- Email: [email protected]
M transition
seminars in
CANCER BIOLOGY, Vol 6, 1995: pp 203–209
The role of protein phosphatase type-2A in the Xenopus cell cycle: initiation of the G2/M transition Tina H. Lee
Regulation of the cell cycle by phosphorylation
The last several years has seen an explosion in the identification of a multiplicity of serine/threonine protein kinases with important functions in eukaryotic cell cycle progression. Although the major serine/threonine phosphoprotein phosphatases, that must oppose the action of the kinases, have been identified and extensively characterized for their involvement in metabolic processes, the functions of the phosphatases in cell cycle regulation is less well established. This paper focuses on the role of the type-2A protein phosphatase (PP2A) in the regulation of the G2/M transition in the Xenopus cell cycle. Although a role for PP2A in regulating G2/M has been suggested by studies in various systems, it is the relative simplicity of the in-vitro cell cycle extracts of Xenopus that has allowed the clearest dissection of the mechanism by which PP2A regulates this transition.
The majority of the enzymes that have been identified as regulators of cell cycle transitions are protein kinases and phosphoprotein phosphatases. Phosphorylation has long been recognized as a primary posttranslational regulatory mechanism controlling cell cycle progression. Many proteins undergo cell cycle dependent changes in their steady state level of phosphorylation. Some of these proteins are structural components of the cell, whose regulation is thought to mediate the ‘downstream events’ of the cell cycle, such as chromosome condensation, nuclear envelope breakdown, and spindle formation during M phase. Others are regulatory enzymes that directly phosphorylate or dephosphorylate the structural components of the cell. These ‘intermediary’ kinases and phosphatases are further controlled by enzymes involved in the regulation of the transitions themselves. Although a single enzyme might act at multiple levels, it is useful to view the effectors of cell cycle progression as members of a hierarchy of controlling elements, and the information passed down the hierarchy as changes in the phosphorylated state of the controlling elements (i.e. a kinase cascade). The ‘downstream’ elements in the hierarchy rely on information from ‘above’. For instance, DNA synthesis and other S phase events do not occur until S phase has been initiated. However, information also flows in the opposite direction. For instance, feedback control mechanisms in most somatic cells ensure that M phase initiation is delayed until the successful completion of downstream S phase events. Thus, each pathway in the hierarchy is linked to all others in a network of interdependency relationships.
Key words: cell cycle / protein phosphatase type-2A / cyclin B/cdc2 / G2/M transition / Xenopus ©1995 Academic Press Ltd
THE CELL CYCLE is a sequence of structural and functional rearrangements necessary for cell duplication and division. The events of the cell cycle are ordered with respect to one another by a series of transitions. Each transition, i.e. G1/S or G2/M, is a change in cell physiology that induces a discrete set of events, for example DNA replication during S-phase and spindle assembly during M phase. The correct timing and sequence of events are ensured by both positive and negative regulatory pathways that control the initiation of each transition. Such controls gone awry are thought to contribute to the development of many cancers; thus increased awareness of the biochemical mechanisms of cell cycle control are likely to aid in the development of treatments for cancer. An important step toward unravelling the complex array of cellular controls for division is an understanding of the biochemical pathways underlying the initiation of each cell cycle transition.
The kinases that regulate cell cycle progression Regulation of cell cycle progression by phosphorylation is mediated by the modulation of both multiple protein kinase, and phosphoprotein phosphatase activities. The last 5 years have seen an explosion in
From the Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA ©1995 Academic Press Ltd 1044-579X/95/040203 + 07$12.00/0
203
T. H. Lee phosphatase types, but little functional evidence for this exists as yet.
the identification of a multiplicity of serine/threonine kinases with specific and important functions in cell cycle progression.2-4 Each cell cycle transition appears to be controlled by one or more members of a highly conserved family of protein kinases called cyclindependent kinases (CDKs). As the name implies, each CDK depends on association with a cyclin for its activity. The cyclins themselves comprise a family of proteins whose abundance fluctuates during the cell cycle and whose accumulation, at least in part, limits progression through each cell cycle transition. G1 cyclins accumulate and associate with CDK2 and CDK4 during the G1 phase to promote entry into S phase, while mitotic cyclins accumulate and associate with CDC2 (CDK1) during late S and G2 phases to promote entry into M phase. Thus the cell cycle can be viewed as an orderly progression of events that occur as a consequence of the sequential activation of the appropriate cyclin/CDK complexes.2 The cyclin/ CDK complexes, in turn, are thought to dictate the physiologic state of the cell through differences in their substrate specificities and/or subcellular localization.
The difficulties in determining the function of the phosphatases in cell cycle progression: distinguishing direct from indirect effects As PP1 and PP2A exhibit broad and overlapping substrate specificities in vitro, results from mixing purified phosphatase with purified substrates are difficult to interpret. The availability of specific inhibitors, such as okadaic acid, which inhibits PP2A at low concentrations,10 and PP1 inhibitors-1 and -2,5 as well as mutant alleles of these enzymes in genetically tractable organisms,11-15 enables one to establish the effects of blocking each individual phosphatase either in vivo, or in complex in-vitro systems that are physiological. The power of such an analysis is illustrated by the strikingly distinct phenotypes exhibited by Schizosaccharomyces pombe cells carrying conditional alleles of either PP1 or PP2A catalytic subunits.14 Whereas PP2A mutants are defective in the G2/M transition, PP1 mutants appear to enter M phase normally, but rather fail to exit from M phase. Conditional mutations in genes encoding the catalytic subunit of PP1 impair mitotic progression in Aspergillus nidulans12 and Drosophila,13 as well as in S. pombe.11,14 Cells carrying mutant alleles exhibit defective mitotic spindle organization, excessive chromosome condensation, and abnormal sister chromatid segregation. Such cells fail to exit from mitosis, clearly indicating an important function(s) for PP1 in mitotic progression. However, the primary defects in these cells are difficult to distinguish from defects that occur as an indirect consequence of the lack of PP1 activity, mainly because of the interdependencies that link regulatory events to structural events. The interdependencies allow various different, but equally plausible interpretations of the PP1 mutational analysis.13 In the first scenario, PP1 activity would be primarily required for the initiation of the metaphase to anaphase transition. Failure to initiate the metaphase to anaphase transition would block all the downstream events that accompany the exit from mitosis, such as sister chromatid segregation. Cells would be stuck in the mitotic state and other abnormalities, such as overcondensed chromosomes, would occur as an indirect consequence. Alternatively, PP1 activity would be primarily required for the execution of a structural event, such as the correct
The phosphatases that regulate cell cycle progression Identification of the phosphatases that must oppose the action of these kinases has been hindered largely by the relatively broad substrate specificities of the serine/threonine phosphatases. The serine/threonine phosphatases have been studied, for many years, for their potential involvement in numerous metabolic processes.5,6 These studies have led to the extensive biochemical characterization of the four major types of serine/threonine phosphoprotein phosphatases in eukaryotic cells: Protein phosphatase type-1 (PP1); type-2A (PP2A); type-2B (PP2B); and type-2C (PP2C). Each holoenzyme is distinguished by its subunit composition, metal ion dependency, and in-vitro substrate specificity. Of these, PP1 and PP2A appear to be responsible for most of the serine/ threonine dephosphorylation in whole cell extracts.7,8 Therefore, these two phosphatases may carry out a number of functions important for cell cycle progression. The existence of multiple regulatory subunits of both PP1 and PP2A raise the possibility that distinct holoenzymes may play various roles during the cell cycle,6,9 and lead to speculations that a multiplicity of phosphatases underlie the apparent homogeneity of 204
Role of protein phosphatase type 2A in xenopus cell cycle mitotic cyclin B/cdc2 kinase complex. The remainder of this mini-review will focus on what has been learned about the role of PP2A in the G2/M transition in Xenopus. For information stemming from comparable genetic studies examining the role of PP2A in the fission yeast cell cycle, the reader is referred to Kinoshita et al.23
alignment of chromosomes on the metaphase plate. This defect would lead indirectly to a mitotic block by activation of a feedback control mechanism that delays mitotic progression until the chromosomes have been properly aligned. In summary, although mutational analyses have uncovered an important role for PP1 in the cell cycle, pinpointing the precise substrates and biochemical function of PP1 in mitotic progression may require a system with fewer interdependencies linking cell cycle events.
Regulation of the G2/M transition in Xenopus extracts The Xenopus embryonic cell cycle Entry into mitosis in early embryonic Xenopus cell cycle extracts depends on the translation of cyclin B alone,20 an indication that all other steps occur posttranslationally. However, cyclin B synthesis is not the only rate-limiting step.4,22 Even after the accumulation of a critical threshold level of cyclin B, there is an approximate 20-min lag phase during which inhibitory modifications of the cyclin B/cdc2 complex suppress its activity, until the G2/M transition. The lag phase can be viewed as a mechanism that transforms the continuous synthesis of cyclin B protein into a concerted activation of the cyclin B/cdc2 complex, and consequently an abrupt transition into mitosis. Not only does this provide a mechanism for the proper timing of mitosis, but may ensure that once mitosis is initiated, a sufficient pool of cyclin B/cdc2 precursor has accumulated to induce mitotic events. Furthermore, in the older embryo, when feedback controls are operative, the components of the lag phase may provide a mechanism linking mitotic initiation to the completion of S phase events. As the function of PP2A is to regulate this lag period, the remainder of this discussion will focus on the regulators of the lag as potential targets of PP2A. The mechanism of the lag phase has yet to be precisely understood, but many of the regulators that contribute to the lag have been identified. These factors can be described as a biochemical pathway that lies between the accumulation of cyclin B and the final activation of the cyclin B/cdc2 complex.4,21,22 The role of cyclin A, if any, in the generation of the lag phase, remains to be established. Ablation of cyclin A message appears to interfere with the cryptic feedback control mechanisms that can, in early Xenopus embryos, link the completion of DNA synthesis to cyclin B/cdc2 activation.24 However, the kinetics of cdc2 activation by cyclin B, in the normal embryonic cell cycle, is unchanged when cyclin A is ablated,24 suggesting that cyclin A is not essential for either the
In contrast to that of somatic cells, the early embryonic cell cycle of Xenopus lacks the feedback controls that normally link the completion of downstream events to cell cycle transitions. Blocking DNA synthesis or the assembly of the mitotic spindle, would lead to a G2 or mitotic delay, respectively, in most somatic cells,16 but has no effect on the cycling between S phase and M phase observed in the early embryonic cell cycle of Xenopus.17 This apparent lack of higher order controls appears not to reflect a fundamental difference in the cell cycle machinery of Xenopus, so much as the unusually small nuclear to cytoplasmic ratio of the egg, since increasing the number of nuclei per volume of cytoplasm in in-vitro eggs extracts reveals cryptic feedback control mechanisms.18,19 The relative simplicity of this embryonic cell cycle can be recapitulated in vitro,20 providing a useful system for dissecting the basic regulatory network that is required for the interconversion between interphase and mitotic states. Biochemical experiments in Xenopus extracts, combined with genetic analyses in yeast, have laid the foundation for a mechanistic understanding of the G2/M transition.4 Essential regulators, many of which were first identified in yeast,21 have been identified and extensively characterized in Xenopus. Furthermore, the simplicity of the Xenopus system has led to a fairly detailed kinetic formulation of the G2/M transition.22 In this in-vitro system and consistent with genetic studies in S. pombe PP2A definitely functions as a negative regulator of the G2/M transition. As described above, the Xenopus embryonic cell cycle differs from the yeast cell cycle in that it lacks feedback control mechanisms that link the execution of structural and regulatory events in most somatic cells; therefore, it has allowed the clearest dissection of the mechanism by which PP2A controls the key event in mitotic induction, the activation of the 205
T. H. Lee mik1, in the highly activated state,22,25 and the maintenance of the phosphatase that removes the inhibitory phosphates, cdc25, in the inactivated state, prior to the transition.22,26,27 The clear importance of these enzymes in regulating the transition has made them the subject of intense study. During the transition, changes in the activities of cdc25 and wee1/mik1 seem to be important for the abrupt activation of cdc2 and the sudden transition into mitosis. These changes are brought about by extensive phosphorylations of both cdc2526-29 and wee130-33 Thus, the initiation of the transition has been attributed to the activation of a ‘starter kinase’ that phosphorylates (and/or inactivation of a phosphatase that dephosphorylates) both cdc25 and wee1. That the mitotic phosphorylation of cdc25 and wee1 coincide with the abrupt activation of cdc2/cyclin B has led to the suggestion that a low level of cdc2/cyclin B activity, escaping the inhibitory modifications during interphase, turns off wee1 and
lag phase, nor for the final activation of cdc2 by cyclin B. Activation of cdc2 by cyclin B requires association with cyclin B, ostensibly because cyclin binding allows the phosphorylation of cdc2 on an essential, activating site, threonine 161. However, cyclin binding simultaneously induces the phosphorylation of cdc2 on a pair of adjacent inhibitory sites, threonine 14 and tyrosine 15. The inhibitory phosphorylations maintain the bulk of the cyclin bound cdc2 in an inactive state until the transition. Once cyclin exceeds a critical threshold level, it initiates a series of events that lead ultimately to the sudden removal of the inhibitory phosphorylations on cdc2, leaving only the essential thrl61 phosphorylation. The suppression of cdc2 activity during interphase as well as its abrupt activation at the transition, depends on the maintenance of the kinases that phosphorylate the thr14 and tyr15 residues, wee1 and
Figure 1. Model for the initiation of the G2/M transition, and the mechanism by which PP2A controls the transition. During interphase, PP2A inhibits two pathways, both required for the final activation of cdc2 by cyclin B. One pathway leads to the formation of a low level of the active complex (cyclin B/cdc-T161P), and the other leads to the formation of a second activator (?) that remains to be identified, indicated in the dashed box. The accumulation of a critical threshold level of cyclin B, however, eventually overcomes the block imposed by PP2A, allowing the formation of both rate-limiting activators. The concurrent activation of a threshold level of both of these activators initiates the positive-feedback loop that turns cdc25 activity up and wee1 activity down, leading to further dephosphorylation and activation of the bulk inhibited (cyclin B/cdc2T14,Y15P) complexes. Thus cyclin B/cdc2, its activators, and their cohorts are abruptly switched into the mitotic state, and M phase is initiated.
206
Role of protein phosphatase type 2A in xenopus cell cycle phase, the period of time when the kinetics of the reaction pathways leading to the activation of cdc2 evaluate either the sufficiency or insufficiency of the cyclin level. The rates of phosphorylation and dephosphorylation of both the inhibitory (thr14, tyr15) and activating sites on cdc2 (thr161) were examined, during the lag phase, prior to the transition.35 Somewhat surprisingly, the rates of phosphorylation and dephosphorylation of the inhibitory sites were unaffected by levels of PP2A that ultimately blocked the transition. This indicated that although the state of cdc25 and weel activities was highly dependent on the levels of PP2A and cyclin in the system, this effect was an indirect consequence of the modulation of an upstream regulator. An analysis of the rates of phosphorylation and dephosphorylation of the thr161 site during the lag, however, yielded a different result. PP2A inhibited the initial rate of accumulation of phosphate at that site. Furthermore, the inhibition was due to an effect on the phosphorylation, rather than the dephosphorylation rate of thr161 (there was no measurable dephosphorylation of thr161), ruling out the earlier model that PP2A regulated the activation of cdc2 by directly dephosphorylating the essential, activating site.36 Whether the inhibition of phosphorylation occurs through the modulation of the enzyme that catalyses thr161 phosphorylation,4 remains to be determined. In summary, the only pathway directly affected by PP2A was that controlling the phosphorylation of thr161.
turns on cdc25, which then feeds back to activate the bulk thr14, tyr15 phosphorylated cdc2.22-28 The initiating event, in this positive feedback loop model, is the accumulation of a critical threshold level of the thr161 phosphorylated, active cdc2/cyclin B complex (Figure 1).
The role of PP2A in the regulation of the G2/M transition in Xenopus The kinetic parameters governing cyclin activation of cdc2 can be modulated by PP2A. Felix et al34 and Solomon et al22 observed that the addition of okadaic acid, a potent PP2A inhibitor, to Xenopus interphase extracts induced the premature activation of cdc2 kinase. Thus PP2A appeared to function as a direct inhibitor of cdc2 activation. The specific effects of altering the level of PP2A activity on the activation process indicated that the length of the lag phase was determined by PP2A. In addition, the level of PP2A activity determined the amount of cyclin B protein required to initiate cdc2 kinase activation in the interphase extract. In summary, PP2A activity appeared to play an essential role in restraining cdc2 activation during interphase, and thus in maintaining the interphase state. PP1 inhibitors, on the other hand, had no detectable effect on the interphase to mitosis transition.34
Potential mechanisms of PP2A action Is thr161 phosphorylation the relevant target of PP2A?
The effect of PP2A on the timing of mitotic initiation, as well as its relationship to cyclin B, strongly suggested that PP2A might oppose cyclin in the control of a rate-limiting step for the initiation of the transition. Thus the identification of the target of PP2A during interphase promised to reveal the initiating event for the interphase to M phase transition. However, the analysis was once again complicated by interdependencies. That is, increased levels of PP2A inhibit the transition and all of the events that normally accompany the transition, such as the activation of cdc25 and the inactivation of wee1. But not all transitional events may be targets of PP2A; rather, they may occur as an indirect consequence of some other, upstream, initiating event. Therefore, a distinction between direct and indirect effects had to be made. In order to isolate the initiating event from the transitional events, analysis was focussed on the lag
The thr161 phosphorylation pathway was sensitively modulated by the ratio of cyclin B and PP2A, raising the possibility that this reaction was the rate-limiting event for entry into M phase. Such a hypothesis fit nicely with the positive feedback loop model which postulated that the accumulation of a low but critical threshold level of active cdc2/cyclin B complexes was required to trigger a switch in the balance of cdc25 and wee1 activities, which would lead to further activation of the bulk of cdc2/cyclin B complexes held inactive by the inhibitory phosphorylations.22,28 By delaying the onset of thr161 phosphorylation, PP2A would allow the inhibitory phosphorylations to outcompete thr161 phosphorylation, and active complexes would fail to form. A simple test of the model, however, proved otherwise.35 The addition of a suprathreshold level of active cdc2/cyclin B complexes to 207
T. H. Lee past and present members of the Kirschner lab, for many stimulating discussions, and M. Cyert for helpful comments on the manuscript. A grant from the NIH supported the writing of this manuscript.
interphase extracts did not substitute for the ratelimiting event. Instead, the complex was rapidly inactivated, indicating that at least one other unidentified pathway critical for mitotic initiation must exist. Furthermore, the presence of okadaic acid prevented the inactivation of the complex, suggesting that this unidentified pathway is regulated by PP2A.
References 1. Hartwell LH, Weinert TA (1989) Checkpoints: Controls that ensure the order of cell cycle events. Science 246:629–633 2. Sherr C (1993) Mammalian G1 cyclins. Cell 73:1059–1065 3. Heichman KA, Roberts JM (1994) Rules to replicate by. Cell 79:557–562 4. King RW, Jackson PK, Kirschner MW (1994) Mitosis in transition. Cell 79:563–571 5. Cohen P (1989) The structure and regulation of protein phosphatases. Annu Rev Biochem 58:485–497 6. Walter G, Mumby M (1993) Protein serine/threonine phosphatases: Structure, regulation, and function in cell growth. Physiol Rev 73:673–699 7. Ingebritsen TS, Stewart AA, Cohen P (1983) The protein phosphatases involved in cellular regulation: Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. Eur J Biochem 132:297–307 8. Labib K, Nurse P (1993) Bring on the phosphatases. Curr Biol 3:164–166 9. Mayer-Jaekel RE, Hemmings BA (1994) Protein phosphatase 2A — a ‘menage a trois’. Trends Cell Biol 4:287–291 10. Schonthal A (1992) Okadaic acid — a valuable new tool for the study of signal transduction and cell cycle regulation? New Biologist 4:16–21 11. Ohkura H, Kinoshita N, Miyatani S, Toda T, Yanagida M (1989) The fission yeast dis2 + gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell 57:997–1007 12. Doonan JH, Morris NR (1989) The bim G gene of Asperigillus nidulans, which is required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1. Cell 57:987–996 13. Axton JM, Dombradi B, Cohen PTW, Glover DM (1990) One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell 63:33–46 14. Kinoshita N, Ohkura H, Yanagida M (1990) Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle. Cell 63:405–415 15. Healy AM, Zolnierowicz S, Stapleton AE, Goebl M, DePaoliRoach AA, Pringle JR (1991) CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization and homology to the B subunit of mammalian type 2A protein phosphatase. Mol Cell Biol 11:5767–5780 16. Nurse P (1994) Ordering S phase and M phase in the cell cycle. Cell 79:547–550 17. Hara K, Tydeman P, Kirschner M (1980) A cytoplasmic clock with the same period as the division cycle in Xenopus eggs. Proc Natl Acad Sci USA 77:462–466 18. Dasso M, Newport JW (1990) Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus. Cell 61:811–823 19. Minshull J, Sun H, Tonks NK, Murray AW (1994) A MAP kinasedependent spindle assembly checkpoint in Xenopus egg extracts. Cell 79:475–486 20. Murray AW, Kirschner MW (1989) Cyclin synthesis drives the early embryonic cell cycle. Nature 339:275–280 21. Nurse P (1990) Universal control mechanism regulating onset of M-phase. Nature 344:503–508
The mechanism of mitotic initiation remains unsolved Thus, the direct target of PP2A in the control of cdc2/ cyclin B activation has yet to be determined. However, PP2A has proved to be a useful probe for dissecting the mechanism of mitotic initiation. The studies described here have led to the conclusion that pathways that actually trigger the G2/M phase transition remain to be identified, and have led us to search for new regulators. Along these lines, recent studies have identified at least two potentially novel regulators of cdc2/cyclin B in Xenopus extracts. One acts as a stoichiometric inhibitor of the kinase complex, and seems to be required to maintain the interphase state37 The second is a kinase(s), other than cdc2, that phosphorylates cdc25 and wee1. Mitotic cdc25 has recently been shown to possess a highly conserved, M phase specific phosphoepitope recognized by the MPM-2 antibody,38,39 suggesting that the kinase that phosphorylates this MPM-2 epitope plays an important role in mitotic induction. Similarly, the mitotic phosphorylation of wee1 has been shown to be due to a kinase other than cdc2.32 Interesting, each of these activities, which remains to be identified, is sensitive to PP2A.32,37,39
Concluding remarks The targets of PP2A in cell cycle progression are likely to be numerous, comprising both regulatory and structural elements of the dividing cell. If the study of the mechanism by which PP2A controls mitotic initiation has told us anything, it is that the broad substrate specificity phosphatases, far from being uninteresting, provide handles for investigating difficult cell biological problems.
Acknowledgements I would like to thank M. Kirschner and J. Kuang, as well as
208
Role of protein phosphatase type 2A in xenopus cell cycle 31. Parker LL, Piwnica-Worms H (1992) Inactivation of the p34cdc2 cyclin B complex by the human wee1 tyrosine kinase. Science 257:1955–1957 32. Tang Z, Coleman TR, Dunphy WG (1993) Two distinct mechanisms for negative regulation of the Wee1 protein kinase. EMBO J 12:3427–3436 33. Wu L, Russell P (1993) Nim 1 kinase promotes mitosis by inactivating Wee1 tyrosine kinase. Nature 363:738–741 34. Felix MA, Cohen P, Karsenti E (1990) Cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid. EMBO J 9:675–683 35. Lee TH, Turck C, Kirschner MW (1994) Inhibition of cdc2 activation by INH/PP2A. Mol Biol Cell 5:323–338 36. Lee TH, Solomon MJ, Mumby MC, Kirschner MW (1991) INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell 64:415–423 37. Lee TH, Kirschner MW (1994) Evidence for a stoichiometric inhibitor of MPF that regulates the G2/M transition in Xenopus. Manuscript submitted. 38. Davis FM, Tsao TY, Fowler SK, Rao PN (1983) Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci USA 80:2926–2930 39. Kuang J, Ashorn CL, Gonzalez-Kuyvenhoven M, Penkala JE (1994) Cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor. Mol Biol Cell 5:135–145
22. Solomon MJ, Glotzer M, Lee TH, Philippe M, Kirschner MW (1990) Cyclin activation of p34 cdc2. Cell 63:1013–1024 23. Kinoshita N, Yamano H, Niwa H, Yoshida T, Yanagida M (1993) Negative regulation of mitosis by the fission yeast protein phosphatase ppa2. Genes & Dev 7:1059–1071 24. Walker DH, Maller JL (1991) Role for cyclin A in the dependence of mitosis on completion of DNA replication. Nature 354:314–317 25. Smythe C, Newport JW (1992) Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34.cdc2 Cell 68:787–797 26. Izumi T, Walker DH, Maller JL (1992) Periodic changes in phosphorylation of the Xenopus Cdc25 phosphatase regulates its activity. Mol Biol Cell 3:927–939 27. Kumagai A, Dunphy WG (1992) Regulation of the Cdc25 protein during the cell cycle in Xenopus extracts. Cell 70:139–151 28. Hoffman I, Clarke PR, Marcote MJ, Karsenti E, Draetta G (1993) Phosphorylation and activation of human Cdc25-C by cdc2-cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J 12:53–63 29. Izumi T, Maller JL (1993) Elimination of cdc2 phosphorylation sites in the Cdc25 phosphatase blocks initiation of M-Phase. Mol Biol Cell 4:1337–1350 30. Coleman TR, Tang TZ, Dunphy WG (1993) Negative regulation of the wee1 protein kinase by direct action of the nim1/cdr1 mitotic inducer. Cell 72:919–929
209
CANCER BIOLOGY, Vol 6, 1995: pp 203–209
The role of protein phosphatase type-2A in the Xenopus cell cycle: initiation of the G2/M transition Tina H. Lee
Regulation of the cell cycle by phosphorylation
The last several years has seen an explosion in the identification of a multiplicity of serine/threonine protein kinases with important functions in eukaryotic cell cycle progression. Although the major serine/threonine phosphoprotein phosphatases, that must oppose the action of the kinases, have been identified and extensively characterized for their involvement in metabolic processes, the functions of the phosphatases in cell cycle regulation is less well established. This paper focuses on the role of the type-2A protein phosphatase (PP2A) in the regulation of the G2/M transition in the Xenopus cell cycle. Although a role for PP2A in regulating G2/M has been suggested by studies in various systems, it is the relative simplicity of the in-vitro cell cycle extracts of Xenopus that has allowed the clearest dissection of the mechanism by which PP2A regulates this transition.
The majority of the enzymes that have been identified as regulators of cell cycle transitions are protein kinases and phosphoprotein phosphatases. Phosphorylation has long been recognized as a primary posttranslational regulatory mechanism controlling cell cycle progression. Many proteins undergo cell cycle dependent changes in their steady state level of phosphorylation. Some of these proteins are structural components of the cell, whose regulation is thought to mediate the ‘downstream events’ of the cell cycle, such as chromosome condensation, nuclear envelope breakdown, and spindle formation during M phase. Others are regulatory enzymes that directly phosphorylate or dephosphorylate the structural components of the cell. These ‘intermediary’ kinases and phosphatases are further controlled by enzymes involved in the regulation of the transitions themselves. Although a single enzyme might act at multiple levels, it is useful to view the effectors of cell cycle progression as members of a hierarchy of controlling elements, and the information passed down the hierarchy as changes in the phosphorylated state of the controlling elements (i.e. a kinase cascade). The ‘downstream’ elements in the hierarchy rely on information from ‘above’. For instance, DNA synthesis and other S phase events do not occur until S phase has been initiated. However, information also flows in the opposite direction. For instance, feedback control mechanisms in most somatic cells ensure that M phase initiation is delayed until the successful completion of downstream S phase events. Thus, each pathway in the hierarchy is linked to all others in a network of interdependency relationships.
Key words: cell cycle / protein phosphatase type-2A / cyclin B/cdc2 / G2/M transition / Xenopus ©1995 Academic Press Ltd
THE CELL CYCLE is a sequence of structural and functional rearrangements necessary for cell duplication and division. The events of the cell cycle are ordered with respect to one another by a series of transitions. Each transition, i.e. G1/S or G2/M, is a change in cell physiology that induces a discrete set of events, for example DNA replication during S-phase and spindle assembly during M phase. The correct timing and sequence of events are ensured by both positive and negative regulatory pathways that control the initiation of each transition. Such controls gone awry are thought to contribute to the development of many cancers; thus increased awareness of the biochemical mechanisms of cell cycle control are likely to aid in the development of treatments for cancer. An important step toward unravelling the complex array of cellular controls for division is an understanding of the biochemical pathways underlying the initiation of each cell cycle transition.
The kinases that regulate cell cycle progression Regulation of cell cycle progression by phosphorylation is mediated by the modulation of both multiple protein kinase, and phosphoprotein phosphatase activities. The last 5 years have seen an explosion in
From the Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA ©1995 Academic Press Ltd 1044-579X/95/040203 + 07$12.00/0
203
T. H. Lee phosphatase types, but little functional evidence for this exists as yet.
the identification of a multiplicity of serine/threonine kinases with specific and important functions in cell cycle progression.2-4 Each cell cycle transition appears to be controlled by one or more members of a highly conserved family of protein kinases called cyclindependent kinases (CDKs). As the name implies, each CDK depends on association with a cyclin for its activity. The cyclins themselves comprise a family of proteins whose abundance fluctuates during the cell cycle and whose accumulation, at least in part, limits progression through each cell cycle transition. G1 cyclins accumulate and associate with CDK2 and CDK4 during the G1 phase to promote entry into S phase, while mitotic cyclins accumulate and associate with CDC2 (CDK1) during late S and G2 phases to promote entry into M phase. Thus the cell cycle can be viewed as an orderly progression of events that occur as a consequence of the sequential activation of the appropriate cyclin/CDK complexes.2 The cyclin/ CDK complexes, in turn, are thought to dictate the physiologic state of the cell through differences in their substrate specificities and/or subcellular localization.
The difficulties in determining the function of the phosphatases in cell cycle progression: distinguishing direct from indirect effects As PP1 and PP2A exhibit broad and overlapping substrate specificities in vitro, results from mixing purified phosphatase with purified substrates are difficult to interpret. The availability of specific inhibitors, such as okadaic acid, which inhibits PP2A at low concentrations,10 and PP1 inhibitors-1 and -2,5 as well as mutant alleles of these enzymes in genetically tractable organisms,11-15 enables one to establish the effects of blocking each individual phosphatase either in vivo, or in complex in-vitro systems that are physiological. The power of such an analysis is illustrated by the strikingly distinct phenotypes exhibited by Schizosaccharomyces pombe cells carrying conditional alleles of either PP1 or PP2A catalytic subunits.14 Whereas PP2A mutants are defective in the G2/M transition, PP1 mutants appear to enter M phase normally, but rather fail to exit from M phase. Conditional mutations in genes encoding the catalytic subunit of PP1 impair mitotic progression in Aspergillus nidulans12 and Drosophila,13 as well as in S. pombe.11,14 Cells carrying mutant alleles exhibit defective mitotic spindle organization, excessive chromosome condensation, and abnormal sister chromatid segregation. Such cells fail to exit from mitosis, clearly indicating an important function(s) for PP1 in mitotic progression. However, the primary defects in these cells are difficult to distinguish from defects that occur as an indirect consequence of the lack of PP1 activity, mainly because of the interdependencies that link regulatory events to structural events. The interdependencies allow various different, but equally plausible interpretations of the PP1 mutational analysis.13 In the first scenario, PP1 activity would be primarily required for the initiation of the metaphase to anaphase transition. Failure to initiate the metaphase to anaphase transition would block all the downstream events that accompany the exit from mitosis, such as sister chromatid segregation. Cells would be stuck in the mitotic state and other abnormalities, such as overcondensed chromosomes, would occur as an indirect consequence. Alternatively, PP1 activity would be primarily required for the execution of a structural event, such as the correct
The phosphatases that regulate cell cycle progression Identification of the phosphatases that must oppose the action of these kinases has been hindered largely by the relatively broad substrate specificities of the serine/threonine phosphatases. The serine/threonine phosphatases have been studied, for many years, for their potential involvement in numerous metabolic processes.5,6 These studies have led to the extensive biochemical characterization of the four major types of serine/threonine phosphoprotein phosphatases in eukaryotic cells: Protein phosphatase type-1 (PP1); type-2A (PP2A); type-2B (PP2B); and type-2C (PP2C). Each holoenzyme is distinguished by its subunit composition, metal ion dependency, and in-vitro substrate specificity. Of these, PP1 and PP2A appear to be responsible for most of the serine/ threonine dephosphorylation in whole cell extracts.7,8 Therefore, these two phosphatases may carry out a number of functions important for cell cycle progression. The existence of multiple regulatory subunits of both PP1 and PP2A raise the possibility that distinct holoenzymes may play various roles during the cell cycle,6,9 and lead to speculations that a multiplicity of phosphatases underlie the apparent homogeneity of 204
Role of protein phosphatase type 2A in xenopus cell cycle mitotic cyclin B/cdc2 kinase complex. The remainder of this mini-review will focus on what has been learned about the role of PP2A in the G2/M transition in Xenopus. For information stemming from comparable genetic studies examining the role of PP2A in the fission yeast cell cycle, the reader is referred to Kinoshita et al.23
alignment of chromosomes on the metaphase plate. This defect would lead indirectly to a mitotic block by activation of a feedback control mechanism that delays mitotic progression until the chromosomes have been properly aligned. In summary, although mutational analyses have uncovered an important role for PP1 in the cell cycle, pinpointing the precise substrates and biochemical function of PP1 in mitotic progression may require a system with fewer interdependencies linking cell cycle events.
Regulation of the G2/M transition in Xenopus extracts The Xenopus embryonic cell cycle Entry into mitosis in early embryonic Xenopus cell cycle extracts depends on the translation of cyclin B alone,20 an indication that all other steps occur posttranslationally. However, cyclin B synthesis is not the only rate-limiting step.4,22 Even after the accumulation of a critical threshold level of cyclin B, there is an approximate 20-min lag phase during which inhibitory modifications of the cyclin B/cdc2 complex suppress its activity, until the G2/M transition. The lag phase can be viewed as a mechanism that transforms the continuous synthesis of cyclin B protein into a concerted activation of the cyclin B/cdc2 complex, and consequently an abrupt transition into mitosis. Not only does this provide a mechanism for the proper timing of mitosis, but may ensure that once mitosis is initiated, a sufficient pool of cyclin B/cdc2 precursor has accumulated to induce mitotic events. Furthermore, in the older embryo, when feedback controls are operative, the components of the lag phase may provide a mechanism linking mitotic initiation to the completion of S phase events. As the function of PP2A is to regulate this lag period, the remainder of this discussion will focus on the regulators of the lag as potential targets of PP2A. The mechanism of the lag phase has yet to be precisely understood, but many of the regulators that contribute to the lag have been identified. These factors can be described as a biochemical pathway that lies between the accumulation of cyclin B and the final activation of the cyclin B/cdc2 complex.4,21,22 The role of cyclin A, if any, in the generation of the lag phase, remains to be established. Ablation of cyclin A message appears to interfere with the cryptic feedback control mechanisms that can, in early Xenopus embryos, link the completion of DNA synthesis to cyclin B/cdc2 activation.24 However, the kinetics of cdc2 activation by cyclin B, in the normal embryonic cell cycle, is unchanged when cyclin A is ablated,24 suggesting that cyclin A is not essential for either the
In contrast to that of somatic cells, the early embryonic cell cycle of Xenopus lacks the feedback controls that normally link the completion of downstream events to cell cycle transitions. Blocking DNA synthesis or the assembly of the mitotic spindle, would lead to a G2 or mitotic delay, respectively, in most somatic cells,16 but has no effect on the cycling between S phase and M phase observed in the early embryonic cell cycle of Xenopus.17 This apparent lack of higher order controls appears not to reflect a fundamental difference in the cell cycle machinery of Xenopus, so much as the unusually small nuclear to cytoplasmic ratio of the egg, since increasing the number of nuclei per volume of cytoplasm in in-vitro eggs extracts reveals cryptic feedback control mechanisms.18,19 The relative simplicity of this embryonic cell cycle can be recapitulated in vitro,20 providing a useful system for dissecting the basic regulatory network that is required for the interconversion between interphase and mitotic states. Biochemical experiments in Xenopus extracts, combined with genetic analyses in yeast, have laid the foundation for a mechanistic understanding of the G2/M transition.4 Essential regulators, many of which were first identified in yeast,21 have been identified and extensively characterized in Xenopus. Furthermore, the simplicity of the Xenopus system has led to a fairly detailed kinetic formulation of the G2/M transition.22 In this in-vitro system and consistent with genetic studies in S. pombe PP2A definitely functions as a negative regulator of the G2/M transition. As described above, the Xenopus embryonic cell cycle differs from the yeast cell cycle in that it lacks feedback control mechanisms that link the execution of structural and regulatory events in most somatic cells; therefore, it has allowed the clearest dissection of the mechanism by which PP2A controls the key event in mitotic induction, the activation of the 205
T. H. Lee mik1, in the highly activated state,22,25 and the maintenance of the phosphatase that removes the inhibitory phosphates, cdc25, in the inactivated state, prior to the transition.22,26,27 The clear importance of these enzymes in regulating the transition has made them the subject of intense study. During the transition, changes in the activities of cdc25 and wee1/mik1 seem to be important for the abrupt activation of cdc2 and the sudden transition into mitosis. These changes are brought about by extensive phosphorylations of both cdc2526-29 and wee130-33 Thus, the initiation of the transition has been attributed to the activation of a ‘starter kinase’ that phosphorylates (and/or inactivation of a phosphatase that dephosphorylates) both cdc25 and wee1. That the mitotic phosphorylation of cdc25 and wee1 coincide with the abrupt activation of cdc2/cyclin B has led to the suggestion that a low level of cdc2/cyclin B activity, escaping the inhibitory modifications during interphase, turns off wee1 and
lag phase, nor for the final activation of cdc2 by cyclin B. Activation of cdc2 by cyclin B requires association with cyclin B, ostensibly because cyclin binding allows the phosphorylation of cdc2 on an essential, activating site, threonine 161. However, cyclin binding simultaneously induces the phosphorylation of cdc2 on a pair of adjacent inhibitory sites, threonine 14 and tyrosine 15. The inhibitory phosphorylations maintain the bulk of the cyclin bound cdc2 in an inactive state until the transition. Once cyclin exceeds a critical threshold level, it initiates a series of events that lead ultimately to the sudden removal of the inhibitory phosphorylations on cdc2, leaving only the essential thrl61 phosphorylation. The suppression of cdc2 activity during interphase as well as its abrupt activation at the transition, depends on the maintenance of the kinases that phosphorylate the thr14 and tyr15 residues, wee1 and
Figure 1. Model for the initiation of the G2/M transition, and the mechanism by which PP2A controls the transition. During interphase, PP2A inhibits two pathways, both required for the final activation of cdc2 by cyclin B. One pathway leads to the formation of a low level of the active complex (cyclin B/cdc-T161P), and the other leads to the formation of a second activator (?) that remains to be identified, indicated in the dashed box. The accumulation of a critical threshold level of cyclin B, however, eventually overcomes the block imposed by PP2A, allowing the formation of both rate-limiting activators. The concurrent activation of a threshold level of both of these activators initiates the positive-feedback loop that turns cdc25 activity up and wee1 activity down, leading to further dephosphorylation and activation of the bulk inhibited (cyclin B/cdc2T14,Y15P) complexes. Thus cyclin B/cdc2, its activators, and their cohorts are abruptly switched into the mitotic state, and M phase is initiated.
206
Role of protein phosphatase type 2A in xenopus cell cycle phase, the period of time when the kinetics of the reaction pathways leading to the activation of cdc2 evaluate either the sufficiency or insufficiency of the cyclin level. The rates of phosphorylation and dephosphorylation of both the inhibitory (thr14, tyr15) and activating sites on cdc2 (thr161) were examined, during the lag phase, prior to the transition.35 Somewhat surprisingly, the rates of phosphorylation and dephosphorylation of the inhibitory sites were unaffected by levels of PP2A that ultimately blocked the transition. This indicated that although the state of cdc25 and weel activities was highly dependent on the levels of PP2A and cyclin in the system, this effect was an indirect consequence of the modulation of an upstream regulator. An analysis of the rates of phosphorylation and dephosphorylation of the thr161 site during the lag, however, yielded a different result. PP2A inhibited the initial rate of accumulation of phosphate at that site. Furthermore, the inhibition was due to an effect on the phosphorylation, rather than the dephosphorylation rate of thr161 (there was no measurable dephosphorylation of thr161), ruling out the earlier model that PP2A regulated the activation of cdc2 by directly dephosphorylating the essential, activating site.36 Whether the inhibition of phosphorylation occurs through the modulation of the enzyme that catalyses thr161 phosphorylation,4 remains to be determined. In summary, the only pathway directly affected by PP2A was that controlling the phosphorylation of thr161.
turns on cdc25, which then feeds back to activate the bulk thr14, tyr15 phosphorylated cdc2.22-28 The initiating event, in this positive feedback loop model, is the accumulation of a critical threshold level of the thr161 phosphorylated, active cdc2/cyclin B complex (Figure 1).
The role of PP2A in the regulation of the G2/M transition in Xenopus The kinetic parameters governing cyclin activation of cdc2 can be modulated by PP2A. Felix et al34 and Solomon et al22 observed that the addition of okadaic acid, a potent PP2A inhibitor, to Xenopus interphase extracts induced the premature activation of cdc2 kinase. Thus PP2A appeared to function as a direct inhibitor of cdc2 activation. The specific effects of altering the level of PP2A activity on the activation process indicated that the length of the lag phase was determined by PP2A. In addition, the level of PP2A activity determined the amount of cyclin B protein required to initiate cdc2 kinase activation in the interphase extract. In summary, PP2A activity appeared to play an essential role in restraining cdc2 activation during interphase, and thus in maintaining the interphase state. PP1 inhibitors, on the other hand, had no detectable effect on the interphase to mitosis transition.34
Potential mechanisms of PP2A action Is thr161 phosphorylation the relevant target of PP2A?
The effect of PP2A on the timing of mitotic initiation, as well as its relationship to cyclin B, strongly suggested that PP2A might oppose cyclin in the control of a rate-limiting step for the initiation of the transition. Thus the identification of the target of PP2A during interphase promised to reveal the initiating event for the interphase to M phase transition. However, the analysis was once again complicated by interdependencies. That is, increased levels of PP2A inhibit the transition and all of the events that normally accompany the transition, such as the activation of cdc25 and the inactivation of wee1. But not all transitional events may be targets of PP2A; rather, they may occur as an indirect consequence of some other, upstream, initiating event. Therefore, a distinction between direct and indirect effects had to be made. In order to isolate the initiating event from the transitional events, analysis was focussed on the lag
The thr161 phosphorylation pathway was sensitively modulated by the ratio of cyclin B and PP2A, raising the possibility that this reaction was the rate-limiting event for entry into M phase. Such a hypothesis fit nicely with the positive feedback loop model which postulated that the accumulation of a low but critical threshold level of active cdc2/cyclin B complexes was required to trigger a switch in the balance of cdc25 and wee1 activities, which would lead to further activation of the bulk of cdc2/cyclin B complexes held inactive by the inhibitory phosphorylations.22,28 By delaying the onset of thr161 phosphorylation, PP2A would allow the inhibitory phosphorylations to outcompete thr161 phosphorylation, and active complexes would fail to form. A simple test of the model, however, proved otherwise.35 The addition of a suprathreshold level of active cdc2/cyclin B complexes to 207
T. H. Lee past and present members of the Kirschner lab, for many stimulating discussions, and M. Cyert for helpful comments on the manuscript. A grant from the NIH supported the writing of this manuscript.
interphase extracts did not substitute for the ratelimiting event. Instead, the complex was rapidly inactivated, indicating that at least one other unidentified pathway critical for mitotic initiation must exist. Furthermore, the presence of okadaic acid prevented the inactivation of the complex, suggesting that this unidentified pathway is regulated by PP2A.
References 1. Hartwell LH, Weinert TA (1989) Checkpoints: Controls that ensure the order of cell cycle events. Science 246:629–633 2. Sherr C (1993) Mammalian G1 cyclins. Cell 73:1059–1065 3. Heichman KA, Roberts JM (1994) Rules to replicate by. Cell 79:557–562 4. King RW, Jackson PK, Kirschner MW (1994) Mitosis in transition. Cell 79:563–571 5. Cohen P (1989) The structure and regulation of protein phosphatases. Annu Rev Biochem 58:485–497 6. Walter G, Mumby M (1993) Protein serine/threonine phosphatases: Structure, regulation, and function in cell growth. Physiol Rev 73:673–699 7. Ingebritsen TS, Stewart AA, Cohen P (1983) The protein phosphatases involved in cellular regulation: Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. Eur J Biochem 132:297–307 8. Labib K, Nurse P (1993) Bring on the phosphatases. Curr Biol 3:164–166 9. Mayer-Jaekel RE, Hemmings BA (1994) Protein phosphatase 2A — a ‘menage a trois’. Trends Cell Biol 4:287–291 10. Schonthal A (1992) Okadaic acid — a valuable new tool for the study of signal transduction and cell cycle regulation? New Biologist 4:16–21 11. Ohkura H, Kinoshita N, Miyatani S, Toda T, Yanagida M (1989) The fission yeast dis2 + gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell 57:997–1007 12. Doonan JH, Morris NR (1989) The bim G gene of Asperigillus nidulans, which is required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1. Cell 57:987–996 13. Axton JM, Dombradi B, Cohen PTW, Glover DM (1990) One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell 63:33–46 14. Kinoshita N, Ohkura H, Yanagida M (1990) Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle. Cell 63:405–415 15. Healy AM, Zolnierowicz S, Stapleton AE, Goebl M, DePaoliRoach AA, Pringle JR (1991) CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization and homology to the B subunit of mammalian type 2A protein phosphatase. Mol Cell Biol 11:5767–5780 16. Nurse P (1994) Ordering S phase and M phase in the cell cycle. Cell 79:547–550 17. Hara K, Tydeman P, Kirschner M (1980) A cytoplasmic clock with the same period as the division cycle in Xenopus eggs. Proc Natl Acad Sci USA 77:462–466 18. Dasso M, Newport JW (1990) Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus. Cell 61:811–823 19. Minshull J, Sun H, Tonks NK, Murray AW (1994) A MAP kinasedependent spindle assembly checkpoint in Xenopus egg extracts. Cell 79:475–486 20. Murray AW, Kirschner MW (1989) Cyclin synthesis drives the early embryonic cell cycle. Nature 339:275–280 21. Nurse P (1990) Universal control mechanism regulating onset of M-phase. Nature 344:503–508
The mechanism of mitotic initiation remains unsolved Thus, the direct target of PP2A in the control of cdc2/ cyclin B activation has yet to be determined. However, PP2A has proved to be a useful probe for dissecting the mechanism of mitotic initiation. The studies described here have led to the conclusion that pathways that actually trigger the G2/M phase transition remain to be identified, and have led us to search for new regulators. Along these lines, recent studies have identified at least two potentially novel regulators of cdc2/cyclin B in Xenopus extracts. One acts as a stoichiometric inhibitor of the kinase complex, and seems to be required to maintain the interphase state37 The second is a kinase(s), other than cdc2, that phosphorylates cdc25 and wee1. Mitotic cdc25 has recently been shown to possess a highly conserved, M phase specific phosphoepitope recognized by the MPM-2 antibody,38,39 suggesting that the kinase that phosphorylates this MPM-2 epitope plays an important role in mitotic induction. Similarly, the mitotic phosphorylation of wee1 has been shown to be due to a kinase other than cdc2.32 Interesting, each of these activities, which remains to be identified, is sensitive to PP2A.32,37,39
Concluding remarks The targets of PP2A in cell cycle progression are likely to be numerous, comprising both regulatory and structural elements of the dividing cell. If the study of the mechanism by which PP2A controls mitotic initiation has told us anything, it is that the broad substrate specificity phosphatases, far from being uninteresting, provide handles for investigating difficult cell biological problems.
Acknowledgements I would like to thank M. Kirschner and J. Kuang, as well as
208
Role of protein phosphatase type 2A in xenopus cell cycle 31. Parker LL, Piwnica-Worms H (1992) Inactivation of the p34cdc2 cyclin B complex by the human wee1 tyrosine kinase. Science 257:1955–1957 32. Tang Z, Coleman TR, Dunphy WG (1993) Two distinct mechanisms for negative regulation of the Wee1 protein kinase. EMBO J 12:3427–3436 33. Wu L, Russell P (1993) Nim 1 kinase promotes mitosis by inactivating Wee1 tyrosine kinase. Nature 363:738–741 34. Felix MA, Cohen P, Karsenti E (1990) Cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid. EMBO J 9:675–683 35. Lee TH, Turck C, Kirschner MW (1994) Inhibition of cdc2 activation by INH/PP2A. Mol Biol Cell 5:323–338 36. Lee TH, Solomon MJ, Mumby MC, Kirschner MW (1991) INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell 64:415–423 37. Lee TH, Kirschner MW (1994) Evidence for a stoichiometric inhibitor of MPF that regulates the G2/M transition in Xenopus. Manuscript submitted. 38. Davis FM, Tsao TY, Fowler SK, Rao PN (1983) Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci USA 80:2926–2930 39. Kuang J, Ashorn CL, Gonzalez-Kuyvenhoven M, Penkala JE (1994) Cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor. Mol Biol Cell 5:135–145
22. Solomon MJ, Glotzer M, Lee TH, Philippe M, Kirschner MW (1990) Cyclin activation of p34 cdc2. Cell 63:1013–1024 23. Kinoshita N, Yamano H, Niwa H, Yoshida T, Yanagida M (1993) Negative regulation of mitosis by the fission yeast protein phosphatase ppa2. Genes & Dev 7:1059–1071 24. Walker DH, Maller JL (1991) Role for cyclin A in the dependence of mitosis on completion of DNA replication. Nature 354:314–317 25. Smythe C, Newport JW (1992) Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34.cdc2 Cell 68:787–797 26. Izumi T, Walker DH, Maller JL (1992) Periodic changes in phosphorylation of the Xenopus Cdc25 phosphatase regulates its activity. Mol Biol Cell 3:927–939 27. Kumagai A, Dunphy WG (1992) Regulation of the Cdc25 protein during the cell cycle in Xenopus extracts. Cell 70:139–151 28. Hoffman I, Clarke PR, Marcote MJ, Karsenti E, Draetta G (1993) Phosphorylation and activation of human Cdc25-C by cdc2-cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J 12:53–63 29. Izumi T, Maller JL (1993) Elimination of cdc2 phosphorylation sites in the Cdc25 phosphatase blocks initiation of M-Phase. Mol Biol Cell 4:1337–1350 30. Coleman TR, Tang TZ, Dunphy WG (1993) Negative regulation of the wee1 protein kinase by direct action of the nim1/cdr1 mitotic inducer. Cell 72:919–929
209