- Email: [email protected]
Plugging it in: signaling circuits and the yeast cell cycle
Plugging it in: signaling circuits and the yeast cell cycle Curt Wittenberg* and Steven I Reed? Signal transduction pathways provide the means to transmit information and elicit specific responses. Modulation of the cell cycle machinery is one such response. Molecular genetic approaches with budding yeast have been
progression, it will become clear to the reader that the mechanisms by which those effects are exerted are, in most cases, only poorly understood. As a consequence we pose more questions than we present answers.
instrumental
in elucidating the components of these complex signaling pathways and the inter-relationships among these components. Recent progress has revealed pathways that link extracellular signals with the machinery governing both cell cycle progression and morphogenesis. The nature of the interface
between
cell cycle apparatus
nutritional and checkpoint
signals with the
is just now emerging.
Addresses ‘Department of Molecular Biology, MB-3, The Scripps Research Institute, 10666 N Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: [email protected] TDepartment of Molecular Biology, MB-7, The Scripps Research institute, 10666 N Torrey Pines Road, La Jolla, CA 92037, USA Current Opinion in Cell Biology 1996, 8:223-230 0 Current Biology Ltd ISSN 0955-0674 Abbreviations CDK cyclin-dependent kinase GAP GTPase-activating protein GEF guanyl nucleotide exchange factor MAPK mitogen-activated protein kinase PKA protein kinase A
Introduction Information is the key to sound decisions. Accordingly, cells must collect and interpret many types of information in order to respond appropriately to the situations with which they are faced. Information is processed via signal transduction pathways to elicit a response. Stimuli can be placed into two classes: external stimuli that evoke adaptive responses to the external environment, and internal stimuli that coordinate intracellular processes. Responses to both types of stimuli can involve alterations in cell cycle progression. The mechanisms by which the signal transduction pathways interact with the cell cycle machinery have been a major focus of cell cycle research. In this review, we concern ourselves with signals that influence cell cycle progression in budding yeast. Despite its relative simplicity, &z~-&~~~yces cemisiae exhibits a full range of responses to both internal and external stimuli. As a free-living organism, it must monitor many aspects of the extracellular environment while at the same time participating in the cell-cell interactions required for its sexual cycle. Finally, as in other cells, internal checkpoint mechanisms are essential for the orderly progression of cell cycle events. Examples of each of these classes of stimuli and signaling pathways are considered here. Although each was chosen because of its known effect on ceil cycle
The yeast mating pheromone
response
The classical signaling system which modulates the cell cycle in budding yeast is the mating pheromone response. Via this pathway, cells of complementary mating types are induced to embark on a physiological and developmental program that promotes sexual conjugation. One element of this program is the arrest of mating partners in the G1 phase of the cell cycle, presumably to ensure synchrony during zygote formation. Genetic analysis has elucidated many elements of the mating pheromone signaling pathway and revealed much about their function (see Fig. 1; reviewed in [1,2]). These elements include the peptide pheromones that initiate the response, the mating type specific receptors that bind the pheromones, and the heterotrimeric G proteins that couple the receptors to downstream signaling elements. A cascade of protein kinases which are homologous to vertebrate mitogen-activated protein kinases (MAPKs) and their activating enzymes has also been shown to be essential for transmission of the pheromone signal. However, one region of this wiring diagram has, until recently, stubbornly resisted elucidation: the segment between the receptor-coupled G protein and the protein kinase cascade. In addition, the precise nature of the interaction between the activated MAPK homologs and the cell cycle regulatory machinery remains somewhat ambiguous. The most significant recent conceptual advance in understanding mating pheromone signaling has been in linking the receptor-coupled G protein to the downstream protein kinase cascade. In the past year, evidence has been gathered implicating the small Rho family GTPase Cdc42 as a signaling element in this segment of the pheromone-response pathway. Firstly, temperature-sensitive mutations in CDC42, or the gene encoding its guanyl nucleotide exchange factor, CDC24, block pheromone-responsive signaling at the restrictive temperature [3**,4”]. In addition, mutations in RGAI, a gene encoding a putative GTPase-activating protein (GAP) for Cdc42, confer constitutive activation of the pheromone-response pathway [S”]. Secondly, activated (GTP-bound) Cdc42 has been shown to activate the protein kinase activity of Ste20 in U~WO [3”]. This protein kinase, a homolog of the mammalian PAK65 [6,7], has, on the basis of genetics, been placed at the top of the MAPK-homologous cascade [S]. Thus, a potential link has been established between activation of Cdc42 and downstream signaling elements that have
224
Ceil regulation
Figure 1 A diagramatic representation of the currently accepted model of the yeast pheromone-response pathway. The identities of the elements as indicated either by their homology with known proteins or by their demonstrated functions are shown (left). Although we have tried to be accurate, not all associations depicted have been directly demonstrated. MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MAPKKKK, MAPKKK kinase.
identity of element
Mating pheromone signaling pathway
Peptide pheromone Seven transmembrane domain receptor Heterotrimeric G protein
GEF GAP/small G protein/ polarity establishment protein 1 MAPKKKK Scaffold protein
MAPKKK
’
MAPKK 3
MAPWMAPK
CDK inhibitor/transcription
factor
AL
8
a Mating-specific genes
Cln
CDK
Cdc28
Ceil cycle arrest -@
Phosphorylation 0
been characterized previously. This is surprising because Cdc4.2 (and Cdc24) have central roles in modulating the polarity of the actin cytoskeleton (reviewed in [9]) which is essential for morphogenesis during the normal cell division cycle; hence the issue is raised of how these two apparently disparate functions are regulated within an individual cell. However, as conjugation requires Cdc42mediated morphogenetic events, it is possible that a duality of Cdc42 function allows coordination of morphogenesis with other pheromone responses. In this regard, it is interesting that Beml, a protein important for establishment of cell polarity, can interact with actin and with both Ste20 and Ste5 [lo*]. Several issues, however, concerning the role of Cdc42 in mating pheromone signaling remain unresolved. First, how is the pheromone signal transmitted from the receptor-coupled heterotrimeric G protein to this small GTPase? A potential explanation is provided by the recent demonstration, in the two-hybrid system, of an interaction between the B subunit of the trimeric G protein (Ste4) and the guanyl nucleotide exchange factor (GEF) for Cdc42, Cdc24 [4**]. As the signaling moiety of the
1996 Current Opinion in Cell Biology
pheromone-responsive G protein in S. cerzvi,isiae is its By subunit, this observation suggests that free &r released in response to pheromone binding may activate GTP exchange on Cdc42 by direct or indirect interaction with the GEF Cdc24. A second issue concerns the relationship of the Cdc4Zactivated kinase Ste20 with kinases of the MAPK-homologous cascade. Although it has been demonstrated that Ste20 can phosphorylate the MAPK kinase kinase, Stell, in vitro, it is not clear yet what the functional implications of this phosphorylation are for Stell activity. Finally, Ste20, in conjunction with the MAPK-activating kinases (but not the MAPK homologs themselves), has an additional role in regulating an alternative foraging growth mode (pseudohyphal growth [ll]; see Fig. 2). How are these regulatory networks segregated in an individual cell? The most likely explanation comes from the identification of an apparent ‘scaffold’ protein that physically constrains elements of the signaling circuits. Ste.5, initially identified in a screen for pheromone-unresponsive mutants, has been shown, on the basis of co-immunoprecipitation and two-hybrid analyses, to physically interact with multiple elements of the MAPK-homologous cascade [12-151. In addition, recent evidence indicates an interaction between
Plugging it in: signaling circuits and the yeast cell cycle Wittenberg and Reed
Ste5 and Ste4-Stel8, the G&y moiety of the transducing G protein [16]. Therefore, it appears that most of the known pathway elements are physically sequestered in the context of mating pheromone signaling. The molecular basis for signaling at the other end of the pheromone-response pathway has also remained elusive. Although it is clear that a cyclin dependent kinase inhibitor, Farl, forms a crucial link between the cell cycle apparatus and the upstream MAPK cascade, it has been difficult to precisely establish the relevant aspects of that interaction. The levels of Far1 increase in response to pheromone signaling [17], and Far1 has recently been shown to be the only target of the Ste12 transcription factor that is required for pheromone induction of G1 arrest (LJWM Oehlen, FR Cross, personal communication). However, transcriptional activation of Far1 is not sufficient to mediate cell cycle arrest [18]; Far1 is also phosphorylated in response to treatment with mating pheromone [19,20]. The accepted model has Fus3, the MAPK homolog, at the bottom of the cascade, where it phosphorylates Farl, activating its function as an inhibitor of cyclin-dependent kinases (CDKs) [19,20]. However, the relationship between phosphorylation by Fus3 and activation of Far1 remains to demonstrated in V&O. In addition, at least a portion of phosphorylation of Far1 results from its association with its target, the Cdc28 kinase [19,21]. However, Cdc28-dependent phosphorylation of Far1 is more likely to signal its inactivation or destabilization than it is to signal its activation as a CDK inhibitor [21].
Pseudohyphal
and haploid invasive growth
The propensity to proliferate as free-living cells has resulted in the classification of S. cemisiue as a yeast. However, under appropriate nutrient conditions (i.e. limiting nitrogen), diploid cells of the so-called dimorphic strains of S. cemisiae can adopt a filamentous mode of growth that is in many ways reminiscent of fungal hyphae [22]. In the pseudohyphal mode, cells become elongated, adopt an altered pattern of budding, fail to abscise following cytokinesis, and attain the ability to penetrate agar [22,23’]. Filamentous forms of haploid cells, referred to as haploid invasive cells, also have the capacity to penetrate agar [24,25**]. Although the haploid and diploid filamentous forms appear to differ in significant ways and are induced by distinct stimuli, both responses depend, at least in part, upon elements of the same signaling pathway [ 11,25**]. Morphogenesis and the cell cycle machinery
Two properties of pseudohyphal growth are consistent with altered regulation of the cell cycle machinery. First, and most noticeably, pseudohyphal cells exhibit a hyperpolarized pattern of growth resulting in an elongated cell morphology ([23*]; Fig. 2a). In these cells, the period of hyperpolarized growth extends from late G1 phase to well into the G2 phase of the cell cycle, whereas only
225
a brief period of hyperpolarized growth associated with the emergence of the bud is observed in yeast-form cells, during late G1 and early S phase [23*]. This elongated morphology, although less dramatic, is also apparent during haploid invasive growth [24,25”]. In addition to this morphological alteration, pseudohyphal cells lack the characteristic mother-daughter asynchrony observed during growth in the yeast form and, as a consequence, budding is synchronous ([23*]; Fig. 2a). This is likely to result from an increase in the period of bud growth during Gz phase, producing newborn daughter cells that require little or no growth prior to reaching the minimum size required for budding. In contrast, the asynchrony characteristic of yeast-form cells results from the fact that daughter, but not mother, cells require significant growth to attain the minimum size for budding. The mechanism governing this G1 phase size requirement remains obscure. What is the source of these morphological perturbations? Although it is likely that modulation of cyclin-CDK activities is involved, no systematic investigation of either the expression or influence of cyclins during pseudohyphal differentiation has been reported. It is tempting to speculate that the hyperpolarized phenotype of pseudohyphal cells is a consequence of extended expression or stabilization of G1 cyclins (Clns) or of delayed expression of mitotic B-type cyclins (Clbs) (see below). Indeed, polarized growth during G1 phase in the yeast form is a consequence of activation of the CDK Cdc28 by Clns, whereas a largely depolarized mode of growth, characteristic of the remainder of the cell cycle, is promoted by Clbs [26]. Constitutive overexpression of the G1 cyclins Clnl or Cln2 is known to promote hyperpolarized growth and delay abscission of daughter cells ([26]; S Reed, C Witcenberg, unpublished data). Therefore, failure to activate the Clb-associated forms of the Cdc28 kinase might explain both the persistent hyperpolarized growth and the delay in the execution of mitosis that is characteristic of filamentous growth. Intriguingly, girl mutants, which display a constellation of phenotypes reminiscent of pseudohyphal growth and exhibit enhanced pseudohyphal differentiation in response to nitrogen limitation [24,27], have been shown to hyperaccumulate Clnl protein [28’]. Although this is consistent with the hypothesis that pseudohyphal growth is associated with persistent G1 cyclin expression, the pleiotropic effects of girl mutations [27,29] preclude a simple interpretation of this result. Alterations in the levels of CDKs may be sufficient to explain the morphogenetic properties of pseudohyphal cells, but it is clear that they are insufficient to explain the full complement of pseudohyphal and haploid invasive phenotypes.
Signaling and filamentous growth
The signals responsible for the induction of pseudohyphal and haploid invasive growth are poorly understood, but may include a complex assortment of nutritional and mechanical signals. So far, the Ras/cAMP pathway and
226
Cell regulation
Figure 2
Low nitrogen Other stimuli?
(a) Yeast =>
(b)
Low nitrogen
Pseudohyphal
Other stimuli?
\lY
Stel2
0
a Unknown targets
Pseudohyphal
J& growth
Haploid invasive growth
W
0
PKA
V
0 1996 Current Opinion in Cell Biology
CAMP
Pseudohyphal and haploid invasive growth. (a) Mother and daughter cells from each division initiate a new bud synchronously during pseudohyphal growth (right) (redrawn after a figure in (23’1). In contrast, mothers and daughters growing in the yeast form bud asynchronously (left). This is a consequence of the inherent asymmetry generated during division of yeast-form cells, coupled with the existence of a G1 checkpoint which monitors cell size in the daughter cells. The periods of polarized bud growth (single arrow within a cell) and isotropic bud growth (multiple arrows within a cell) observed for each growth form are also indicated [23’,26]. (b) The putative transduction pathway for generation of haploid invasive and pseudohyphal cells. Both pathways require elements of the MAPK cascade in addition to the Stel2 transcription factor, which is also used by the mating pheromone signaling pathway (see Fig. 1). However, neither the MAPK homologs nor the putative scaffolding protein are common to both pathways. The upstream and downstream components of the pathway are currently unknown. The RaslcAMP pathway is known to have opposite effects on the two responses. However, the mechanism by which these effects are exerted remains to be elucidated.
a novel pathway utilizing some elements of the mating pheromone MAPK cascade (Fig. 2b) have been shown to be important. The involvement of the Ras/cAMP pathway is complex as it has opposite effects on pseudohyphal versus haploid invasive growth [22,30*]. In contrast, the Ste20, Ste7, and Stell protein kinases, elements of the mating pheromone induced MAPK-homologous cascade, and the Stel2 transcription factor, a putative target of that cascade, have been shown to be essential for both the pseudohyphal and haploid invasive phenotypes [ 11,25**]. Conspicuously absent from the list are Fus3 and Kssl, the two MAPK homologs activated by this cascade in
response to mating pheromone. This finding, together with the observation that the Ste.5 protein inferred to serve as a scaffold for this cascade is also unnecessary in this context, has led to the hypothesis that the essential elements for pseudohyphal growth are sequestered into a distinct complex containing an as yet unidentified MAPK by a scaffold protein analogous to Ste5 [25**]. This might explain how a single pathway could be used by alternative stimuli to evoke distinct responses (haploid invasive growth and the mating response) in the same cell. A further distinction in these pathways must also occur at the level of the Ste12 transcription factor such that
Plugging it in: signaling circuits and the yeast cell cycle Wittenberg
this shared element can generate distinct transcriptional outputs. The resolution of these issues will provide much needed insight into the mechanisms by which eukaryotic cells segregate intracellular signals that are transduced by common or closely related elements.
Nutrients
and the CAMP pathway
The status of nutrients in the environment is of primary importance for cellular growth and differentiation. As such, specific nutrients must be monitored, and met with an appropriate response. The Ras/cAMP pathway is probably the best understood of the nutrient-regulated signaling pathways (reviewed in [31]). Increased CAMP accumulation is known to occur in response to glucose and other rapidly fermented carbon sources. However, CAMP is a prerequisite for growth and proliferation on any carbon source. For instance, CAMP influences the
and Reed
227
protein synthetic machinery by modulating the expression of specific ribosomal components [32]. Consistent with the importance of CAMP for growth and proliferation, a number of elements of the Ras/cAMP pathway have been shown to be essential for progression through Gr phase of the cell cycle. These include adenylate cyclase (encoded by CDC35/CYRI) which generates CAMP, its regulators (encoded by CDC25, RASl or RASZ), and at least one of three redundant CAMP-dependent protein kinases (encoded by TPKZ, TPKZ or TPK3) which are the sole known targets of CAMP (Fig. 3a). Hyperactivation of CAMP-dependent protein kinase (also known as A kinase and protein kinase A) results in failure to arrest during Gr phase in response to nutrient limitation. Despite increasing knowlege of the elements of this pathway, the nutrient sensor and the downstream targets relevant to growth and proliferation remain issues of conjecture.
Flgure 3 The RaslcAMP
pathway and cell cycle
regulation. (a) Elements of the RaslcAMP pathway. Cdc25 is a GAP for the small G proteins Rasl and Ras2 which, in S. cerevisiae, activate adenylate cyclase (Cdc35Kyrl). CAMP promotes the disociation of Bcyl, the negative regulatory subunit, from Tpkl, Tpk2. and Tpk3, three PKA enzymes. The glucose sensor for this pathway has been proposed to be a subunit of the transporter (see [31]). (b) Influences of CAMP on the expression
ATP
CAMP
of Gf cyclins.
Two antagonistic effects of CAMP on the expression of CLNl and CLN2 have been described. First, addition of glucose to starved cells induces CLN3
Other effects
gene
Inactive
expression independently of the presence of CAMP (left; 1331). However, timely activation of CLN7 and CLN2 gene expression depends upon both Cln3 and CAM!? In contrast to this positive effect of CAMP on CLNl the accumulation
and CLN2
expression,
(b)
Glucose
Glucose
(other carbon source?)
n
of CAMP that occurs in
response to addition of glucose to cells growing on other non-repressing carbon sources
is associated
with a delay in
CLN7 and CLN2
gene expression (right; [35**,41 ‘*I) and a concomitant increase in the minimum cell size required for budding.
CLN7/2
expression
Cell cycle progression
CLN1/2
expression
Gr delay (resetting of minimum size for budding) 0 1996 Current Opinion in Cell Biology
228
Cell regulation
Recent studies of the role of the Ras/cAMP pathway have begun to shed some light on the link between nutrient availability and cell proliferation. Two apparently antagonistic effects of CAMP on the cell cycle apparatus have been described. First, CAMP has been shown to be necessary for efficient transcriptional activation of the Gr cyclin genes, CLNI and CLNZ [33]. CLN3, on the other hand, is expressed in a nutrient-dependent, CAMP-independent manner. Stimulation of CLNZ and CLNZ expression in response to CAMP depends upon Cln3 (Fig. 3b), consistent with its importance for the timely activation of CLNZ and CLNZ transcription in cycling cells [34,35]. Whether this effect of CAMP results from the action of protein kinase A (PKA) on some element of the CLN transcriptional apparatus, or whether it affects CLN expression via a more general mechanism, remains to be established. Although a direct effect of PKA on the activity of transcription factors has been demonstrated in other systems (reviewed in [36]), there is as yet no such demonstration in yeast. In contrast to the positive role described above, CAMP also mediates a negative effect on cell cycle progression. In response to nutritional upshift from a non-fermentable carbon source to glucose, cells experience a rapid burst of CAMP accumulation [37,38”]. This burst is associated with a transient lag in Gr phase and a coincident upward readjustment of the minimal cell size required for passage through a Gr-size checkpoint. The glucose-induced Gr lag depends upon the Ras/cAMP pathway (Fig. 3b; [39]) and can be effectively induced by exogenously supplied CAMP [40]. Recent reports demonstrate that the cell cycle lag coincides with the transcriptional downregulation of CLNl and, to a lesser extent, of CLN2 and a number of other Gr-specific genes [38”,41”]. Mutational inactivation of CLNl and CLN,?, but not of CLN3, largely abrogates the minimum budding size readjustment, consistent with the idea that those cyclins are the relevant targets of this negative signal. Assuming that the effects of CAMP are exerted solely through the activation of PKA, how does CAMP mediate these apparently antagonistic effects? One possibility is that one or both of these effects is non-specific, in the sense that it is more closely related to either growth rate or metabolic conditions than it is to PKA-dependent phosphorylation of specific elements of the transcriptional machinery. Such explanations will be difficult to exclude because of the absence of a defined PKA-mediated regulatory effect on a transcription factor or other element of the cell cycle regulatory apparatus.
Signaling and cell cycle control by checkpoints Checkpoint controls are internal regulatory systems that prevent cell cycle progression if ‘prerequisites for progression have not yet occurred ([42]; reviewed in [43]).
Such controls are thought to protect cells from damage or death resulting as a consequence of the occurrence of events out of order. The presence of checkpoint controls in yeast is revealed by the identification of checkpoint mutations that release cells from the dependency of events upon prior conditions. Within this class of mutations are those that release the dependency of mitosis upon the repair of damaged DNA, the completion of DNA replication, the formation of a functional spindle and (the most recent discovery) the formation of a bud [42,43]. Several salient observations have emerged from investigation of these regulatory systems. One of these is that whereas the checkpoints involving DNA damage, DNA replication and spindle formation confer cell cycle arrest in Gz, they apparently do not do so by preventing activation of the CdcB-Clb kinases responsible for mitotic induction [44-47]. Instead, these checkpoints block cell cycle progression either by inhibiting an event downstream of Cdc28 kinase activation, or by inhibiting a parallel pathway essential for mitotic events. The exception is the newly identified checkpoint that couples bud emergence to mitotic events [48’]. In this case, the Cdc28-Clb kinases are inhibited when budding is prevented either mutationally or by external conditions. Although all Cdc28-Clb kinases become phosphorylated at Tyr19 in Cdc28 (equivalent to Tyr15 in mammalian Cdkl) when budding is prevented, accounting for much of the inhibition, transcription of CLBZ, the gene encoding the principle mitotic cyclin, is also inhibited. Therefore, either mutation of Tyr19 of Cdc28 or overexpression of mitotic Clbs overrides this checkpoint. Regulation of mitotic kinase activity by negative phosphorylation of CDKs is observed in other organisms [27], but in the context of different checkpoints, namely those associated with DNA replication and DNA damage (reviewed in [43]). The molecular basis of signaling the presence or absence of a bud to the cell cycle machinery remains to be elucidated.
Perhaps the most intriguing observation to emerge in the checkpoint field in the past year is the recognition that the elements that signal DNA damage are extraordinarily conserved from yeast to man. The yeast gene MECZ was identified from a genetic screen for mutations that conferred sensitivity to DNA damage [49”,50**,51]. The encoded product is a large polypeptide with what appears to be a lipid kinase domain. Surprisingly, this protein is homologous to the product of the human ataxia telangiectasia (A7’M) gene, mutations in which confer sensitivity to DNA damage, among other related phenotypes [52]. The homology extends well past the lipid kinase domain, suggesting a true functional relationship. These proteins, therefore, are likely to constitute part of a conserved mechanism that signals DNA damage so that cell cycle and repair responses can be implemented. The lipid kinase domain suggests that phosphorylation of phosphatidylinositol or a derivative is a component of the signal resulting from the damage. However, it is not
Plugging it in: signaling circuits and 9
clear that lipids are the authentic targets of these kinases, and other substrates such as tyrosines of proteins have not been excluded. Furthermore, although signaling via the ATM gene product appears to interact with the mammalian cell cycle machinery via the ~53 tumor suppressor protein [53-S], it is not at all clear how Mecl-mediated DNA damage signaling impinges on cell cycle control in yeast.
Acknowledgements The authors would like to acknowledge members of their research groups for heloful discussions. and Stefan Lanker and David Stuart for comments on the manuscript. Thank you to Fred Cross, George Sprague and Stephen Garrett for communications of results prior to publication. Finally, we would like to thank those of you who communicated results and manuscripts that we were unable to use in the context of this review. Curt Wittenberg was supported by Public Health Service (PHS) grants GM43487 and GM46006, and Steven I Reed was supported by PHS grant GM38328.
References
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . l
*
and Reed
229
Establishes that elem nts inbolved in udding and cytoskeletal polarity are also components of tie mating phero)) one signaling pathway, filling in the final gaps between th recebtor and t gets of the pathway. See also [4*1. 4. ..
Zhao ZS, Leung T, Manser E, I;im L: Pheromone signailing in Seccharomyces ceretisiae raulres the small GTP-binding protein Cdc42p and its activator CDC24. MO/ Cell Biol 1995, 15:5246-5257. See annotation [3**1. 5. ..
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Conclusions Although circuits of signal transduction that impact the cell cycle in yeast have been increasingly elucidated, the mechanistic links to the cell cycle machinery remain frustratingly vague. The unique exception is the mating pheromone response in which the CDK inhibitor Far1 is mobilized both transcriptionally and post-translationally to inhibit the G1 form of Cdc28. Even here, however, the nature of the phosphorylation events that are crucial for Far1 activation have eluded characterization. For other pathways, the mechanism of interface with the known elements of cell cycle control can only be speculated. For the Ras/cAMP pathway, transcriptional control of G1 cyclins by PKA is implicated, but no known transcription factor of this system has been shown to be regulated by PKA. For pseudohyphal and haploid invasive growth, it is clear that elements of the cell ‘cycle machinery must be regulated to generate the observed alterations in polarity and timing of events. Yet no mechanism for this regulation has been presented. The most puzzling unresolved issue is the link between the DNA-replication and DNA-damage checkpoints and the cell cycle. Whereas in other systems there is evidence that these checkpoints impact directly on the activation of the mitotic CDK, in budding yeast, cell cycle control in these contexts is mediated independently of activation of Cdc28. Clearly, the interface between signal transduction pathways and cell cycle control will remain a research frontier of great importance until the remaining questions can be resolved.
yeast cell c$cle Wittenberg
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and GLN2
and a number of other
41. ..
Baroni MD, Monti P, Alberghina L: Repression of growthregulated Gl cyclin expression by cyclic AMP in budding yeast Nature 1994, 371:339-342. Shows that addition of exogenous CAMP to cycling cells acts via PKA to regulate the expression of CLNI and CLN2, and shows that CLNI and CLN2 are the relevant taroets for the cell size readiustment that occurs in response to an increase in-CAMP 42.
Hartwell LH, Weinert TA: Checkpoints: controls that ensure the order of cell cycle events. Science 1989, 248:629-834.
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Murray AW: The genetics of cell cycle checkpoints. Genet Dev 1995, 5:5-l 1.
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Hadwiger JA. Wittenberg C, Mendenhall MD, Reed SI: The S8ccharomyces cerevisiae CKSl gene, a homolog of the Schizoseccheromyces pombe sucl+ gene, encodes a subunit of the Cdc28 protein kinase complex. MO/ Cell Biol 1989, 9:2034-2041.
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Amon A. Surana U. lvor M. Nasmvth K: Reaulation of 034-z* tyrosine phospho&ation’ is not’required?or entry l&o mitosis in S. cerevisiae. Nature 1992, 355:388-394.
Erickson JR, Johnston M: Suppressors reveal two classes of glucose repression genes in the yeast Saccheromyces cerevisiee. Genetics 1993, 138:1271-l 278.
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Sorger PK, Murray AW: S-phase feedback control in budding yeast independent of tyrosine phosphoryletion of p34ck2*. Nature 1992, 355:365-368.
Ward M, Gimeno CJ, Fink GR, Garrett S: SOK2 may regulate cyclic AMP-dependent protein kinase-stimulated growth and pseudohyphal development by repressing transcription. MO/ Cell Biol 1995, 15:6854-6863. Describes a putative PKA-regulated transcriptional repressor that may play a role in governing the switch between unicellular and filamentous growth.
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Stueland CS, Lew DJ, Cismowski MJ, Reed SI: Full activation of p34CDC28 histone Hl kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast S8CCh8rOmyCeS cerevisise. MO/ Cell Biol 1993, 13:3744-3755.
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Kim YJ, Francisco L, Chen GC, Marcotte E, Chan CS: Control of cellular morphogenesis by the Ipl2IBem2 GTPase-activating protein: possible role of protein phosphorylation. J Cell Biol 1994, 127:1381-1394.
Barral Y, Jentsch S, Mann C: Gl cyclin turnover and nutrient uptake are controlled by a common pathway In yeast Genes Dev 1995, 9:399-409. Shows that Clnl is stabilized and hyperaccumulated in grrl mutants (which are unable to respond properly to glucose), suggesting a link between nutrient availability and G, cyclin activity. 28. .
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Neuman-Silberberg FS, Bhattacharya S, Broach JR: Nutrient availability and the RAS/cyclic AMP pathway both induce expression of ribosomal protein genes in Saccharomyces cerevisise but by different mechanisms. MO/ Cell Biol 1994, 15:3187-3196.
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Dirick L, Bohm T, Nasmyth K: Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of S8CCh8rOmyCeS cerevisiae. EMBO J 1995, 14:4803-4813.
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Stuart D, Wittenberg C: CLN3, not positive feedback, determines the timing of CLN2 gene expression in cycling cells. Genes Dev 1995, 9:2780-2793.
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Karin M, Hunter T: Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr Bioll995, 5:747-757.
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Tokiwa G, Tyers M, Volpe T, Futcher B: Inhibition of Gl cyclin activity by the Ras/cAMP pathway in yeast Nature 1994, 371:342-345. Shows that the adjustment of cell size caused by addition of glucose or of exogenous CAMP, or by hyperactivation of PKA, is associated with a transient
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Lew DJ, Reed SI: A cell cycle checkpoint monitors cell morphogenesis in budding yeast J Cell Biol 1995, 129:739-749. Defines a new cell cycle checkpoint in S. cerevisiae at which successful morphogenesis is monitored, and mitosis is controlled, by regulating the activity of the Cdc28 protein kinase.
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Weinert TA, Kiser GL, Hartwell LH: Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev 1994. 8:652-665. Shows th’at a yeast gene.(MEC7) involved in monitoring DNA damage is structurally related to the human ATM gene which is required for a related function. See also [50”1. 60. ..
Morrow DM, Tagle DA, Shiloh Y, Collins FS, Hieter P: TELI, an S. cerevisiee homolog of the human gene mutated in atexia telangiectesia, is functionally related to the yeast checkpoint gene MECI. Cell 1995, 82:831-840. See annotation [49**1. 51.
Paulovich AG, Hartwell LH: A checkpoint regulates the rate of progression through S phase in S. cerevisiee in response to DNA damage. Cell 1995,82:841-847.
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Savitsky K, Bar SA, Gilad S, Rotman G, Ziv Y, Vanagaite L, Tagle DA, Smith S, Uziel T, Sfez S et a/.: A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 1995, 268:1749-l 753.
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Canman CE, Wolff AC, Chen CY, Fornace AJ, Kastan MB: The p53-dependent Gl cell cycle checkpoint pathway and atexiatelangiectesia. Cancer Res 1994, 54:5054-5058.
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Kastan MB, Zhan Q, El Diery W, Carrier F, Jacks T, Walsh WV, Plunkett BS, Vogelstein 8, Fornace Al: A mammalian cell cycle checkpoint pathway utilizing ~53 and GADD45 is defective in ataxia-telangiectasia. Cell 1992, 71:587-597.
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Khanna KK, Beamish H, Yan J, Hobson K, Williams R, Dunn I, Lavin MF: Nature of Gl/S cell cycle checkpoint defect in ataxia-telangiectasia. Oncogene 1995, 11:609-618.
38. ..
progression, it will become clear to the reader that the mechanisms by which those effects are exerted are, in most cases, only poorly understood. As a consequence we pose more questions than we present answers.
instrumental
in elucidating the components of these complex signaling pathways and the inter-relationships among these components. Recent progress has revealed pathways that link extracellular signals with the machinery governing both cell cycle progression and morphogenesis. The nature of the interface
between
cell cycle apparatus
nutritional and checkpoint
signals with the
is just now emerging.
Addresses ‘Department of Molecular Biology, MB-3, The Scripps Research Institute, 10666 N Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: [email protected] TDepartment of Molecular Biology, MB-7, The Scripps Research institute, 10666 N Torrey Pines Road, La Jolla, CA 92037, USA Current Opinion in Cell Biology 1996, 8:223-230 0 Current Biology Ltd ISSN 0955-0674 Abbreviations CDK cyclin-dependent kinase GAP GTPase-activating protein GEF guanyl nucleotide exchange factor MAPK mitogen-activated protein kinase PKA protein kinase A
Introduction Information is the key to sound decisions. Accordingly, cells must collect and interpret many types of information in order to respond appropriately to the situations with which they are faced. Information is processed via signal transduction pathways to elicit a response. Stimuli can be placed into two classes: external stimuli that evoke adaptive responses to the external environment, and internal stimuli that coordinate intracellular processes. Responses to both types of stimuli can involve alterations in cell cycle progression. The mechanisms by which the signal transduction pathways interact with the cell cycle machinery have been a major focus of cell cycle research. In this review, we concern ourselves with signals that influence cell cycle progression in budding yeast. Despite its relative simplicity, &z~-&~~~yces cemisiae exhibits a full range of responses to both internal and external stimuli. As a free-living organism, it must monitor many aspects of the extracellular environment while at the same time participating in the cell-cell interactions required for its sexual cycle. Finally, as in other cells, internal checkpoint mechanisms are essential for the orderly progression of cell cycle events. Examples of each of these classes of stimuli and signaling pathways are considered here. Although each was chosen because of its known effect on ceil cycle
The yeast mating pheromone
response
The classical signaling system which modulates the cell cycle in budding yeast is the mating pheromone response. Via this pathway, cells of complementary mating types are induced to embark on a physiological and developmental program that promotes sexual conjugation. One element of this program is the arrest of mating partners in the G1 phase of the cell cycle, presumably to ensure synchrony during zygote formation. Genetic analysis has elucidated many elements of the mating pheromone signaling pathway and revealed much about their function (see Fig. 1; reviewed in [1,2]). These elements include the peptide pheromones that initiate the response, the mating type specific receptors that bind the pheromones, and the heterotrimeric G proteins that couple the receptors to downstream signaling elements. A cascade of protein kinases which are homologous to vertebrate mitogen-activated protein kinases (MAPKs) and their activating enzymes has also been shown to be essential for transmission of the pheromone signal. However, one region of this wiring diagram has, until recently, stubbornly resisted elucidation: the segment between the receptor-coupled G protein and the protein kinase cascade. In addition, the precise nature of the interaction between the activated MAPK homologs and the cell cycle regulatory machinery remains somewhat ambiguous. The most significant recent conceptual advance in understanding mating pheromone signaling has been in linking the receptor-coupled G protein to the downstream protein kinase cascade. In the past year, evidence has been gathered implicating the small Rho family GTPase Cdc42 as a signaling element in this segment of the pheromone-response pathway. Firstly, temperature-sensitive mutations in CDC42, or the gene encoding its guanyl nucleotide exchange factor, CDC24, block pheromone-responsive signaling at the restrictive temperature [3**,4”]. In addition, mutations in RGAI, a gene encoding a putative GTPase-activating protein (GAP) for Cdc42, confer constitutive activation of the pheromone-response pathway [S”]. Secondly, activated (GTP-bound) Cdc42 has been shown to activate the protein kinase activity of Ste20 in U~WO [3”]. This protein kinase, a homolog of the mammalian PAK65 [6,7], has, on the basis of genetics, been placed at the top of the MAPK-homologous cascade [S]. Thus, a potential link has been established between activation of Cdc42 and downstream signaling elements that have
224
Ceil regulation
Figure 1 A diagramatic representation of the currently accepted model of the yeast pheromone-response pathway. The identities of the elements as indicated either by their homology with known proteins or by their demonstrated functions are shown (left). Although we have tried to be accurate, not all associations depicted have been directly demonstrated. MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MAPKKKK, MAPKKK kinase.
identity of element
Mating pheromone signaling pathway
Peptide pheromone Seven transmembrane domain receptor Heterotrimeric G protein
GEF GAP/small G protein/ polarity establishment protein 1 MAPKKKK Scaffold protein
MAPKKK
’
MAPKK 3
MAPWMAPK
CDK inhibitor/transcription
factor
AL
8
a Mating-specific genes
Cln
CDK
Cdc28
Ceil cycle arrest -@
Phosphorylation 0
been characterized previously. This is surprising because Cdc4.2 (and Cdc24) have central roles in modulating the polarity of the actin cytoskeleton (reviewed in [9]) which is essential for morphogenesis during the normal cell division cycle; hence the issue is raised of how these two apparently disparate functions are regulated within an individual cell. However, as conjugation requires Cdc42mediated morphogenetic events, it is possible that a duality of Cdc42 function allows coordination of morphogenesis with other pheromone responses. In this regard, it is interesting that Beml, a protein important for establishment of cell polarity, can interact with actin and with both Ste20 and Ste5 [lo*]. Several issues, however, concerning the role of Cdc42 in mating pheromone signaling remain unresolved. First, how is the pheromone signal transmitted from the receptor-coupled heterotrimeric G protein to this small GTPase? A potential explanation is provided by the recent demonstration, in the two-hybrid system, of an interaction between the B subunit of the trimeric G protein (Ste4) and the guanyl nucleotide exchange factor (GEF) for Cdc42, Cdc24 [4**]. As the signaling moiety of the
1996 Current Opinion in Cell Biology
pheromone-responsive G protein in S. cerzvi,isiae is its By subunit, this observation suggests that free &r released in response to pheromone binding may activate GTP exchange on Cdc42 by direct or indirect interaction with the GEF Cdc24. A second issue concerns the relationship of the Cdc4Zactivated kinase Ste20 with kinases of the MAPK-homologous cascade. Although it has been demonstrated that Ste20 can phosphorylate the MAPK kinase kinase, Stell, in vitro, it is not clear yet what the functional implications of this phosphorylation are for Stell activity. Finally, Ste20, in conjunction with the MAPK-activating kinases (but not the MAPK homologs themselves), has an additional role in regulating an alternative foraging growth mode (pseudohyphal growth [ll]; see Fig. 2). How are these regulatory networks segregated in an individual cell? The most likely explanation comes from the identification of an apparent ‘scaffold’ protein that physically constrains elements of the signaling circuits. Ste.5, initially identified in a screen for pheromone-unresponsive mutants, has been shown, on the basis of co-immunoprecipitation and two-hybrid analyses, to physically interact with multiple elements of the MAPK-homologous cascade [12-151. In addition, recent evidence indicates an interaction between
Plugging it in: signaling circuits and the yeast cell cycle Wittenberg and Reed
Ste5 and Ste4-Stel8, the G&y moiety of the transducing G protein [16]. Therefore, it appears that most of the known pathway elements are physically sequestered in the context of mating pheromone signaling. The molecular basis for signaling at the other end of the pheromone-response pathway has also remained elusive. Although it is clear that a cyclin dependent kinase inhibitor, Farl, forms a crucial link between the cell cycle apparatus and the upstream MAPK cascade, it has been difficult to precisely establish the relevant aspects of that interaction. The levels of Far1 increase in response to pheromone signaling [17], and Far1 has recently been shown to be the only target of the Ste12 transcription factor that is required for pheromone induction of G1 arrest (LJWM Oehlen, FR Cross, personal communication). However, transcriptional activation of Far1 is not sufficient to mediate cell cycle arrest [18]; Far1 is also phosphorylated in response to treatment with mating pheromone [19,20]. The accepted model has Fus3, the MAPK homolog, at the bottom of the cascade, where it phosphorylates Farl, activating its function as an inhibitor of cyclin-dependent kinases (CDKs) [19,20]. However, the relationship between phosphorylation by Fus3 and activation of Far1 remains to demonstrated in V&O. In addition, at least a portion of phosphorylation of Far1 results from its association with its target, the Cdc28 kinase [19,21]. However, Cdc28-dependent phosphorylation of Far1 is more likely to signal its inactivation or destabilization than it is to signal its activation as a CDK inhibitor [21].
Pseudohyphal
and haploid invasive growth
The propensity to proliferate as free-living cells has resulted in the classification of S. cemisiue as a yeast. However, under appropriate nutrient conditions (i.e. limiting nitrogen), diploid cells of the so-called dimorphic strains of S. cemisiae can adopt a filamentous mode of growth that is in many ways reminiscent of fungal hyphae [22]. In the pseudohyphal mode, cells become elongated, adopt an altered pattern of budding, fail to abscise following cytokinesis, and attain the ability to penetrate agar [22,23’]. Filamentous forms of haploid cells, referred to as haploid invasive cells, also have the capacity to penetrate agar [24,25**]. Although the haploid and diploid filamentous forms appear to differ in significant ways and are induced by distinct stimuli, both responses depend, at least in part, upon elements of the same signaling pathway [ 11,25**]. Morphogenesis and the cell cycle machinery
Two properties of pseudohyphal growth are consistent with altered regulation of the cell cycle machinery. First, and most noticeably, pseudohyphal cells exhibit a hyperpolarized pattern of growth resulting in an elongated cell morphology ([23*]; Fig. 2a). In these cells, the period of hyperpolarized growth extends from late G1 phase to well into the G2 phase of the cell cycle, whereas only
225
a brief period of hyperpolarized growth associated with the emergence of the bud is observed in yeast-form cells, during late G1 and early S phase [23*]. This elongated morphology, although less dramatic, is also apparent during haploid invasive growth [24,25”]. In addition to this morphological alteration, pseudohyphal cells lack the characteristic mother-daughter asynchrony observed during growth in the yeast form and, as a consequence, budding is synchronous ([23*]; Fig. 2a). This is likely to result from an increase in the period of bud growth during Gz phase, producing newborn daughter cells that require little or no growth prior to reaching the minimum size required for budding. In contrast, the asynchrony characteristic of yeast-form cells results from the fact that daughter, but not mother, cells require significant growth to attain the minimum size for budding. The mechanism governing this G1 phase size requirement remains obscure. What is the source of these morphological perturbations? Although it is likely that modulation of cyclin-CDK activities is involved, no systematic investigation of either the expression or influence of cyclins during pseudohyphal differentiation has been reported. It is tempting to speculate that the hyperpolarized phenotype of pseudohyphal cells is a consequence of extended expression or stabilization of G1 cyclins (Clns) or of delayed expression of mitotic B-type cyclins (Clbs) (see below). Indeed, polarized growth during G1 phase in the yeast form is a consequence of activation of the CDK Cdc28 by Clns, whereas a largely depolarized mode of growth, characteristic of the remainder of the cell cycle, is promoted by Clbs [26]. Constitutive overexpression of the G1 cyclins Clnl or Cln2 is known to promote hyperpolarized growth and delay abscission of daughter cells ([26]; S Reed, C Witcenberg, unpublished data). Therefore, failure to activate the Clb-associated forms of the Cdc28 kinase might explain both the persistent hyperpolarized growth and the delay in the execution of mitosis that is characteristic of filamentous growth. Intriguingly, girl mutants, which display a constellation of phenotypes reminiscent of pseudohyphal growth and exhibit enhanced pseudohyphal differentiation in response to nitrogen limitation [24,27], have been shown to hyperaccumulate Clnl protein [28’]. Although this is consistent with the hypothesis that pseudohyphal growth is associated with persistent G1 cyclin expression, the pleiotropic effects of girl mutations [27,29] preclude a simple interpretation of this result. Alterations in the levels of CDKs may be sufficient to explain the morphogenetic properties of pseudohyphal cells, but it is clear that they are insufficient to explain the full complement of pseudohyphal and haploid invasive phenotypes.
Signaling and filamentous growth
The signals responsible for the induction of pseudohyphal and haploid invasive growth are poorly understood, but may include a complex assortment of nutritional and mechanical signals. So far, the Ras/cAMP pathway and
226
Cell regulation
Figure 2
Low nitrogen Other stimuli?
(a) Yeast =>
(b)
Low nitrogen
Pseudohyphal
Other stimuli?
\lY
Stel2
0
a Unknown targets
Pseudohyphal
J& growth
Haploid invasive growth
W
0
PKA
V
0 1996 Current Opinion in Cell Biology
CAMP
Pseudohyphal and haploid invasive growth. (a) Mother and daughter cells from each division initiate a new bud synchronously during pseudohyphal growth (right) (redrawn after a figure in (23’1). In contrast, mothers and daughters growing in the yeast form bud asynchronously (left). This is a consequence of the inherent asymmetry generated during division of yeast-form cells, coupled with the existence of a G1 checkpoint which monitors cell size in the daughter cells. The periods of polarized bud growth (single arrow within a cell) and isotropic bud growth (multiple arrows within a cell) observed for each growth form are also indicated [23’,26]. (b) The putative transduction pathway for generation of haploid invasive and pseudohyphal cells. Both pathways require elements of the MAPK cascade in addition to the Stel2 transcription factor, which is also used by the mating pheromone signaling pathway (see Fig. 1). However, neither the MAPK homologs nor the putative scaffolding protein are common to both pathways. The upstream and downstream components of the pathway are currently unknown. The RaslcAMP pathway is known to have opposite effects on the two responses. However, the mechanism by which these effects are exerted remains to be elucidated.
a novel pathway utilizing some elements of the mating pheromone MAPK cascade (Fig. 2b) have been shown to be important. The involvement of the Ras/cAMP pathway is complex as it has opposite effects on pseudohyphal versus haploid invasive growth [22,30*]. In contrast, the Ste20, Ste7, and Stell protein kinases, elements of the mating pheromone induced MAPK-homologous cascade, and the Stel2 transcription factor, a putative target of that cascade, have been shown to be essential for both the pseudohyphal and haploid invasive phenotypes [ 11,25**]. Conspicuously absent from the list are Fus3 and Kssl, the two MAPK homologs activated by this cascade in
response to mating pheromone. This finding, together with the observation that the Ste.5 protein inferred to serve as a scaffold for this cascade is also unnecessary in this context, has led to the hypothesis that the essential elements for pseudohyphal growth are sequestered into a distinct complex containing an as yet unidentified MAPK by a scaffold protein analogous to Ste5 [25**]. This might explain how a single pathway could be used by alternative stimuli to evoke distinct responses (haploid invasive growth and the mating response) in the same cell. A further distinction in these pathways must also occur at the level of the Ste12 transcription factor such that
Plugging it in: signaling circuits and the yeast cell cycle Wittenberg
this shared element can generate distinct transcriptional outputs. The resolution of these issues will provide much needed insight into the mechanisms by which eukaryotic cells segregate intracellular signals that are transduced by common or closely related elements.
Nutrients
and the CAMP pathway
The status of nutrients in the environment is of primary importance for cellular growth and differentiation. As such, specific nutrients must be monitored, and met with an appropriate response. The Ras/cAMP pathway is probably the best understood of the nutrient-regulated signaling pathways (reviewed in [31]). Increased CAMP accumulation is known to occur in response to glucose and other rapidly fermented carbon sources. However, CAMP is a prerequisite for growth and proliferation on any carbon source. For instance, CAMP influences the
and Reed
227
protein synthetic machinery by modulating the expression of specific ribosomal components [32]. Consistent with the importance of CAMP for growth and proliferation, a number of elements of the Ras/cAMP pathway have been shown to be essential for progression through Gr phase of the cell cycle. These include adenylate cyclase (encoded by CDC35/CYRI) which generates CAMP, its regulators (encoded by CDC25, RASl or RASZ), and at least one of three redundant CAMP-dependent protein kinases (encoded by TPKZ, TPKZ or TPK3) which are the sole known targets of CAMP (Fig. 3a). Hyperactivation of CAMP-dependent protein kinase (also known as A kinase and protein kinase A) results in failure to arrest during Gr phase in response to nutrient limitation. Despite increasing knowlege of the elements of this pathway, the nutrient sensor and the downstream targets relevant to growth and proliferation remain issues of conjecture.
Flgure 3 The RaslcAMP
pathway and cell cycle
regulation. (a) Elements of the RaslcAMP pathway. Cdc25 is a GAP for the small G proteins Rasl and Ras2 which, in S. cerevisiae, activate adenylate cyclase (Cdc35Kyrl). CAMP promotes the disociation of Bcyl, the negative regulatory subunit, from Tpkl, Tpk2. and Tpk3, three PKA enzymes. The glucose sensor for this pathway has been proposed to be a subunit of the transporter (see [31]). (b) Influences of CAMP on the expression
ATP
CAMP
of Gf cyclins.
Two antagonistic effects of CAMP on the expression of CLNl and CLN2 have been described. First, addition of glucose to starved cells induces CLN3
Other effects
gene
Inactive
expression independently of the presence of CAMP (left; 1331). However, timely activation of CLN7 and CLN2 gene expression depends upon both Cln3 and CAM!? In contrast to this positive effect of CAMP on CLNl the accumulation
and CLN2
expression,
(b)
Glucose
Glucose
(other carbon source?)
n
of CAMP that occurs in
response to addition of glucose to cells growing on other non-repressing carbon sources
is associated
with a delay in
CLN7 and CLN2
gene expression (right; [35**,41 ‘*I) and a concomitant increase in the minimum cell size required for budding.
CLN7/2
expression
Cell cycle progression
CLN1/2
expression
Gr delay (resetting of minimum size for budding) 0 1996 Current Opinion in Cell Biology
228
Cell regulation
Recent studies of the role of the Ras/cAMP pathway have begun to shed some light on the link between nutrient availability and cell proliferation. Two apparently antagonistic effects of CAMP on the cell cycle apparatus have been described. First, CAMP has been shown to be necessary for efficient transcriptional activation of the Gr cyclin genes, CLNI and CLNZ [33]. CLN3, on the other hand, is expressed in a nutrient-dependent, CAMP-independent manner. Stimulation of CLNZ and CLNZ expression in response to CAMP depends upon Cln3 (Fig. 3b), consistent with its importance for the timely activation of CLNZ and CLNZ transcription in cycling cells [34,35]. Whether this effect of CAMP results from the action of protein kinase A (PKA) on some element of the CLN transcriptional apparatus, or whether it affects CLN expression via a more general mechanism, remains to be established. Although a direct effect of PKA on the activity of transcription factors has been demonstrated in other systems (reviewed in [36]), there is as yet no such demonstration in yeast. In contrast to the positive role described above, CAMP also mediates a negative effect on cell cycle progression. In response to nutritional upshift from a non-fermentable carbon source to glucose, cells experience a rapid burst of CAMP accumulation [37,38”]. This burst is associated with a transient lag in Gr phase and a coincident upward readjustment of the minimal cell size required for passage through a Gr-size checkpoint. The glucose-induced Gr lag depends upon the Ras/cAMP pathway (Fig. 3b; [39]) and can be effectively induced by exogenously supplied CAMP [40]. Recent reports demonstrate that the cell cycle lag coincides with the transcriptional downregulation of CLNl and, to a lesser extent, of CLN2 and a number of other Gr-specific genes [38”,41”]. Mutational inactivation of CLNl and CLN,?, but not of CLN3, largely abrogates the minimum budding size readjustment, consistent with the idea that those cyclins are the relevant targets of this negative signal. Assuming that the effects of CAMP are exerted solely through the activation of PKA, how does CAMP mediate these apparently antagonistic effects? One possibility is that one or both of these effects is non-specific, in the sense that it is more closely related to either growth rate or metabolic conditions than it is to PKA-dependent phosphorylation of specific elements of the transcriptional machinery. Such explanations will be difficult to exclude because of the absence of a defined PKA-mediated regulatory effect on a transcription factor or other element of the cell cycle regulatory apparatus.
Signaling and cell cycle control by checkpoints Checkpoint controls are internal regulatory systems that prevent cell cycle progression if ‘prerequisites for progression have not yet occurred ([42]; reviewed in [43]).
Such controls are thought to protect cells from damage or death resulting as a consequence of the occurrence of events out of order. The presence of checkpoint controls in yeast is revealed by the identification of checkpoint mutations that release cells from the dependency of events upon prior conditions. Within this class of mutations are those that release the dependency of mitosis upon the repair of damaged DNA, the completion of DNA replication, the formation of a functional spindle and (the most recent discovery) the formation of a bud [42,43]. Several salient observations have emerged from investigation of these regulatory systems. One of these is that whereas the checkpoints involving DNA damage, DNA replication and spindle formation confer cell cycle arrest in Gz, they apparently do not do so by preventing activation of the CdcB-Clb kinases responsible for mitotic induction [44-47]. Instead, these checkpoints block cell cycle progression either by inhibiting an event downstream of Cdc28 kinase activation, or by inhibiting a parallel pathway essential for mitotic events. The exception is the newly identified checkpoint that couples bud emergence to mitotic events [48’]. In this case, the Cdc28-Clb kinases are inhibited when budding is prevented either mutationally or by external conditions. Although all Cdc28-Clb kinases become phosphorylated at Tyr19 in Cdc28 (equivalent to Tyr15 in mammalian Cdkl) when budding is prevented, accounting for much of the inhibition, transcription of CLBZ, the gene encoding the principle mitotic cyclin, is also inhibited. Therefore, either mutation of Tyr19 of Cdc28 or overexpression of mitotic Clbs overrides this checkpoint. Regulation of mitotic kinase activity by negative phosphorylation of CDKs is observed in other organisms [27], but in the context of different checkpoints, namely those associated with DNA replication and DNA damage (reviewed in [43]). The molecular basis of signaling the presence or absence of a bud to the cell cycle machinery remains to be elucidated.
Perhaps the most intriguing observation to emerge in the checkpoint field in the past year is the recognition that the elements that signal DNA damage are extraordinarily conserved from yeast to man. The yeast gene MECZ was identified from a genetic screen for mutations that conferred sensitivity to DNA damage [49”,50**,51]. The encoded product is a large polypeptide with what appears to be a lipid kinase domain. Surprisingly, this protein is homologous to the product of the human ataxia telangiectasia (A7’M) gene, mutations in which confer sensitivity to DNA damage, among other related phenotypes [52]. The homology extends well past the lipid kinase domain, suggesting a true functional relationship. These proteins, therefore, are likely to constitute part of a conserved mechanism that signals DNA damage so that cell cycle and repair responses can be implemented. The lipid kinase domain suggests that phosphorylation of phosphatidylinositol or a derivative is a component of the signal resulting from the damage. However, it is not
Plugging it in: signaling circuits and 9
clear that lipids are the authentic targets of these kinases, and other substrates such as tyrosines of proteins have not been excluded. Furthermore, although signaling via the ATM gene product appears to interact with the mammalian cell cycle machinery via the ~53 tumor suppressor protein [53-S], it is not at all clear how Mecl-mediated DNA damage signaling impinges on cell cycle control in yeast.
Acknowledgements The authors would like to acknowledge members of their research groups for heloful discussions. and Stefan Lanker and David Stuart for comments on the manuscript. Thank you to Fred Cross, George Sprague and Stephen Garrett for communications of results prior to publication. Finally, we would like to thank those of you who communicated results and manuscripts that we were unable to use in the context of this review. Curt Wittenberg was supported by Public Health Service (PHS) grants GM43487 and GM46006, and Steven I Reed was supported by PHS grant GM38328.
References
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . l
*
and Reed
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Establishes that elem nts inbolved in udding and cytoskeletal polarity are also components of tie mating phero)) one signaling pathway, filling in the final gaps between th recebtor and t gets of the pathway. See also [4*1. 4. ..
Zhao ZS, Leung T, Manser E, I;im L: Pheromone signailing in Seccharomyces ceretisiae raulres the small GTP-binding protein Cdc42p and its activator CDC24. MO/ Cell Biol 1995, 15:5246-5257. See annotation [3**1. 5. ..
Stevenson BJ, Ferguson B, De Virgilio C, Bi E, Pringle JR, Ammerer G, Sprague GF: Mutation of RGAI, which encodes
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Conclusions Although circuits of signal transduction that impact the cell cycle in yeast have been increasingly elucidated, the mechanistic links to the cell cycle machinery remain frustratingly vague. The unique exception is the mating pheromone response in which the CDK inhibitor Far1 is mobilized both transcriptionally and post-translationally to inhibit the G1 form of Cdc28. Even here, however, the nature of the phosphorylation events that are crucial for Far1 activation have eluded characterization. For other pathways, the mechanism of interface with the known elements of cell cycle control can only be speculated. For the Ras/cAMP pathway, transcriptional control of G1 cyclins by PKA is implicated, but no known transcription factor of this system has been shown to be regulated by PKA. For pseudohyphal and haploid invasive growth, it is clear that elements of the cell ‘cycle machinery must be regulated to generate the observed alterations in polarity and timing of events. Yet no mechanism for this regulation has been presented. The most puzzling unresolved issue is the link between the DNA-replication and DNA-damage checkpoints and the cell cycle. Whereas in other systems there is evidence that these checkpoints impact directly on the activation of the mitotic CDK, in budding yeast, cell cycle control in these contexts is mediated independently of activation of Cdc28. Clearly, the interface between signal transduction pathways and cell cycle control will remain a research frontier of great importance until the remaining questions can be resolved.
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