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A conserved kinase cascade for MAP kinase activation in yeast
A conserved kinase cascade for MAP kinase activation in yeast Beverly Errede and David E. Levin University of North Carolina, Chapel Hill, and Johns Hopkins University, Baltimore, USA Mitogen-activated protein kinases are regulators of proliferation and differentiation in many eukaryotes. Studies during the last year have revealed that functionally distinct signal pathways in yeast use related protein kinase cascades for mitogen-activated protein kinase activation. These cascades act as intracellular signaling modules that are likely to be conserved from yeast to mammals. Current Opinion in Cell Biology 1993, 5:254-260 Introduction Mitogen-activated protein (MAP) kinases comprise a family of serine/threonine-specific protein kinases [1.,2] that mediate intracellular phosphorylation events linking receptor activation to the control of cell proliferation and differentiation. A broad spectrum of extracellular signals can stimulate the catalytic activity of members of this family [3]. Transmission of these signals is variously dependent on receptor tyrosine kinases, protein kinase C, G proteins and RAS [1,,2,4q5]. An understanding of the subsequent signaling events that lead to MAP kinase activation is just beginning to emerge. Activation of MAP kinases requires both threonine and tyrosine phosphorylation, a property unique to these enzymes [7,8]. A dual specificity protein kinase responsible for both phosphorylation events has been isolated from several species [9",10-13]. This MAP kinase activator also requires phosphorylation for its activation, consistent with another protein kinase acting upstream of this enzyme (NG Ahn, R Seger, AL Jensen, EG Krebs, unpublished data) [14-16]. Recent evidence implicates raf as an upstream kinase in mammalian cells [17-19]. Yeast MAP kinase family members function in at least four distinct signal transduction pathways. These homologs are part of protein kinase cascades that constitute the intracellular signal transduction apparatus of each pathway. The striking discovery of this past year is that structurally and functionally related protein kinases comprise each tier of these cascades. This brief review summarizes the evidence that resolved the order of protein kinases in the mating response, protein kinase C mediated and high osmolarity glycerol (HOG) response pathways of Saccharomyces cerevisiae. Additionally, we point out the functional and structural conservation in the mating response pathways of S. cere-
visiae and Schizosaccharomyces pombe. The reiteration represented here suggests that a common module of protein kinases has been conserved for intracellular signal transmission. Based on recent indications that MAP kinase activators are related to yeast counterparts, we speculate that the architecture of the yeast signal transduction modules is likely to be predictive of MAP kinase activation pathways in vertebrates.
Yeast mating response pathways The S. cerevisiae pathway as a paradigm for a mitogen-activated protein kinase signal transduction module The two haploid cell types of S. cerevisiae (a and ~) differentiate into mating-competent forms by inducing transcription of mating genes and blocking progression of the cell cycle at G 1. The binding of peptide pheromones from one cell type to specific receptors on the opposite cell type activates a signal transduction pathway that prepares cells for mating. Figure 1 gives an outline of this pathway and includes the probable targets that regulate the transcriptional and G 1 arrest responses. There are several recent reviews that summarize the evidence supporting this general scheme [20,21",22.]. We focus on recent results that clarified the order of the protein kinase cascade leading to activation of MAP kinase family members. We have known for some time that four protein kinases (STEll, STE7, FUS3 and KSS1) function in signal transduction between the G protein and the STE12 transcription factor [23-28,29"]. The STE20 kinase is a newly identified component of this pathway [30"]. STE20, STE11 and STE7 are each required for signal transmission [24,30"*,31]. In contrast, the FUS3 and KSS1 kinases, which are members of the MAP kinase family, are functionally redundant [29"].
Abbreviations BCK--bypass of C kinase; HOG high osmolarity glycerol; MAP--mitogen-activated protein; MKK--MAP kinase kinase; MPK--MAP kinase; PKC--protein kinase C. 254
O Current Biology Ltd ISSN 0955-0674
A conserved kinase cascade for MAP kinase activation in yeast Errede and Levin 255
Saccharomyces cere~siae
Cell wall construction/ proliferation
? o
Schizosaccharomyces pombe
Mating (z-factor ~2' STE2 receptor
a-factor ~2' STE3 receptor
or
M-factor ~7 MAP3 receptor
STE,8
Gc~-GPA' ~ PKC1 kinase
Mating and meiosis
J)
Gff-STE4
STE20 kinase
P-factor ~7 MAM2 receptor
or
, RAS1
Gc~-GPA1
STE5
BCK1 kinase ]
[ STE11 kinase I
] BYR2 kinase I
[ MKK1/MKK2 kinases ]
MPK1 kinase
? o
? o
STE12 transcription factor
Activation/stimulation
CLN3 CLN2 ~ CLN1 CDC28 kinase
, FAR1
STE11 transcription factor
=::=[] Inhibition/repression
Fig. 1. Comparison of signal transduction pathways of S. cerevisiae and S. pombe. Recent studies on the FUS3 and KSS1 kinases provided direct evidence that protein phosphorylation transmits the pheromone-induced signal. Similar to other MAP kinases, the pheromone-induced signal stimulates phosphorylation at threonine and tyrosine residues in FUS3 and KSS1 [32"']. Substitution mutations introduced at either of two positions of FUS3 (Thr180 and Tyr182) cause complete loss of function in vivo and loss of phosphotransferase activity in vitro [32**,33"]. These phosphorylations are not autocatalytic because a catalytically inactive substitution mutant of FUS3 is still phosphorylated on both Thrl80 and Tyr182. Rather, FUS3 phosphorylation requires functions provided by the STE11 and STE7 kinases [32".]. These observations suggested that a kinase cascade is responsible for signal transmission. Studies reviewed below resolve the order of this cascade. Constitutive alleles of the STE 11 kinase that are presumed to encode hyperactive or unregulated forms of the kinase were identified [34-°,35]. The alleles made it possible to position STEll on the pathway by genetic epistasis experiments. These experiments use strains that combine alleles with distinguishable phenotypes to deduce
the order in which the two gene products function on a common pathway. For example, a strain expressing a hyperactive STE11 (e.g. STEll-I) has a constitutive response, whereas a strain with a deletion of STE5 (steSA), a protein of unknown function, does not respond to pheromone. A strain that combines both mutations has a constitutive response phenotype. This result is consistent with STEll acting after or at the same level as STE5. Similar logic applied to other double mutant strains showed that STE11 also acts after the G[3 subunit but before the STE7, FUS3/KSS1 and STE12 steps in signal transmission [34.°,35]. Additionally, the phosphorylation states of STE7 and FUS3 corroborated the placement of STEll on the pathway. Hyperactive STE11 caused phosphorylation of STE7 and FUS3, even in the absence of pheromone, whereas absence of STEll blocked their phosphorylation [32",34*.,36.-]. Biochemical experiments resolved the order for STE7 and FUS3/KSS1 function by showing that pheromone induces STE7 activity in the absence of FUS3 and KSS1 [36**]. This result is consistent with STE7 functioning before the FUS3/KSS1 step in signal transmission and made
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Cell regulation Comparison of S. pombe and S. cerevisiae mating response pathways In S. pombe, both nutritional cues (starvation) and pheromone-receptor interactions stimulate the two haploid cell types (P and M) to mate with each other [38]. The S. cerevisiae and S. pombe mating response pathways are strikingly analogous (Fig. 1) [38-47]. One functional difference is that in S. cerevisiae, the G6~, complex transmits the signal, whereas in S. pombe, the Gu-subunit transmits the signal [21",421. Also different from S. cerevisiae, the S. pombe mating response pathway integrates the pheromone-induced signal with one that is dependent on an activity provided by a RAS homolog, RAS1 [48,49"].
STE7 a good candidate for the MAP kinase activator. In vitro kinase assays and reconstitution experiments confirmed this role. STE7 is a dual specificity kinase that modifies FUS3 at the appropriate threonine and tyrosine sites [33"1. Further, these in vitro modifications stimulate the ability of FUS3 to phosphorylate one of its physiological substrates, FAR1 (M Peter, A Garmer, G Ammerer, I Herskowitz, unpublished data) [33°.,37-]. Once activated, FUS3 also phosphorylates STE7 in vitro [33"]. This finding is consistent with the observation that pheromone-induced phosphorylation of STE7 in vivo depends on activity of STEll and either FUS3 or KSS1 [36"]. Because STE7 is a more active kinase when isolated from a fus3A ksslA strain compared with a wildtype strain, this reciprocal phosphorylation of STE7 by FUS3 could be part of an adaptive response [33"'].
The BYR2, BYR1 and SPK1 predicted protein kinases of the S. pombe pathway are structurally related to the STE11, STE7 and FUS3/KSS1 protein kinases, respectively, of the S. cerevisiae pathway (Fig. 2) [43-46]. Epistasis relationships and complementation studies place the structurally related kinases at analogous positions in their respective pathways [43,44,50"]. For most pairs, high level expression of the heterologous protein kinase in the corresponding mutant strain is sufficient for complementation. A notable exception is suppression of the S. cerevisiae stell mutant. This suppression requires expression of both the BYR1 and BYR2 proteins of S. pombe, suggesting that BYR2 and BYR1 interact cooperatively [50"]. The implied interaction and the deduced order of function are consistent with the possibility that BYR2 is the direct activator of BYR1.
The STE20 predicted protein kinase is a newly identified component of the pathway. Its catalytic domain has some similarity to that of mammalian protein kinase C (PKC) subtypes (27-33 % identity) {30"]. Notably, the aminoterminal regulatory domain of STE20 is not characteristic of PKC and is not similar to any other known protein. STE20 placement on the pathway was deduced from double mutant strains that combined a deletion of STE20 (ste2OA), which blocks pheromone response, with constitutive alleles of other pathway components. These epistasis experiments showed that STE20 acts after or at the same level as the G protein, but before STE5 and STEll [30"].
(a) PKC1 family PKC1 YPK1
(b) STE11 family
YPK1
YPK2
43
90
YPK2
PKC-~
52
43
43
PKC-~
52
44
43
STE11 PKC-E 68
(c) STE7 family
BYR2 ~
BYR2
BCK1
47
(d) MAP kinsase family
STE7 BYR1
Fig. 2. Per cent identities for the catalytic
FUS3 BYR1
KSS1
MKK1
42
42
MKK1
MKK2
42
43
80
MKK2
PBS2
42
51
44
42
PBS~
MEK
49
53
44
43
55
KSSI
SPK1
61
66
SPK1
MPK1
51
52
53
MPK1
HOG1
51
52
53
50
HO(
ERK2
56
57
60
54
53
domains of protein kinase subfamilies. Sequence comparisons were limited to the region bounded by the conserved GXGXXG motif of subdomain I (where X is any amino acid) and conserved R of subdomian Xl [64]. The percentage of identical amino acid residues was calculated for each pair using the GCG BestFit program to maximize the number of matches. Sources of sequences used for these comparisons are referenced in the text except for PKC [65], PCK-8 [66] and ERK2 [67].
A conserved kinase cascade for MAP kinase activation in yeast Errede and kevin Additional pathways in S. cerevisiae use related protein kinase modules for intracellular signal transduction The PKCl-mediated signal transduction pathway The S. cerevisiae PKCI gene encodes a homolog of the a - , [3- and y-subtypes of mammalian PKC [51]. Preliminary biochemical studies of PKC1 suggest that it behaves similarly to its metazoan counterparts (P Chen, DE Levin, unpublished data). Loss of PKC 1 function results in a cell lysis defect that is due to a deficiency in cell wall construction. Addition of osmotic stabilizing agents to the growth medium compensates for the defect and allows cell proliferation [52%53]. Although we do not know the signal to which PKC 1 responds, its mutant phenotype was exploited to identify downstream components of the PKC1 pathway. This approach uncovered four genes that, when mutationally activated or expressed at high levels, suppressed the cell-lysis defect associated with pathway inactivation [ 5 4 " - 5 6 " ] . The four genes encode predicted protein kinases that are structurally analogous to those on the yeast mating response pathways (Fig. 2). BCK1 (bypass of C kinase) is most similar to STE11 and BYR2 [54"]. MKK1 and MKK2 (MAP kinase kinase), are related to each other and to STE7 and BYR1 [55"]. MPK1 (MAP kinase) is a member of the MAP kinase family and thus related to FUS3/KSS1 and SPK1 ]50"]. The phenotypes of deletion mutants are consistent with all four gene products acting on the same pathway. BCK1 and MPK1 each provide a unique function because bcklA and mpkIA single deletion mutants have a temperature-dependent cell-lysis defect that is suppressed by osmotic stabilizers [54",56"°]. MKK1 and K K 2 are redundant because only the mkklA mkk2A double mutant shows this phenotype [55"]. While the pkcl mutant has an unconditional lysis phenotype, cell lysis is temperature-dependent in deletion mutants of the other pathway components. The less severe defect is consistent with BCK1, MKK1/MKK2 and MPK1 operating on one branch of a pathway that bifurcates after PKCl (Fig. 1) [54"]. Genetic epistasis analyses revealed the order of components on this branch of the PKCl pathway (Fig. 1). Mutationally activated BCK1 kinase transmits a signal even in the absence of PKCl [54°.]. As either mpklz~ or mkklA mkk2A mutations block this constitutive signal, MPK1 and MKK1/MKK2 function downstream of BCK1 [55"%56"]. The use of a mutationally activated MKK1 confirmed its position downstream of BCK1. The ability of overexpressed MPK1 to suppress the mkklA mkk2A mutations is consistent with MPK1 acting after MKK1/MKK2. This result suggests that MPK1 is activated by a mechanism similar to that of other MAP kinases. This notion has some support because MPK1 function is severely impaired by phenylalanine or alanine substitutions at the respective tyrosine or threonine residues that correspond to sites of phosphorylation associated with the activation of other MAP kinase family members
[56,,].
In S. cerevisiae both the PKCl pathway and the pheromone-induced pathway use a kinase cascade with the same architecture. This striking conservation suggests that a common module of protein kinases has evolved to mediate different physiological responses in yeast. We suggest that this module extends minimally between BCK1 (or STEll) and MPK1 (or FUS3/KSS1). Presumably to avoid confusion of signals, the components of one module do not normally interact productively with components of the other. The finding that a Xenopus MAP kinase expressed in yeast complements mpkIA mutants but notfus3AkssIA mutants is consistent with each module having specificity for interacting members [56"]. However, it is possible to create conditions under which components of one pathway will interact with those of another. For example, expression of either STEll or BYR2 at high levels can suppress loss of BCK1 function (KS Lee, DE Levin, unpublished data).
s. cerevisiae may use related kinase modules for other
signaling pathways The HOG accumulation pathway responds to increases in extracellular osmolarity by increasing intracellular glycerol production. The signal pathway leading to this response involves at least two predicted protein kinases, HOG1 and PBS2 [57",58]. HOG1 is most closely related to members of the MAP kinase family and PBS2 is most closely related to members of the STE7 family (Fig. 2). The rapid, PBS2-dependent, tyrosine phosphorylation of HOG1 in response to increases in extracellular osmolarity is consistent with the functional analogies implied by their respective structural similarities to STE7 and FUS3. We predict that upstream protein kinases will be uncovered in this pathway and speculate that one will be structurally analogous to BCK1/STEll. The possibility of still another conserved module in S. cerevisiae is suggested by the recent finding of a redundant pair of PKC-related proteins, YPK1 and YPK2, that are essential for vegetative growth (Fig. 2) [59].
Conservation of protein kinase modules from yeast to mammals cDNA clones encoding MAP kinase activators have been isolated recently from Xenopus [60], mouse [61], rat [62] and human [63] cells. The sequences of these genes reveal that they encode enzymes that are most closely related to members of the STE7 family of protein kinases. This observation suggests the possibility that the entire module of protein kinases used for yeast signaling pathways may be conserved throughout metazoan evolution. The yeast paradigm presented here suggests a STE1l-like protein kinase in vertebrates may act as the MAP kinase kinase activator. Yet the vertebrate pathway may be more complex. For example, raf, which is not closely related to members of the STEll family, has been implicated as an activator of the MAP kinase kinase [17-19]. One possi-
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Cell regulation bility is that there may be multiple activators at this level. Considering the wide array of extracellular agents that activate vertebrate MAP kinases, a multiplicity of MAP kinase kinase activators may have evolved to integrate signals from different stimuli. An alternative possibility is that raf will turn out to be the activator of a STEll family member [19].
Conclusion The conserved modules for yeast signal transduction pathways, as presently defined, begin a phosphorylation cascade with a STEll-like enzyme, and end with the activation of a MAP kinase. The modules described here provide a road map for the dissection of signaling pathways leading to MAP kinase activation in vertebrates. These pathways must amplify and integrate signals from different extracellular stimuli. We suggest the yeast module may have been preserved not only because its design allows for signal amplification, but also because it provides multiple sites for regulatory inputs and signal integration. The yeast PKC1 pathway provides the paradigm for PKC-dependent MAP kinase activation in mammals. The conserved modules in other yeast pathways involve G protein mediated processes and may use other protein kinases. These precedents suggest mechanisms by which mammalian MAP kinases could be activated through PKC-independent pathways.
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32. ••
33. ••
ERREDEB, GARTNERA, ZHOU Z, N&SMYTHK, AMMERERG: MAP Kinase-Related FUS3 from S. cerevisiae is Activated by STE7 in vitro. Nature 1993, in press. Presents evidence from in vitro phosphorylation assays and reconstitution experiments that STE7 is the FUS3 activator. STE7 is a dual specificity kinase that directly phosphorylates FUS3 on the appropri ate threonine and tyrosine residues. The STE7 modification of FUS3 stinmlates the ability of FUS3 to phosphorylate FAR1 and STE7. 34. ••
STEVENSONBJ, RHODES N, ERREDE B, SPRAGUE GF, JR: Constitutive Mutants of the Protein Kinase STEll Activate the Yeast Pheromone Response Pathway in the Absence of the G Protein. Genes Dev 1992, 6:1293 1304. Describes alleles of STE11 that cause a constitutive pheromone response consistent with them encoding hyperactive or unregulated forms of the kinase. This paper includes epistasis analyses that place STE 11 on the pathway before STE7 and FUS3/KSS1. Additional evidence shows that the hyperactive STE11 causes phosphorylation of STE7 in the absence of added pheromone. 35.
36. ••
CAIRNSBR, RAMER SW, KORNBERG RD: Order of Action of Components in the Yeast Pheromone Response Pathway Revealed with a Dominant ALlele of the STEll Kinase and the Multiple Phosphorylation of the STE7 Kinase. Genes Dev 1992, 6:1305 1318. ZHOU Z, GARTNER A, CADE R, AMMERER G, ERREDE B: Pheromone Induced Signal Transduction in S. cerevisiae
49. •
NIELSEN O, DAVEY J, EGEL R: The rasl Function of Schizosaccharomyces p o m b e Mediates Pheromone-induced Transcription. EMBO J 1992, 11:1391 1395. Shows that activation of the RAS1 protein is not sufficient to activate the mating response pathway. Activation still requires nutritional cues and pheromone induction. 50. ••
NEIMAN Am, STEVENSON BJ, XU H-P, SPRAGUE GF JR, HERSKOW1TZ I, WIGLER M, MARCUS S: Functional Homology of Protein Kinases Required for Sexual Differentiation in Sehizosaccharomyces p o m b e and Saccharomyces cerevisiae Suggests a Conserved Signal Transduction Module in Eukaryotic Organisms. Mol Biol Cell 1993, 4:10~120.
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LE~NDE, BARTLETP-HEUBUSCHE: Mutants in the S. cerevisiae PKC1 Gene Display a Cell Cycle-specific Osmotic Stability Defect. J Cell Biol 1992, 116:1221-1229. Demonstrates that pkcl mutants undergo cell lysis. Mutant cells proliferate in osmotically stabilized medium.
KOSAKOH, NISHIDAE, GOTOH Y: cDNA Cloning of MAP Kinase Kinase Reveals Kinase Cascade Pathways in Yeast to Vertebrates. EMBO J 1993, 12:787-794.
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CREWSCM, ALESSANDPdNIA, ERIKSONRE: The Primary Structure of MEK, a Protein Kinase that Phosphorylates the ERKGene Product. Science 1992, 258:478-480.
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LEE KS, LEV1NDE: Dominant Mutations in a Gene Encoding
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BOULTONTG, NYE SH, ROBBINS DJ, IP NY, RADZIEJEWSKA E, MORGENBESSER SD, DEPINHO RA, PANAYOTATOS N, COBB MH, YANCOPOULOS GD: ERKs: A Family of Protein-Serine/Threonine Kinases that are Activated and Tyrosine Phosphorylated in Response to Insulin and NGF. Cell 1991, 65:663~575.
Presents evidence that three predicted protein kinases of S. pombe, BYR2, BYR1 and SPK1, that are structurally related to the S. cerevisiae kinases STEll, STE7 and FUS3, respectively, are also functionally related. This paper also cites epistasis results that are consistent with the RASl-dependent signal intersecting the pheromone-induced pathway at a step before or at the G~-subunit. 51.
LEVINDE, FIELDS FD, KUNISAWAR, BISHOPJM, THORNERJA: A Candidate Protein Kinase C Gene, PKC1, Is Required for the S. cerevisiae Ceil Cycle. Cell 1990, 62:213-224.
52. •
••
a Putative Protein Kinase (BCK1) Bypass the Requirement for a Saccharomyces cerevisiae Protein Kinase C Homolog. Mol Cell Biol 1992, 12:172-182. Describes the isolation and sequence of the BCK1 gene, whose mutational activation suppresses the loss of PKC1 function. Deletion of BCK1 results in a temperature dependent cell lysis defect. 55. ••
IRIE K, TAKASE M, LEE KS, LEV1N DE, ARAKI H, MATSUMOTO K, OSHIMAY: MKK1 and MKK2, encoding Saccharomyces cerevisiae MAP Kinase-kinase Homologs, Function in the Pathway Mediated by Protein Kinase C. Mol Cell Biol 1993, in press. Describes the isolation of the MKK1/2 genes, whose deletion results in temperature-dependent cell lysis. Demonstrates that overexpression of MKK1 suppresses loss of BCK1. 56. ••
LEE KS, IRIE K, GOTOH Y, WATANABEY, NISHIDAE, MATSUMOTO K, LEV1NDE: A Yeast MAP Kinase Homolog (MPK1) Mediates Signalling by Protein Kinase C. Mol Cell Biol 1993, in press. Describes the isolation of the MPKI gene, whose overexpression suppresses the loss of BCKI function. Deletion of MPK1 results in temperature-dependent cell lysis, which is complemented by Xenopus MAP kinase. BREWSTERJL, DE VALOIRT, DWYER ND, WINTER E, GUSTIN MC: An Osmo-sensing Signal Transduction Pathway in Yeast. Science 1993, in press. Describes the isolation of mutants in HOG1 and PBS2 using a genetic screen for osmotic sensitivity. Demonstrates PBS2 dependent tyrosine phosphorylation of the MAP kinase homolog, HOG1. 57. ••
B Errede, Department of Chemistry, CB#3290, University of North Carolina, Chapel Hill, North Carolina 27599, USA. DE Levin, Department of Biochemistry, John Hopkins University, School of Public Health, Baltimore, Maryland 21205, USA.
visiae and Schizosaccharomyces pombe. The reiteration represented here suggests that a common module of protein kinases has been conserved for intracellular signal transmission. Based on recent indications that MAP kinase activators are related to yeast counterparts, we speculate that the architecture of the yeast signal transduction modules is likely to be predictive of MAP kinase activation pathways in vertebrates.
Yeast mating response pathways The S. cerevisiae pathway as a paradigm for a mitogen-activated protein kinase signal transduction module The two haploid cell types of S. cerevisiae (a and ~) differentiate into mating-competent forms by inducing transcription of mating genes and blocking progression of the cell cycle at G 1. The binding of peptide pheromones from one cell type to specific receptors on the opposite cell type activates a signal transduction pathway that prepares cells for mating. Figure 1 gives an outline of this pathway and includes the probable targets that regulate the transcriptional and G 1 arrest responses. There are several recent reviews that summarize the evidence supporting this general scheme [20,21",22.]. We focus on recent results that clarified the order of the protein kinase cascade leading to activation of MAP kinase family members. We have known for some time that four protein kinases (STEll, STE7, FUS3 and KSS1) function in signal transduction between the G protein and the STE12 transcription factor [23-28,29"]. The STE20 kinase is a newly identified component of this pathway [30"]. STE20, STE11 and STE7 are each required for signal transmission [24,30"*,31]. In contrast, the FUS3 and KSS1 kinases, which are members of the MAP kinase family, are functionally redundant [29"].
Abbreviations BCK--bypass of C kinase; HOG high osmolarity glycerol; MAP--mitogen-activated protein; MKK--MAP kinase kinase; MPK--MAP kinase; PKC--protein kinase C. 254
O Current Biology Ltd ISSN 0955-0674
A conserved kinase cascade for MAP kinase activation in yeast Errede and Levin 255
Saccharomyces cere~siae
Cell wall construction/ proliferation
? o
Schizosaccharomyces pombe
Mating (z-factor ~2' STE2 receptor
a-factor ~2' STE3 receptor
or
M-factor ~7 MAP3 receptor
STE,8
Gc~-GPA' ~ PKC1 kinase
Mating and meiosis
J)
Gff-STE4
STE20 kinase
P-factor ~7 MAM2 receptor
or
, RAS1
Gc~-GPA1
STE5
BCK1 kinase ]
[ STE11 kinase I
] BYR2 kinase I
[ MKK1/MKK2 kinases ]
MPK1 kinase
? o
? o
STE12 transcription factor
Activation/stimulation
CLN3 CLN2 ~ CLN1 CDC28 kinase
, FAR1
STE11 transcription factor
=::=[] Inhibition/repression
Fig. 1. Comparison of signal transduction pathways of S. cerevisiae and S. pombe. Recent studies on the FUS3 and KSS1 kinases provided direct evidence that protein phosphorylation transmits the pheromone-induced signal. Similar to other MAP kinases, the pheromone-induced signal stimulates phosphorylation at threonine and tyrosine residues in FUS3 and KSS1 [32"']. Substitution mutations introduced at either of two positions of FUS3 (Thr180 and Tyr182) cause complete loss of function in vivo and loss of phosphotransferase activity in vitro [32**,33"]. These phosphorylations are not autocatalytic because a catalytically inactive substitution mutant of FUS3 is still phosphorylated on both Thrl80 and Tyr182. Rather, FUS3 phosphorylation requires functions provided by the STE11 and STE7 kinases [32".]. These observations suggested that a kinase cascade is responsible for signal transmission. Studies reviewed below resolve the order of this cascade. Constitutive alleles of the STE 11 kinase that are presumed to encode hyperactive or unregulated forms of the kinase were identified [34-°,35]. The alleles made it possible to position STEll on the pathway by genetic epistasis experiments. These experiments use strains that combine alleles with distinguishable phenotypes to deduce
the order in which the two gene products function on a common pathway. For example, a strain expressing a hyperactive STE11 (e.g. STEll-I) has a constitutive response, whereas a strain with a deletion of STE5 (steSA), a protein of unknown function, does not respond to pheromone. A strain that combines both mutations has a constitutive response phenotype. This result is consistent with STEll acting after or at the same level as STE5. Similar logic applied to other double mutant strains showed that STE11 also acts after the G[3 subunit but before the STE7, FUS3/KSS1 and STE12 steps in signal transmission [34.°,35]. Additionally, the phosphorylation states of STE7 and FUS3 corroborated the placement of STEll on the pathway. Hyperactive STE11 caused phosphorylation of STE7 and FUS3, even in the absence of pheromone, whereas absence of STEll blocked their phosphorylation [32",34*.,36.-]. Biochemical experiments resolved the order for STE7 and FUS3/KSS1 function by showing that pheromone induces STE7 activity in the absence of FUS3 and KSS1 [36**]. This result is consistent with STE7 functioning before the FUS3/KSS1 step in signal transmission and made
256
Cell regulation Comparison of S. pombe and S. cerevisiae mating response pathways In S. pombe, both nutritional cues (starvation) and pheromone-receptor interactions stimulate the two haploid cell types (P and M) to mate with each other [38]. The S. cerevisiae and S. pombe mating response pathways are strikingly analogous (Fig. 1) [38-47]. One functional difference is that in S. cerevisiae, the G6~, complex transmits the signal, whereas in S. pombe, the Gu-subunit transmits the signal [21",421. Also different from S. cerevisiae, the S. pombe mating response pathway integrates the pheromone-induced signal with one that is dependent on an activity provided by a RAS homolog, RAS1 [48,49"].
STE7 a good candidate for the MAP kinase activator. In vitro kinase assays and reconstitution experiments confirmed this role. STE7 is a dual specificity kinase that modifies FUS3 at the appropriate threonine and tyrosine sites [33"1. Further, these in vitro modifications stimulate the ability of FUS3 to phosphorylate one of its physiological substrates, FAR1 (M Peter, A Garmer, G Ammerer, I Herskowitz, unpublished data) [33°.,37-]. Once activated, FUS3 also phosphorylates STE7 in vitro [33"]. This finding is consistent with the observation that pheromone-induced phosphorylation of STE7 in vivo depends on activity of STEll and either FUS3 or KSS1 [36"]. Because STE7 is a more active kinase when isolated from a fus3A ksslA strain compared with a wildtype strain, this reciprocal phosphorylation of STE7 by FUS3 could be part of an adaptive response [33"'].
The BYR2, BYR1 and SPK1 predicted protein kinases of the S. pombe pathway are structurally related to the STE11, STE7 and FUS3/KSS1 protein kinases, respectively, of the S. cerevisiae pathway (Fig. 2) [43-46]. Epistasis relationships and complementation studies place the structurally related kinases at analogous positions in their respective pathways [43,44,50"]. For most pairs, high level expression of the heterologous protein kinase in the corresponding mutant strain is sufficient for complementation. A notable exception is suppression of the S. cerevisiae stell mutant. This suppression requires expression of both the BYR1 and BYR2 proteins of S. pombe, suggesting that BYR2 and BYR1 interact cooperatively [50"]. The implied interaction and the deduced order of function are consistent with the possibility that BYR2 is the direct activator of BYR1.
The STE20 predicted protein kinase is a newly identified component of the pathway. Its catalytic domain has some similarity to that of mammalian protein kinase C (PKC) subtypes (27-33 % identity) {30"]. Notably, the aminoterminal regulatory domain of STE20 is not characteristic of PKC and is not similar to any other known protein. STE20 placement on the pathway was deduced from double mutant strains that combined a deletion of STE20 (ste2OA), which blocks pheromone response, with constitutive alleles of other pathway components. These epistasis experiments showed that STE20 acts after or at the same level as the G protein, but before STE5 and STEll [30"].
(a) PKC1 family PKC1 YPK1
(b) STE11 family
YPK1
YPK2
43
90
YPK2
PKC-~
52
43
43
PKC-~
52
44
43
STE11 PKC-E 68
(c) STE7 family
BYR2 ~
BYR2
BCK1
47
(d) MAP kinsase family
STE7 BYR1
Fig. 2. Per cent identities for the catalytic
FUS3 BYR1
KSS1
MKK1
42
42
MKK1
MKK2
42
43
80
MKK2
PBS2
42
51
44
42
PBS~
MEK
49
53
44
43
55
KSSI
SPK1
61
66
SPK1
MPK1
51
52
53
MPK1
HOG1
51
52
53
50
HO(
ERK2
56
57
60
54
53
domains of protein kinase subfamilies. Sequence comparisons were limited to the region bounded by the conserved GXGXXG motif of subdomain I (where X is any amino acid) and conserved R of subdomian Xl [64]. The percentage of identical amino acid residues was calculated for each pair using the GCG BestFit program to maximize the number of matches. Sources of sequences used for these comparisons are referenced in the text except for PKC [65], PCK-8 [66] and ERK2 [67].
A conserved kinase cascade for MAP kinase activation in yeast Errede and kevin Additional pathways in S. cerevisiae use related protein kinase modules for intracellular signal transduction The PKCl-mediated signal transduction pathway The S. cerevisiae PKCI gene encodes a homolog of the a - , [3- and y-subtypes of mammalian PKC [51]. Preliminary biochemical studies of PKC1 suggest that it behaves similarly to its metazoan counterparts (P Chen, DE Levin, unpublished data). Loss of PKC 1 function results in a cell lysis defect that is due to a deficiency in cell wall construction. Addition of osmotic stabilizing agents to the growth medium compensates for the defect and allows cell proliferation [52%53]. Although we do not know the signal to which PKC 1 responds, its mutant phenotype was exploited to identify downstream components of the PKC1 pathway. This approach uncovered four genes that, when mutationally activated or expressed at high levels, suppressed the cell-lysis defect associated with pathway inactivation [ 5 4 " - 5 6 " ] . The four genes encode predicted protein kinases that are structurally analogous to those on the yeast mating response pathways (Fig. 2). BCK1 (bypass of C kinase) is most similar to STE11 and BYR2 [54"]. MKK1 and MKK2 (MAP kinase kinase), are related to each other and to STE7 and BYR1 [55"]. MPK1 (MAP kinase) is a member of the MAP kinase family and thus related to FUS3/KSS1 and SPK1 ]50"]. The phenotypes of deletion mutants are consistent with all four gene products acting on the same pathway. BCK1 and MPK1 each provide a unique function because bcklA and mpkIA single deletion mutants have a temperature-dependent cell-lysis defect that is suppressed by osmotic stabilizers [54",56"°]. MKK1 and K K 2 are redundant because only the mkklA mkk2A double mutant shows this phenotype [55"]. While the pkcl mutant has an unconditional lysis phenotype, cell lysis is temperature-dependent in deletion mutants of the other pathway components. The less severe defect is consistent with BCK1, MKK1/MKK2 and MPK1 operating on one branch of a pathway that bifurcates after PKCl (Fig. 1) [54"]. Genetic epistasis analyses revealed the order of components on this branch of the PKCl pathway (Fig. 1). Mutationally activated BCK1 kinase transmits a signal even in the absence of PKCl [54°.]. As either mpklz~ or mkklA mkk2A mutations block this constitutive signal, MPK1 and MKK1/MKK2 function downstream of BCK1 [55"%56"]. The use of a mutationally activated MKK1 confirmed its position downstream of BCK1. The ability of overexpressed MPK1 to suppress the mkklA mkk2A mutations is consistent with MPK1 acting after MKK1/MKK2. This result suggests that MPK1 is activated by a mechanism similar to that of other MAP kinases. This notion has some support because MPK1 function is severely impaired by phenylalanine or alanine substitutions at the respective tyrosine or threonine residues that correspond to sites of phosphorylation associated with the activation of other MAP kinase family members
[56,,].
In S. cerevisiae both the PKCl pathway and the pheromone-induced pathway use a kinase cascade with the same architecture. This striking conservation suggests that a common module of protein kinases has evolved to mediate different physiological responses in yeast. We suggest that this module extends minimally between BCK1 (or STEll) and MPK1 (or FUS3/KSS1). Presumably to avoid confusion of signals, the components of one module do not normally interact productively with components of the other. The finding that a Xenopus MAP kinase expressed in yeast complements mpkIA mutants but notfus3AkssIA mutants is consistent with each module having specificity for interacting members [56"]. However, it is possible to create conditions under which components of one pathway will interact with those of another. For example, expression of either STEll or BYR2 at high levels can suppress loss of BCK1 function (KS Lee, DE Levin, unpublished data).
s. cerevisiae may use related kinase modules for other
signaling pathways The HOG accumulation pathway responds to increases in extracellular osmolarity by increasing intracellular glycerol production. The signal pathway leading to this response involves at least two predicted protein kinases, HOG1 and PBS2 [57",58]. HOG1 is most closely related to members of the MAP kinase family and PBS2 is most closely related to members of the STE7 family (Fig. 2). The rapid, PBS2-dependent, tyrosine phosphorylation of HOG1 in response to increases in extracellular osmolarity is consistent with the functional analogies implied by their respective structural similarities to STE7 and FUS3. We predict that upstream protein kinases will be uncovered in this pathway and speculate that one will be structurally analogous to BCK1/STEll. The possibility of still another conserved module in S. cerevisiae is suggested by the recent finding of a redundant pair of PKC-related proteins, YPK1 and YPK2, that are essential for vegetative growth (Fig. 2) [59].
Conservation of protein kinase modules from yeast to mammals cDNA clones encoding MAP kinase activators have been isolated recently from Xenopus [60], mouse [61], rat [62] and human [63] cells. The sequences of these genes reveal that they encode enzymes that are most closely related to members of the STE7 family of protein kinases. This observation suggests the possibility that the entire module of protein kinases used for yeast signaling pathways may be conserved throughout metazoan evolution. The yeast paradigm presented here suggests a STE1l-like protein kinase in vertebrates may act as the MAP kinase kinase activator. Yet the vertebrate pathway may be more complex. For example, raf, which is not closely related to members of the STEll family, has been implicated as an activator of the MAP kinase kinase [17-19]. One possi-
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Cell regulation bility is that there may be multiple activators at this level. Considering the wide array of extracellular agents that activate vertebrate MAP kinases, a multiplicity of MAP kinase kinase activators may have evolved to integrate signals from different stimuli. An alternative possibility is that raf will turn out to be the activator of a STEll family member [19].
Conclusion The conserved modules for yeast signal transduction pathways, as presently defined, begin a phosphorylation cascade with a STEll-like enzyme, and end with the activation of a MAP kinase. The modules described here provide a road map for the dissection of signaling pathways leading to MAP kinase activation in vertebrates. These pathways must amplify and integrate signals from different extracellular stimuli. We suggest the yeast module may have been preserved not only because its design allows for signal amplification, but also because it provides multiple sites for regulatory inputs and signal integration. The yeast PKC1 pathway provides the paradigm for PKC-dependent MAP kinase activation in mammals. The conserved modules in other yeast pathways involve G protein mediated processes and may use other protein kinases. These precedents suggest mechanisms by which mammalian MAP kinases could be activated through PKC-independent pathways.
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AHN NG, SEGER R, KREBS EG: T h e MAP Kinase Activator. Curr Opin Cell Biol 1992, 4:992-999. Reviews the MAP kinase activator, its purification from different organisms, its characterization, its regulation by phosphorylation, and its structural relationship to the STE7 family of protein kinases. 9. •
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MATSUDAS, KOSAKAH, TAKENAKAK, MORIYAMAK, SAKA1H, AKIYAMAT, GOTOH Y, N1SH1DA E: X e n o p u s MAP Kinase Activator: Identification and Function as a Key Intermediate in the Phosphorylation Cascade. EMBO J 1992, 11:973-982.
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29. •
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Requires the Sequential Function of Three Protein Kinases. Mol Cell Biol 1993, in press. Shows that pheromone induces hypeFphosphorylation of STE7 on serine and threonine residues. Because a catalytically inactive form of STE7 is still modified in response to pheromone, the modifications are not due to intramolecular autocatalysis. Because STE7 activiW is induced under conditions where it is not h3qgerphospho~4ated, the modificatkms are a consequence of its actiw~tion, but not the determining event. CHANG F, HERSKOWITZ I: Phosphorylation of FAR1 in Response to ~-Factor: A Possible Requirement for Cell-cycle Arrest. Mol Biol Cell 1992, 3:445-450. Shows that transcriptional induction of FAR1 from a heterologous promoter is not suft]cient for it to inhibit CLN2. Presents data showing that FAR1 is phosphorylated in response to pheromone. It is proposed that this phosphorylation may be required for FAR1 activity. 37. .
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STEVENSONBJ, RHODES N, ERREDE B, SPRAGUE GF, JR: Constitutive Mutants of the Protein Kinase STEll Activate the Yeast Pheromone Response Pathway in the Absence of the G Protein. Genes Dev 1992, 6:1293 1304. Describes alleles of STE11 that cause a constitutive pheromone response consistent with them encoding hyperactive or unregulated forms of the kinase. This paper includes epistasis analyses that place STE 11 on the pathway before STE7 and FUS3/KSS1. Additional evidence shows that the hyperactive STE11 causes phosphorylation of STE7 in the absence of added pheromone. 35.
36. ••
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Presents evidence that three predicted protein kinases of S. pombe, BYR2, BYR1 and SPK1, that are structurally related to the S. cerevisiae kinases STEll, STE7 and FUS3, respectively, are also functionally related. This paper also cites epistasis results that are consistent with the RASl-dependent signal intersecting the pheromone-induced pathway at a step before or at the G~-subunit. 51.
LEVINDE, FIELDS FD, KUNISAWAR, BISHOPJM, THORNERJA: A Candidate Protein Kinase C Gene, PKC1, Is Required for the S. cerevisiae Ceil Cycle. Cell 1990, 62:213-224.
52. •
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a Putative Protein Kinase (BCK1) Bypass the Requirement for a Saccharomyces cerevisiae Protein Kinase C Homolog. Mol Cell Biol 1992, 12:172-182. Describes the isolation and sequence of the BCK1 gene, whose mutational activation suppresses the loss of PKC1 function. Deletion of BCK1 results in a temperature dependent cell lysis defect. 55. ••
IRIE K, TAKASE M, LEE KS, LEV1N DE, ARAKI H, MATSUMOTO K, OSHIMAY: MKK1 and MKK2, encoding Saccharomyces cerevisiae MAP Kinase-kinase Homologs, Function in the Pathway Mediated by Protein Kinase C. Mol Cell Biol 1993, in press. Describes the isolation of the MKK1/2 genes, whose deletion results in temperature-dependent cell lysis. Demonstrates that overexpression of MKK1 suppresses loss of BCK1. 56. ••
LEE KS, IRIE K, GOTOH Y, WATANABEY, NISHIDAE, MATSUMOTO K, LEV1NDE: A Yeast MAP Kinase Homolog (MPK1) Mediates Signalling by Protein Kinase C. Mol Cell Biol 1993, in press. Describes the isolation of the MPKI gene, whose overexpression suppresses the loss of BCKI function. Deletion of MPK1 results in temperature-dependent cell lysis, which is complemented by Xenopus MAP kinase. BREWSTERJL, DE VALOIRT, DWYER ND, WINTER E, GUSTIN MC: An Osmo-sensing Signal Transduction Pathway in Yeast. Science 1993, in press. Describes the isolation of mutants in HOG1 and PBS2 using a genetic screen for osmotic sensitivity. Demonstrates PBS2 dependent tyrosine phosphorylation of the MAP kinase homolog, HOG1. 57. ••
B Errede, Department of Chemistry, CB#3290, University of North Carolina, Chapel Hill, North Carolina 27599, USA. DE Levin, Department of Biochemistry, John Hopkins University, School of Public Health, Baltimore, Maryland 21205, USA.