza viruses have exploited a variety of strategies to increase their genome coding capacities. These include unspliced, spliced, alternatively spliced and bicistronic mRNAs, translation from overlapping reading frames and a coupled stop-start translation of tandem cistrons. The influenza A, B and C viruses comprise a family of related enveloped viruses with a segmented singlestranded RNA genome that has b e e n called 'negative stranded' because the viral mRNAs are transcribed from the viral RNA segments (vRNAs) by a virus-encoded RNA-dependent RNA transcriptase. The complete

PYGR VIRION PROTEINS A schematic diagram of the structure of the influenza A virus particle. Three types of HA integral membrane protein - hemagglutinin (Hemagglutinin) (HA), neuraminidase (NA) and small amounts of M2 - are inserted through the lipid bilayer of the viral membrane. The virion membrane protein M] is thought to NA (Neuraminidase) underlie the lipid membrane. Within the envelope are the eight segments of singlestranded genome RNA contained in the form of helical ribonucleoproteins (RNP). Lipid Bilayer The nucleocapsid protein (NP) is associated with approximately every 20 nucleotides of M2 the RNA. Associated with the RNPs are small amounts of the transcriptase complex, consisting of proteins PB1, PB2 and PA. The coding assignments of the eight RNA segTranscriptase ments are also illustrated. RNA segments 7 Complex and 8 each code for more than one protein (M1 and Me, and NS1 and NS 2, respectively). NS1 and NS2 are found only in infected cells and are not thought to be structural comM1 INFECTED CELL PROTEINS (Membrane Protein) ponents of the virus. Influenza B virus does NS 1 and NS 2 not encode an Me integral ,nembrane protein, but the NB glycoprotein encoded by RNA segment 6 (which also encodes NA) is of very similar structure to M2, and it seems possible it will perfoml a similar function in cells and virions. Influenza C virus contains sc',cn RNA segments, lacking an RNA segment for NA. Ho\vcvcr. the l tA also has a neuraminate-O-acetylesterase activity (reviewed in Ref. 1). An equivalent of M, or NB has v~:t to h~: klcntiliccl in influenza C viruses.

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